Activation of Cell Function: Pharmacological Agents Which Degranulate Mast Cells and Cause Skeletal Muscle to Contract Citation Herndier, Brian (1981) Activation of Cell Function: Pharmacological Agents Which Degranulate Mast Cells and Cause Skeletal Muscle to Contract. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9txk-fw43. https://resolver.caltech.edu/CaltechTHESIS:03062018-113544116 Abstract Chapter I This chapter presents a general introduction to the cellular metabolism of calcium with particular reference to the concept of calcium pools. The importance of the ionized calcium pool of the cytosol in determining the specific function of cells and tissues is discussed. A model for stimulus-release coupling in mast cells is presented. Excitation-contraction (EC) coupling in skeletal muscle is defined and the fundamental premises required for a theory on EC coupling are given. The two basic theories of excitation-contraction coupling are briefly reviewed. Chapter II We have discovered a new class of drugs which induce the release of 3 H-serotonin in mast cells. Verapamil, previously described as a "specific calcium antagonist", was found to release 3 H-serotonin from mast cells. Our study investigated and compared verapamil and compound 48/80, a drug previously known to be a potent degranulator of mast cells. This investigation revealed that low concentrations of verapamil inhibited the spontaneous release of 3 H-serotonin and efflux of 86 Rb + in mast cells. High concentration of verapamil caused an increase in the rate of the release of 3 H-serotonin and 86 Rb + from mast cells. Compound 48/80 causes only an increase in the release 3 H-serotonin and 86 Rb + -efflux. The effect of compound 48/80 and verapamil on the rate of 86 Rb + -efflux in mast cells was investigated. The significance of these results is discussed. Verapamil, at low concentrations, is thought to act as a "specific Ca channel blocker". The increase in 86 Rb + efflux from mast cells induced by high concentration of verapamil suggests an increase in concentration of cytosolic Ca ++ . The mobilization of calcium into the cytosolic pool is believed also to trigger the release of 3 H-serotonin from the mast cell. We propose that verapamil is not simply a calcium antagonist in mast cells but also has the ability at higher concentrations to cause an increase in ionic or cytosolic Ca 2+ . Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Chemistry) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Dervan, Peter B. Thesis Committee: Dervan, Peter B. (chair) Richards, John H. Hood, Leroy E. Raftery, Michael Augustine Defense Date: 4 May 1981 Funders: Funding Agency Grant Number National Research Council of Canada UNSPECIFIED Caltech UNSPECIFIED Record Number: CaltechTHESIS:03062018-113544116 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:03062018-113544116 DOI: 10.7907/9txk-fw43 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 10754 Collection: CaltechTHESIS Deposited By: Mel Ray Deposited On: 06 Mar 2018 21:45 Last Modified: 10 Feb 2025 23:26 Thesis Files Preview PDF - Final Version See Usage Policy. 76MB Repository Staff Only: item control page

ACTIVATION OF CELL FUNCTION; PHARMACOLOGICAL AGENTS WHICH DEGRANULATE MAST CELLS AND CAUSE SKELETAL MUSCLE TO CONTRACT Thesis by Brian Herndier In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 1981 (Submitted May 4, 1981) -iiAcknowledgements I would like to gratefully acknowledge my research advisor, Professor John H. Richards, for his friendship, patience and guidance during my stay at Caltech. I am also extremely appreciative of the freedom I was allowed in pursuing my research interests and his help and advice whenever needed, especially during the preparation of this thesis. Scientifically, I acknowledge my partnership with Neal Handly in the experimental work and intellectual develop- ment of the muscle project. I would also like to acknowl- edge Tom Perkins for the difficult NMR characterizations described in this thesis. I also appreciate the help from David Strader during the early phases of our research at Caltech. I wish also to acknowledge the financial support of the National Research Council of Canada and the California Institute of Technology. I enjoyed the environment of the Chemistry Department at Caltech especially working with Professor Michael A. Raft~ry in Bi 110 and playing tennis with Professor Norman Davidson. I also appreciate the assistance of Professor William Dorson in the preparation of this thesis, the production of this thesis by Debbie Chester (unless we don't finish) and the absolutely essential laboratory work of our technician, Betty Aalseth, was greatly appreciated. -iiiI would like to acknowledge the friendship and guidance of Professor Donald E. Brooks during my initial attempts at scientific research as an undergraduate at UBC. Most of all I would like to acknowledge the close friends made in the lab and around campus during my stay at Caltech, for it is these friendships that make life transcend. -iv- ABSTRACTS Chapter I This chapter presents a general introduction to the cellular metabolism of calcium with particular reference to the concept of calcium pools. The importance of the ionized calcium pool of the cytosol in determining the specific function of cells and tissues is discussed. A model for stimulus-release coupling in mast cells is presented. Excitation-contraction (EC) coupling in skeletal muscle is defined and the fundamental premises required for a theory on EC coupling are given. The two basic theories of excitation-contraction coupling are briefly reviewed. Chapter II We have discovered a new class of drugs which induce the release of 3 H-serotonin in mast cells. Verapamil, previously described as a "specific calcium antagonist", was found to release 3 H-serotonin from mast cells. Our study investigated and compared verapamil and compound 48/80, a drug previously known to be a potent degranulator of mast cells. This investigation revealed that low concentrations of verapamil inhibited the spontaneous release of 3 H- -vserotonin and efflux of 86 Rb+ in mast cells. High concen- tration of veraparnil caused an increase in the rate of 3 86 + the release of H-serotonin and Rb from mast cells. Compound 48/80 causes only an increase in the release of 3 H-serotonin and 86 + Rb -efflux. The effect of compound 48/80 and verapamil on the rate of 86 Rb+-efflux in mast cells was investigated. The significance of these results is discussed. Verapamil, at low concentrations, is thought to act as a "specific Ca channel blocker 11 • The increase in 86 Rb+ efflux from mast cells induced by high concentration of verapamil suggests an increase in concentration of . cytosol1c c a ++ . The mobilization of calcium into the cytosolic pool is believed also to trigger the release of 3 H-serotonin from the mast cell. We propose that verapamil is not simply a calcium antagonist in mast cells but also has the ability at higher concentrations to cause an increase in ionic or cytosolic Ca 2 +. -viChapter III A comparative study of the effects of compound 48/80, verapamil, and newly synthesized derivative of verapamil called "crosslinked verapamil" on mast cells has been undertaken with the goal to identify the cellular site of action of the drugs. We have studied the role of crosslinking in the mechanism of action of compound 48/80 and verapamil. Analogies are drawn to our study of the interaction of crosslinked antigen with immunoglobulin E bringing about the degranulation of mast cells. linked verapamil was found to be causing the release of 3 Cross- more potent in H-serotonin from mast cells. Pretreatment of mast cells with neuraminidase was found to inhibit the r~lease of 3 H-serotonin caused by compound 48/80 but does not change the release of 3 H- serotonin caused by verapamil and crosslinked verapamil. We propose that compound 48/80 interacts with components of the cell surface that contain negatively charged sialic acids. In contrast, verapamil and crosslinked verapamil could operate intracellularly or at cell membrane sites that are not affected by pretreatment of the mast cell with neuraminidase. We also examined possible mechanisms for the degranulation of mast cells by verapamil and discussed the kinetics of 3 H-serotonin release from mast cells caused by compound 48/80, crosslinked verapamil and verapamil. -vii- Chapter IV The nature of a contracture in skeletal muscle caused by compound 48/80, a potent agent of degranulation of mast cells, was investigated. The observation that the contracture was inhibited by pretreatment of the muscle indicated that compound 48/80 interacts with cell surface structures that contain neuraminidase-sensitive sialic acids essential for activity. An increase in myoplasmic [Ca++] during contracture caused by compound 48/80 was suggested by the observation that the rate of 86 Rb+- efflux and the rate of protein degradation are increased when the muscle is challenged by the drug. Measurements of 45 ca uptake during pharmacological challenge of the muscle indicated that the increase in the influx of extracellular Ca ++ caused by compound 48/80 was not correlated with contracture, i.e., the increase in 45 ca uptake still occurred in muscle pretreated with neuraminidase but unable to undergo contracture when challenged with compound 48/80. This finding and the observation that contracture could occur in the absence of extracellular calcium indicated that the extracellular medium was not the source of Ca++ required to cause the increase in myoplasmic [Ca++] sufficient to activate the contractile apparatus. -viiiA contracture of skeletal muscle caused by verapamil was also investigated by examining dose-dependent 86 Rb+efflux. This study revealed that verapamil could be mobilizing intramuscular pools of calcium in analogy to its effects on mast cells to cause contracture. A mechanistic model consistent with the experimental results is proposed to explain contracture and increased 45 Ca uptake caused by compound 48/80 and tests of the model are offered. Parallels between stimulus-secretion coupling in mast cells and stimulus-contracture coupling in skeletal muscle are summarized. -ixABBREVIATIONS ATP adenosine DMEM Dulbecco's modified Eagles' medium EC excitation-contraction EDTA ethylenediamine tetraacetic acid EGTA ethyleneglyco-bis(S-aminoethyl ether) triphosphate N,N'-tetraacetic acid IgE immunoglobulin E MCB mast cell buffer (Materials and Methods, Chapter II) PAB protein anabolism buffer (Materials and Methods, Chapter IV) PBS phosphate buffered saline PC phosphatidyl choline PCA passive cutaneous anaphylaxis PE phosphatidyl ethanolamine PS phosphatidyl serine SAM S-adenosyl methionine SR sarcoplasmic reticulum t-tubule transverse tubule TCA trichloracetic acid -x- TABLE OF CONTENTS CHAPTER I: BACKGROUND . Introduction to Mast Cells . . . . . Excitation-Contraction Coupling in Skeletal Muscle . . . . . . . Excitation-Contraction Coupling Theories: The Coupling between the t-tubules and the Sarcoplasmic Reticulum References CHAPTER II: 1 7 . 12 . . 16 . 18 VERAPAMIL; A NEW AGENT FOR ACTIVATION OF MAST CELLS Introduction . . . 22 . 23 Verapamil . . 23 Compound 48/80 . . . . . . . . . . . . 27 Stimulus-Permeability Coupling . . . . . . . . . 29 Materials and Methods . . 30 Isolation of Rat Peritoneal Mast Cells . . . 30 Measurement of 3H-Serotonin Release in Rat Mast Cells . . . . . . . . . . . . . 35 P815 Mastocytoma: Origin and Maintenance . . . 36 Results . 37 Results Establishing the General Nature of the Release of 3H-Serotonin from Mast Cells . 37 86Rb+-3H-Serotonin Double Label Experiments in Mast Cells . . . . . . . . . . . . 43 Discussion . The Effect of Verapamil on Mast Cells Possible Indirect Effects of Verapamil on Blood Pressure . . . . . References . . . . . . . 53 . . 54 . 57 . 59 -xiCHAPTER III: A COMPARATIVE STUDY OF THE THE ACTIVATION OF MAST CELLS BY COMPOUND 48/80 AND VERAPAMIL Introduction 62 63 Crosslinking and Degranulation of Mast Cells; IgE . . . . . . . . . . Crosslinking and Degranulation of Mast Cells; Non-Antibody Reagents . . . Sialic Acids and Neuraminidase . . . . Molecular Weight Determination of Crosslinked Verapamil . . . . NMR Characterization of Crosslinked Verapamil . . . . . . . . . . . . . 63 66 67 69 75 97 Discussion Discussion of Neuraminidase Results . . . . . . 99 Discussion of Possible Mechanisms . 102 Kinetics of the Release of 3H-Serotonin from Mast Cells . . 106 108 References CHAPTER IV; A STUDY OF CONTRACTURE OF SKELETAL MUSCLE CAUSED BY COMPOUND 48/80 . . . 111 Introduction 112 Muscle Contracture As A Model for ExcitationContraction Coupling . 112 Protein Turnover in Muscle . 115 Materials and Methods Isolation of Frog Skeletal Muscles . . Denervation of the Sartorius Muscle Treatment of Frog Muscles with Neuraminidase . . . . . . . . . . . Measurement of the Efflux of 86Rb+ from Frog Muscle . . . . . . . . . . . . Protein Degradation Measurements Origin and Maintenance of LB Myoblsst Cell Line . . . . . . . . . . . . . Protein Degradation in L8 Cells . . . . Measurement of 45Ca Uptake . . 117 . . . . 117 . . . . 118 . . . . 119 . 120 . 121 . . . . 123 . . . . 124 . . . . 125 -xii- Assay of Protein Synthesis in Frog Skeletal Muscle . . . . . . . 126 . 128 Results . . . General . . . . . . . . . . 128 Neuraminidase and Skeletal Muscle . . . . . 130 Efflux of 86Rb+ from Skeletal Muscle . . . . . 131 Protein Degradation in Skeletal Muscle . . 138 Uptake of 45ca in Skeletal Muscles . . . 144 Protein Synthesis in Skeletal Muscle . 150 Discussion . 153 Model for the Contracture of Frog Skeletal Muscle Caused by Compound 48/80 . . . . . Evidence Relevant to Location of "Trigger" Structures in t-Tubules . . . . . . . . . Evidence for an Increase in Myoplasmic [ca++] Possible Source of ca++ for Contracture . . . Conclusion and Further Tests of the Model . . Parallels between Pharmacological Challenge of Rat Mast Cells and Skeletal Muscle. A Summary . . . . . . . . . . . . . . References . 153 . 157 160 . 164 . 169 . 173 . . 175 -xiii- LIST OF FIGURES Chapter Figure Title Page 2 1 The role of energy metabolism in the release of 3H-serotonin from rat peritoneal cells challenged by verapamil . . . . . . 39 The role of energy metabolism in the release of 3H-serotonin from rat peritoneal cells challenged with compound 48/80 41 Double label experiment measuring the initial rates of release of 86Rb+ and 3H-serotonin from rat peritoneal cells challenged with compound 48/80 . 45 Double label experiment measuring the initial rates of release of 86Rt+ and 3 H-serotonin from rat peritoneal cells challenged with verapamil. . . . . . 47 The release of 86Rb+ from P815 mastocytoma cells challenged by compound 48/80 . . . . . 49 The release of 86Rb+ from P815 mastocytoma cells by verapamil 51 Molecular weight determination of crosslinked verapamil: gel filtration on a G-15 Sephadex column . . . . . . . . 71 Molecular weight determination of crosslinked verapamil: gel filtration on a G-25 Sephadex column 73 Proton magnetic resonance (500.13 MHz) spectrum and peak assignments of monomeric verapamil in D20 . . . . . . 77 2 3 4 5 6 3 7 8 9 -xivChapter Figure 10 11 Title Proton magnetic resonance (500.13 MHz) spectrum and peak assignments of crosslinked verapamil in n2 o. . . . . . . . Page . 78 The release of 3H-serotonin from rat peritoneal cells challenged by compound 48/80, crosslinked verapamil and verapamil . . . . . 80 12 Effect of neuraminidase on the release of 3H-serotonin from rat peritoneal cells challenged with compound 48/80, crosslinked verapamil and verapamil . . . . . . 82 13 Effect of calcium concentration on 3H-serotonin release from rat peritoneal cells challenged by compound 48/80 . . . . . . . . . . . 87 14 Effect of calcium concentraion on 3H-serotonin release from rat peritoneal cells challenged with verapamil . . . . . . . . . . . . 89 The effect of lanthanum concentration on calcium uptake in P815 mastocytoma cells . . . . . 91 15 16 Rate of release of 3 H-serotonin fron rat peritoneal cells challenged with high concentrations of verapamil and crosslinked verapamil . . . . . . . . . . . . . 93 17 Rate of release of 3H-serotonin from rat peritoneal cells challenged with crosslinked verapamil, verapamil, and compound 48/80 . . . 95 -xvChapter Figure 4 18 Initial rates of 86 Rb+-efflux from frog skeletal muscle challenged with compound 48/80. . . 132 19 Efflux of 86Rb+ from a frog sartorius muscle challenged with compound 48/80 for different periods of time . . . . . . . . . . 134 20 Initial rates of 86 Rb+-efflux from frog skeletal muscle challenged with verapamil . . . . . 136 21 Protein degradation in frog skeletal muscle: effect of neuraminidase and compound 48/80 . . . . . 140 Effect of compound 48/80: protein degradation in LS myoblast cells. 142 Calcium uptake in frog skeletal muscle: effect of neuraminidase and compound 48/80. . . . . . . . 146 Effect of verapamil and compound 48/80 on calcium uptake in frog skeletal muscle . . . . . . . . . 148 Protein synthesis in frog skeletal muscles: effect of neuraminidase and compound 48/80. . . . . . 151 Schematic of Skeletal Muscle 156 22 23 24 25 26 Tile Page -xvi INDEX OF TABLES Table I Table II Table III Table IV Cellular Events Initiated or Modulated by changes in Cytosolic [ca++] . . . . 6 Effect of Verapamil (or D-600) on Cellular Function . . . . . . . . . 25 Release and Retention of 3H-Serotonin in Mast Cells Preincubated in IgE 14-205 Challenged by CrosslinkedOvalbumin or Ovalbumin. . . . . . . . . . . . . . . 86 Summary of Similar Findings on Rat Mast Cells and Frog Skeletal Muscle of this Thesis . . . . . . . . . . . 174 CHAPTER I BACKGROUND 2 A multicellular organism requires specialization of cellular function which in turn mandates communication between the various types of cells such that the organism can generate an integrated response to environmental change. A common mechanism of cellular communication involves the interaction of an extracellular messenger with specific cellular receptors causing the release of intracellular secondary messengers, thereby triggering the cellular response. An example of an integrated re- sponse that illustrates these points is the immediate hypersensitivity reaction to antigen. An antigen which binds to a complex of immunoglobulin E and a specific IgE-receptor on the mast cell membrane (Metzger, 1977) triggers a change in calcium permeability in the mast cell membrane. This causes a transient rise in the con- centration of calcium, a secondary messenger, in the cytosol o f t h e mast eel 1 . . Ca++ triggers . Th e cytoso 1 ic a series of biochemical events that lead to the degranulation and subsequent release of biogenic amines and chemotactic factors from the cell (Hirata and Axelrod, 1980). In turn the biogenic amines and chemotactic factors continue the integrated response of immediate hypersensitivity by specifically interacting with other target cells and tissues. For example, histamine interacts with target cells of the peripheral 3 vasculature causing vasodilation and increased fluid permeability across the vascular epithelium (Middleton, 1980). Again, these final events of immediate hyper- sensitivity depend on the interaction of an extracellular messenger, histamine, with a specific cellular receptor which in turn leads to the tissue or cellular response. The interaction of extracellular messengers with specific cell surface receptors causing the generation of intracellular secondary messengers mechanisms. has two basic The receptor, such as the IgE receptor (Metzger, 1979),could be at the cell surface and serve via induction of changes in structure and enzymatic activity in the membrane as a generator of intracellular secondary messenger or the intracellular messenger could be transported directly into the cytoplasm, such as the steroid-receptor complex (Bischoff and Bryson , 1977), to interact with target structures to cause the cellular response. Regardless of the site of action of the extracellular messenger the induction of intracellular secondary messengers or even tertiary messengers are central features which, depending on the differentiated cell type, initiate or modulate the particular cellular response. 4 Calcium ions have been implicated as secondary and tertiary messengers in the cellular response to the extra cellular signals in a large number of systems. The mes- senger function of calcium occurs within the framework of the highly complex cellular metabolism of calcium. A useful model for understanding the cellular metabolism of calcium involves the concept of calcium pools. The principal pools of calcium of the "typical" cell are extracellular calcium, calcium bound to the plasma membrane, intramitochondrial calcium, calcium within the endoplasmicreticulum, bound calcium in the cytosol and free ionized calcium in the cytosol. The distribution of calcium in these pools is partially determined by the permeability of calcium across the plasma membrane, the mitochondrial membrane and the membrane of the endo- plasmic reticulum. Also the, ATP-dependent calcium pumps in these membranes play an important role in determining the distribution of calcium in the cellular pools. In muscle the highly specialized sarcoplasmic reticulum determines to a large extent the transient distribution of calcium in this tissue. A cellular response that involves messenger calcium is usually correlated with a change in the concentration of free ionized calcium in the cytosol. For example, a transient increase in the free myoplasmic calcium con- 5 centration from 0.1 µM to 1-2 µM is seen in the excitationcontraction cycles of skeletal muscle (Caputo, 1978). The principal source of the messenger calcium in muscle is the sarcoplamsic reticulum and the target of the messenger is the regulatory troponin contractile apparatus. of the actin-myosin The contractile response is terminated by the removal of the messenger Ca ++ from the myoplasm by the ATP-dependent pumps of the sarcoplasmic reticulum (Hasselbach, 1980). A partial list of cellular events that are known to be initiated or modulated by changes in the concentration of calcium in the important cytosolic pool are given in Table I. Table I, Chapter I - Cellular Events Initiated of Modulated by Changes in Cytosolic [Ca++] Tissue Effect Reference + K -efflux Gardos (1958) Salivary gland differential ion flux and fluid transport Douglas and Poisner (1963) Lacrimal gland differential ion flux and fluid transport Parod and Putney (1978) Exocrive pancreas enzyme release Case (1978) Liver cell a-adrenergia glucose release De Wulf and Keppens (1976) Gastrointestinal epithelium electrolyte and water exchange Frizzell (1977) Adipocytes a-adrenergie activation Perry and Hales (1970) Neutrophils chemotactic responses Naccache et al (1977) Neurons transmitter release Rahamimoff et al (1978) Mast cells IgE-induced degranulation Hirata and Axelrod (1980) Renal cells gluconeogenesis Nagata and Rasmussen (1970) Protozoa ciliary motility Schmidt and Eckert (1976) Pancreatic B-cells secretion of insulin Malaisse et al Egg cell activation Okamota et al (1976) Red cell and secretory ( 1976) CJ) 7 Introduction to Mast Cells Mast cells are granulocytes found in the connective tissue and are characterized by staining with basic dyes (Ehrlich, 1879). Acid mucopolysaccharides, such as heparin, account for the metachromasia of the granules. The mast cell is closely related to the blood-borne basophil. The basophil is round and regular in cell shape, contains a multilobed nucleus similar to other granulocytes and has relatively few granules. The mast cell has irregular cell shapes, has a round nucleus and has numerous delicate granules. The granules of both cells contain histamine and serotonin stored as an amine-protein heparin complex (Bergendorff and Uvnas, 1972). The triggering of the mast cell is the focal point of immediate hypersensitivity. Mast cells when activated release the contents of their granules in a process called degranulation. The granules contain mediators which precipitate either a localized inflammatory reaction or a severe systemic reaction called anaphylactic shock. Mediators, such as histamine, cause the relaxa- tion of smooth muscle of the local vasculature and cause fluid leakage into the surrounding tissue. Also, mast cells release chemotactic factors which attract other leukocytes and enzymes that degrade local tissue, furthering the inflammatory response and possibly laying the groundwork for any subsequent repair. 8 Allergic responses are triggered by allergens interacting with immunoglobulin E bound to the surface of mast cells. Because allergies serve no obvious function, there is considerable debate on what actually is the evolutionary benefit of the mast cell. Kay (1979) believes that the mast cell plays a key role in protection from parasites. Specifically mast cells were shown to degranulate in the presence of IgE specific for shistosomula. The mast cells release a chemotactic factor (ECFA) which attracts eosinophils. Kay (1979) found the eosinophils could cause the death of theshistosomula if the parasites were precoated with IgG and the C3 component of the complement. Many correlations have infections and IgE. been found between parasitic For example, a role for IgE and the mast cells in protection from parasites was inferred in the study of Grove and Forbes (1975) which showed that individuals with increased levels of blood-borne IgE have an increased resistance to hookworm infestation. While the function of the mast cell is not clear, much progress has been made on resolving the mechanism of degranulation. Hirata and Axelrod (1980) proposed a mechanism for the IgE-mediated release of histamine from mast cells that tied together many past observations ondegranulation. For example, Hirata and Axelrod (1980) found that Rocr 2 ROClI 2 ROCH 2 ROCH 2 phosph~tidyl~ serine (PS) 'If co 2 /R CH 2 0-f-OCH 2 CH 2 NH 2 0- phosphatidylethanolamine (PE) PMT I SAM ~ I R Clf -0P-OCH CH NCH 2 2 2H 3 • o- PMT II 2SAM phosphatidyl choline (PC) phosphatidyl N-monomethylethanolamine 9 the central feature in stimulus-secretion coupling in mast cells is the enzymatic transfer of methyl groups to phospholipids. They demonstrated that an early step in the activation of the mast cell was the rapid incorporation of 3 H-methyl group from S-adenosyl methionine (SAM) into the phospholipid fraction of the membrane of the mast cell. Degranulation of the mast cells and the methyltransferase were inhibited by a specific methyltransferase inhibitor (Ishizaka et al, 1980). This result explained the long standing observation that ROC~ 2 ROCH 2 phosph~tidyl~ serine ~ (PS) co 2 /R CH 20-f-OCH 2CH 2 NH 2 0- phospbatidylethanolamine PME I PME II SAM 2SAM phosphatidyl choline (PC) phosphatidyl N-monometbylethanolamine (PE) mast cells required the addition of phosphatidylserine to degranulate in the presence of IgE (Keller, 1962). The methylation of the phospholipids was correlated with an increased influx of 45 ca into the mast cells (Ishizaka et al, 1980). The increased permeability to calcium was thought to be caused by the methylation and subsequent flipping of phospholipids of the inner surface of the membrane to outer surface of the membrane which in turn causes a decrease in membrane viscosity. The increase in membrane 10 fluidity supposedly would allow unknown components of the cell surface to aggregate and thus increase Ca++ permeability. Hirata and Axelrod (1980) proposed that the influx of calcium activated the Ca-dependent enzyme phospholipase A2 . The activated phospholipase A2 converts PC into lysophosphatidylcholine, a substance that pro- motes calcium-dependent cell fusion (Poole et al, 1970) and enhance degranulation of mast cells (Giacobini et al, 1965). Since the release of histamine involves the fusion of membrane enclosed granules with the outer membrane (Goth and Johnson, 1975) the production of lysophosphatidylcholine and the influx of calcium may be important events in the final stages of degranulation. However, the mechanism of fusion of granules with the cell surface is not known. The Hirata and Axelrod (1980) model for IgE-mediated release of histamine may not be completely applicable to other agents that degranulate mast cells. For example, the potent ctegranulating agent, compound 48/80, does not strictly require extracellular calcium for its action (Uvnas and Thon, 1961). However, the Hirata and Axelrod (1980) model which implies that extracellular calcium has a role in degranulation, may be valid if compound 48/80 induces a methylation event that releases calcium 11 from some yet undetermined calcium pool. The addition of phosphatidylserine is not required for the compound 48/80mediated degranulation of mast cells (Goth et al, 1971). There is no simple extension of the model of Hirata and Axelrod (1980) that would both explain the requirement for PS in the degranulation of mast cells by IgE and also account for the apparent lack of this requirement in the degranulation of mast cells by compound 48/80. Since degranulation of mast cells by IgE feature utilization of PS in the plasma membrane this particular membrane may have a specific requirment for PS. In contrast, the degranu- lation of mast cells by compound 48/80 does not necessarily feature changes in phosphilipid methylation in the plasma membrane, but may feature a similar process in the membrane defining the intracellular calcium pools. These intra- cellular calcium pools are thought to be mobilized in the degranulation of mast cells by compound 48/80 (Douglas and 8eda, 1973) and it is possible that the mast cell does not require PS for degranulation by compound 48/80 because the membrane of the intracellular calcium pools have sufficient PS for this process. 12 Excitation-Contraction Coupling in Skeletal Muscle Almost all of the major steps ·in the contractile activation of skeletal muscle are now well-established. The depolarization of the muscle or muscle fibers at the neuromuscular junction is the first step leading to the contraction of the muscle. The action potential of a cholinergic nerve is correlated with the release of vesicles containing the transmitter acetylcholine. The acetylcholine interacts with specific receptors at the neuromuscular junction (Heidman and Changeaux, 1978) including a uniform depolarization of the transmembrane resting potential of the muscle. The depolarization is basically due to an increased permeability of the muscular membrane to sodium ion. A threshold depolarization occurs when sufficient transmitter is released at the neuromuscular junction. The subsequent action potential spreads throughout the muscle via localized changes of ionic conductance. The muscle action potential features a voltage and time-dependent increase in Na+ current and a delayed voltage and time-dependent increase in potassium conductance (Adrian et al, 1970). The resting transmembrane potential is re-established after the action potential + + by the transport of sodium and potassium by Na /K ATPases back across the muscle membrane. 13 The inward spread of the activation of the muscle from the sarcolemma through the transverse tubule sys- tem is the second major step in the contractile mechanism (Constantin, 1975). The coupling between the muscular action potential and contraction could be abolished by treatment of the muscle with hypertonic glycerol (Gage and Eisenberg, 1969). Anatomical studies of the muscles treated with glycerol showed that the t-tubule system was physically pinched off from the sarcolemma by the glycerol treatment. These experiments implied that coupling between excitation and contraction in skeletal muscle required a direct communication of the action potential of the sarcolemrna into the t-tubule system. The third basic step in the sequence for the activation of the muscle at the neuromuscular junction to the activation of the contractile apparatus is the coupling between the activation of the t-tubule with the increase in calcium permeability of the membrane of the sarcoplasmic reticulum. Anatomically, the t-tubules and the sarcoplasmic reticulum are close and form distinct junctions called the SR-t-tubule triadic junctions (Huxley, 1971). The t-tubule action potential has been correlated with the radial spread of the activation of skeletal muscle fibers (Constantin, 1975). Despite the correlation 14 between t-tubule activation and the increase in calcium permeability of the membrane of the sarcoplasmic reticulum, the molecular mechanism of the coupling of these processes is not known. Theories proposed to explain this step in excitation-contraction coupling will be discussed in detail later in this chapter because of their importance to the work reported in this thesis. The final basic step in the contractile sequence is the release of calcium from the sarcoplasmic reticulum into the myoplasm causing the activation of the contractile apparatus. In skeletal muscle, the myoplasmic calcium interacts with a target protein called troponin-C associated with the actin-myosin contractile apparatus; binding of ca 2 + activates an actinomyosin ATPase. The hydrolysis of ATP by the actinomyosin ATPase provides the driving energy for the interdigitation and "sliding" of the actin and myosin filaments during the active shortening of muscle (Katz, 1966). The myoplasmic concentration of free cal- cium is lowered to the resting level ~Y the action of the ATP-dependent calcium pumps of the sarcaoplasmic reticulum Hasselbach, 1980). This process causes relaxation of the contractile apparatus and reduces the tension of the muscle to the resting state. The calcium binding protein calmodulin (Cheung, 1971) is known to directly potentiatethe reuptake of calcium into cardiosarcoplasmic reticulum (Katz and Reintulla, 15 1978; Lopaschuk et al, 1980). Calmodulin may also in- directly cause a cAMP-dependent increase in sarcoplasmic reticuluar Ca++ transport by stimulating cardiac adenylate cyclase activity (Tada and Kirchberger, 1978). However, the role of calmodulin in the relaxation of skeletal muscle is not known. 16 Excitation-Contraction Coupling Theories: The Coupling Between the t-tubules and the Sarcoplasmic Reticulum Several theories have been proposed to explain the communication between the t-tubules and the sarcoplasmic reticulum. An early approach was to assume that the currents in the t-tubule system were transmitted directly to the sarcoplasmic reticulum causing a depolarization of the sarcoplasmic reticulum, thus leading in calcium permeability. to the increase Several studies have shown that insufficient ionic current flows through the SRt-tubule junction to produce a significant depolarization of the sarcoplasmic reticulum (Chandler et al, 1975). Another early approach featured extracellular calcium ions entering the muscle fiber during the action potential and subsequently initiating the contractile response (Frank, 1957; Bianchi and Shanes, 1959). But it was found that the influx of extracellular calcium during a single contraction was insufficient to account for the increase in myoplasmic concentration of calcium associated with the contractile event. Therefore, to account for this discrepancy it was proposed that the small amounts of calcium entering the muscle triggered an intracellular event that would lead to contraction. This mechanism is called the "trigger Ca++" hypothesis of excitationcontraction coupling (Bianchi, 1968). 17 Several experiments have shown that the intracellular event that is triggered by calcium was the release of calcium by the sarcoplasmic reticulum. Direct applica- tion of calcium buffers to skinned skeletal muscle fibers induces the release of calcium by the SR 1970). (Endo et al, A relatively small increase in the concentration of Ca++ can cause a rapid release of calcium from vesicles derived from the sarcoplasmic reticulum (Kupsaw et al, 1980). These two pieces of evidence, repeated in other studies, are essential for support of a mechanism of direct coupling between calcium and calcium release from the sarcoplasmic reticulum of skeletal muscle. Despite the accumulating evidence that the "triggerCa++,, hypothesis has value in explaining the coupling between activation of the t-tubules and release of calcium from the SR, the mechanism of the coupling is unknown. For example, the calcium pool that provides the "trigger. not k nown, a 1 t h oug h ex t race 11 u 1 ar Ca ++ " f or t h e coup l'ing is calcium and calcium bound to the membranes of the t-tubules have been suggested (Frank, 1979). There is no conclusive evidence pertaining to the molecular mechanism or even the molecules involved other than Ca++ in the coupling between activation of thet-tubules and the release of calcium. The mechanism of excitation-coupling remains one of the major ongoing problems in muscle physiology. 18 REFERENCES Adrian, R.H., Chandler, W.K. and Hodgkin, A.L. (1970) J. Physiol. 208, 607. Bergendorff, A. and Uvnas, B. (1972) Acta Physiol. Scand. 84, 320. Bianchi, C.P. (1968) Fed. Proc. 27, 126. Bianchi, C.P. and Shanes, A.M. (1959) J. Gen. Physiol. 42, 803. Bischoff, F. and Bryson, G. (1977) Adv. Lipid Res. 15, 61. Caputo, C. (1978) Ann. Rev. Biophys. Bioeng. Case, R.M. l, 63. (1978) Biol. Rev. 53, 211. Chandler, W.K., Schneider, M.F., Rakowski, R.F. and Adrian, R.H. (1975) Phil. Trans. R. Soc. Lond. B 270, 501. Cheung, W.K. (1971) J. Biol. Chem. 246, 2859. Constantin, U. (1975) Prog. Biophys. Mol. Biol. 29, 197. DeWulf, H. and Keppens, S. (1976) Arch. Int. Physiol. Biochem. 84, 159. Douglas, W.W. and Poisner, A.M. (1963) J. Physiol. (Lond.) 82, 393. Ehrlich, P. (1879) Arch. Anat. Physiol. ~' 116. Endo, M., Tanaka, M. and Ogawa, Y. (1970) Nature (Lond.) 228, 34. Frank, G.B. (1957) Nature (Lond.) 182, 1800. 19 Frank, G.B. (1979) Proc. West Pharmacol. Soc. 22, 309. Frizzell, R.A. (1977) J. Membrane Biol. 35, 175. Gage, P.W. and Eisenberg, R.S. (1969) J. Gen. Physiol. 53, 298. Gardos, G. (1958) Acta Physiol. Acad. Sci. Hung. 14, 1. Giacobini, E., Sedvall, G. and Uvnas, B. (1965) Exp. Cell Res. ·3 7, 368. Goth, A., Adams, H.R. and Knoohnizen, M. (1971) Science 173, 1034. Goth, A. and Johnson, A.R. (1975) Life Sci. 16, 1201. Grove, D.I. and Forbes, K.J. (1975) Med. J. Aust. l, 336. Hasselbach, W. (1980) Basic Res. Cardiol. 75, 2. Heidman, T. and Changeaux, J.P. (1978) Ann. Rev. Biochem. 47, 317. Hirata, F. and Axelrod, J. Huxley, A.F. (1980) Science 209, 1082. (1971) Proc. Roy. Soc. B 178. Ishizaka, T., Hirata, F., Ishizaka, K. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 1903. Katz, B. (1966) "Nerve Muscle, and Synapse," McGrawHill, New York. Katz, S. and Reintulla,M.A. (1978) Biochem. Biophys. Res. Commun. 83, 1373. Kay, A.B. (1979) J. Allergy Clin. Immunol. 64, 90. Keller, R. (1962) Helv. Physiol. Pharmacol. Acta 20, C66. Kupsaw, R., Lewis, C.F. and Katz, A.M. Cardiol. 75, 13. (1980) Basic Res. 20 Lopaschuk, G., Richter, B. and Katz, S. (1980) Biochemistry 19, 5603. Mailaisse, W.J., Devis, G., Pipeleers, D.G. and Somers, G. (1976) Diabetologia Metzger, H. 12, 77. (1977) Ann. Rev. Pharmacol. Toxiol. 19, 427. Middleton, E. (1980) J. Pharm. Sci. 69, 243. Naccache, P.H., Showell, J.H., Becker, E.L. and Shaafi, R.I. (1977) J. Cell Biol. 75, 635. Nagata, N. and Rasmussen, H. (1970) Proc. Natl. Acad. Sci. USA 65, 368_. Okamota, A., Takahasi, K. and Yoshii, M. (1976) J. Physiol. (Lond.) 22, 527. Parod, R.J. and Putney, J.W. (1978) J. Physiol. (Lond.) 281, 359. Perry, M.C. and Hales, C.N. (1970) Biochem. J. 117, 615. Poole, A.R., Howell, J.I. and Lucy, J.A. (1970) Nature 227, 810. Rahaminoff, R., Meiri, H., Erulkar, S.D. and Barenholz, Y. ( 1978) Proc. Natl. Acad. Sci. USA 75, 5214. Schmidt, J.A. and Eckert, R. (1976) Nature 262, 713. Tada, M. and Kirchberger, M.A. (1978) in "Recent Advances in Cardiac Structure and Metabolism" (Kobayashi, T., Shaw, T. and Dhalla, N.S., eds.) Vol. II, ~niversity Park Press, Baltimore, Md) p. 265. 21 Uvnas, B. and Thon, I.L. (1961) Exp. Cell Res. 23, 45. 22 CHAPTER II VERAPAMIL; A NEW AGENT FOR ACTIVATION OF MAST CELLS 23 INTRODUCTION Verapamil The structure of verapamil and its methoxy-derivative, D-600 are given below. OCH 3 OCH 3 Veraparnil CH 30 CH 3 D-600 H3C H C 3 C::N H C::N H HC 3 H C 3 CH CH2 I 2 I ?H2 CH2 CHz CH 2 N-CH N-CH 3 I I I 1- 3 I I CH 2 CH 2 ·cH ~H. I 2 QOCH3 OCH 3 OCH 3 Verapamil and D-600 were found to be highly potent inhibitors of excitation-contraction coupling in the mammalian myocardium(Fleckenstein, 1964) and of excitationcontraction coupling in uterine smooth muscle (Fleckenstein and Grun, 1969). The effects on muscle could be overcome by increased extracellular Ca++, therefore these drugs 24 were designated as calcium antagonists. Since the dis- covery of verapamil and D-600 as calcium antagonists in heart muscles a large number of systems have been investigated. An incomplete list partially compiled from Rosenberger and Triggle (1978) is given in Table II. The first general mechanism proposed for the pharmacology of verapamil was by Fleckenstein (Kohlhart et al, 1972), who noted that verapamil did not affect the action potential of mammalian cardiac fibers. The cardiac action potential is primarily due to an influx of Na ions through what is designated as a fast channel. However, verapamil did inhibit cardiac contraction and the transient calcium current associated with depolarizing membrane potentials. Calcium was found to enter the heart through the fast Nachannel and through its own specific Ca-channel (Mascher and Peper, 1969, Reuter, 1967). through the slow Ca-channel was verapamil. This flux of calcium blocked by Fleckenstein (1977) designated verapamil and D-600 as specific Ca-channel antagonists because of these findings. Sympathetic 6-receptor stimulating agents such as epinephrine andisoproterenol had an opposite effect by causing an increase in the Ca current across the cardiac membrane (Reuter, 1965). Verapamil has been shown effective in a variety of therapeutic situations. For example, the drug has been used as Table II,Chapter II - Effects of Verapamil (or D-600) on Cellular Function Tissue Action Reference Aorta relaxation of norepinephrine (NE) effect Schuman et al (1975) Portal vein decreases spontaneous contractions Golenhofen and Herstein (1975) Ear artery relaxation of NE effect Golenhofen and Weston (1976) Ileum relaxation of acetylcholine effect Triggle et al (1975) Pancreatic islets inhibition of insulin release Malaisse et al, (1976) Neurohypophysis inhibition of mytocin release Dreifus et al, (1975) Pituitary inhibition of ACTH release Eto et al (1974) Adrenal inhibition of catacholamine release Pinto and Trifaro (1976) Pulminary Artery relaxation of K+ stimulation Haeusler (1972) Mesenteric vein relaxation of NE effect Church and Zsoter (1980) l\J CJ1 26 a depressor of a variety of hyperkinetic cardiac phenomena by virtue of its excitation-contraction decoupling properties on cardiac muscle. Moreover, verapamil has immediate positive effects on cardiac arrhythmias (Shamroth et al, 1972). Verapamil has also been used as a long term therapeutic agent. For example, Sandler et al (1968) in a controlled clinical trial found that verapamil caused a decrease in the frequency of angina pectoris in cardiac patients. Vera- pamil has also been suggested as a possible drug for the management of hypertension. verapamil, at The hypotensive action of doses below that required for reduction of cardiac output, has been proposed to be due strictly to changes in peripheral vasodilation (Ross and Jorgensen, 1967). Recently in the popular press, verapamil has been described as a potential "wonder drug" in the treatment of hypertension (Clark and Shapiro, 1981). A possible mechanism for the hypotensive effect of verapamil is given in this study. 27 Compound 48/80 Compound 48/80 was first synthesized by Baltzby et al (1949). The compound was prepared by an acid catalyzed polymerization of p-methoxyphenethylmethylamine with formalin. Morrison et al (1974) radiolabeled the "com125 pound" with 1 and found a very broad peak in gel filtration of approximately 800 molecular weight. Several factors probably contribute to the complexity of the mixture. One factor is the formation of isoquinolines as a side reaction. ¢ 0CH CH3 ~ H'"H H+ 3 Isoquinolines do not account for the potent ability of compound 48/80 to reduce blood pressure in dogs (Baltzby et al, 1949). When tertiary amines were polymerized the hypotensive activity was of maximum potency. Because tertiary amines do not form isoquinoline derivatives Baltzby et al (1949) proposed that the following structure was the active ingredient in mixtures of polymerized 28 methoxyphenalkyamines. Another factor possibly contributing to the complexity of the compound 48/80 preparation is the possibility that the isoquinoline units are crosslinked (Evans, 1981, personal communication). The ability of compound 48/80 to lower blood pressure is probably due to its ability to release histamine from tissue mast cells (Mongar and Schild, 1952). The release of histamine was due to compound 48/80 causing a degranulation of mast cells and basophils. can Compound 48/80 cause the release of histamines from mast cells in the absence of extracellular calcium (Uvnas and Thon, 1961). Mast cells when treated with 2 mM EDTA for three hours do lose their ability to respond to compound 48/80 (Douglas and Ueda, 1973). Douglas and Ueda (1973) suggested that the EDTA treatment of mast cells caused a depletion of the intracellular pools of calcium that are mobilized when the mast cells are challenged by compound 48/80. In contrast, the degranulation of mast cells caused by complexes of IgE and antigen have an absolute requirement for extracellular calcium (Ishizaka et al, 1979). 29 Stimulus-Permeability Coupling Gardos (1958) found that treatment of red cells with EDTA causes a decrease in the possible leakage of potassium from red cells. Whitman (1968) later pro- posed that the decrease in potassium permeability was due to a decrease in the concentration of ionized calcium in the red cell. Lew and Ferreira ( 1978) showed that treatment of red cells with the calcium specific ionophore A23187 caused an increase in potassium permeability. This they ascribed to the activation of a Ca-sensitive potassium channel by the inward movement of extracellular calcium. The mechanism for the activation of such "channels" is unknown (Putney, 1979). The Ca-dependent changes in potassium efflux have been observed in a wide variety of cells and tissues since the discovery of the "Gardos effect" (Putney, 1979). Putney (1979) has coined the term stimulus-permeability coupling to describe this effect. Examples of systems that exhibit stimulus-permeability coupling are activation of salivary glands, stimulation of lymphocytes, hormone action on liver cells, and contraction of smooth muscle such as the ileum. This study has found that efflux of rubidium-86 (an analogue of potassium (Putney, 1979)) in skeletal muscle and mast cells was sensitive to challenges by verapamil and compound 48/80. 30 MATERIALS AND METHODS Isolation of Rat Peritoneal Mast Cells The house bred ratsused in this study were predominantly of the Fisher-344 breed and occasionally of the WisterFurth breed. No differences were observed between the breeds in cell yields and cell responses. Rats between 150 and 300 grams were sacrificed by cervical dislocation with a blunt guillotine. The rats were injected in the peritoneal cavity with either phosphate buffer saline (PBS) or sterile mast cell buffer (MCB). MCB contained 137 mM NaCl, 2.6 mM KCl, 1.0 mM MgC1 , 10 mM Tris (hydro2 methyl)aminomethane, and 1 mg/ml glucose. 1 mg/ml bovine-serum albumin MCB was titrated to pH 7.4 with concentrated HCl. This buffer was modified from Cochrane and Douglas (1976) but omitted heparin for it was found that heparin precipitated compound 48/80. MCB, unless otherwise designated, was used in all the pharmacological studies on rat peritoneal cells and P815 mastocytoma cells. It is very important to note that CaC1 of MCB. The CaC1 2 2 is not a component was omitted when it was found that the spontaneous release of 3 H-serotonin from mast cells isolated in the presence of a calcium containing solution was approximately two-fold higher than cellw isolated in buffer without caclium. Furthermore, rat peritoneal cells 31 isolated in buffers with no calcium were more reactive to the various drugs used in this study. There is a possibility that ++ the presence of Ca during the isolation somewhat desensitizes the mast cells to further 3 H-serotonin release. If Ca++ was required for a drug challenge of the mast cells, a volume of a calcium chloride stock solution was added to the MCB until an appropriate final concentration was obtained (usually 2.0 mM). this addition of CaC1 2 Often, but not reproducibly, caused a small release of 3 H- serotonin from the mast cells. The peritoneal cavity of the rats was massaged manually to dislodge cells from the inner peritoneal wall. The peritoneal cavity was then cut open and the fluid was transferred to a plastic centrifuge tube using a Pasteur pipette. B. L. Aalseth improved the yields of mast cells by developing a variation of the standard technique. The rat peritoneal cavity was widely exposed by forceps. A second person then jetted 10 ml of MCB from a syringe (#22 needle) onto the exposed surface of the peritoneal cavity. This wash was transferred to the centrifuge tube by pipette to complete the first stage of the cell isolation. A typical yield of leukocytes was 5 x 10 5 - 10 6 cells per rat of which approximately 50% were identified as mast cells. Two methods were used to crudely identify mast cells. 32 Under the light microscope, mast cells underwent a profound and quick change (~ 1 minute) when exposed to relatively high concentrations of compound 48/80. The mast cell visibly degranulated and a characteristic halo of vesicles surrounded the cells. Those cells were assumed to be mast cells because of their sensitivity to the classical mast cell degranulating agent; compound 48/80. A staining procedure differentiated mast cells from the other leukocytes. The rat peritoneal cells were exposed to 1% alcian blue and 3% acetic acid in 0.15 M NaCl. The cells were washed by centrifugation and the stain discarded. Then the cells were immediately exposed to 0.1% safranin 0 in 1% acetic acid and 0.15 M NaCl. After five minutes the cells were washed and resuspended in saline. This preparation demonstrated the stages in mast cell differentiation. Small lymphocyte-like mast cells contain only calcium blue-stained granules. Large mature mast cells contain safranin-positive heparin granules and thus stain pink. Generally, approximately 50-70% of the leukocytes (cells not obviously red cells) showed this pink staining. Another 5-10% of the leukocytes showed the characteristic blue staining of the immature mast cell. Purified mast cells used to verify the staining techniques were isolated by the method of Morrison et al (1974). Their method used a lXG gravity separation of 33 rat peritoneal cells in a 1.5% BSA solution of buffered saline. The procedure was modified by centrifuging the syringe at lOOXG for eight minutes. Even with this extra g-force the separation was not as good as described by Morrison et al (1974) and there was no detectable "mast cell" pellet. Nevertheless, this partial isolation . allowed some verification of the above staining procedure. In general mast cells were not isolated from the rat peritoneal cell mixture for several expedient reasons. Morrison et al (1974) showed that compound 48/80, a degranulating agent used in this study, bound exclusively to the mast cell population. More important, the assay for mast cell amine release involved prepacking of the rat peritoneal cells with 3 H-serotonin. Morrison et al (1974) demonstrated that the serotonin was incorporated almost exclusively into mast cells. Furthermore, the 3 H- serotonin behaved the same as the endogeneous histamine. The release of 3 H-serotoin and histamine was identical when measured as a function of dosage of compound 48/80. Thus, for practical purposes the mast cells were not isolated from the rat peritoneal cell mixture. The terms mast cells and rat peritone~lcells are used interchangeably in this study because of the exact functional similarities with regards to elicited release of biogenic amines. 34 The second stage of mast cell preparation involved the loading of 3 H-serotonin into the cells. The cells from one to four rats were suspended at room temperature in 50 ml of MCB containing approximately 20 µCi of 3 Hserotonin (Amersham). After 1-1.5 hours the cells were washed four to five times in 50 ml of PBS or MCB with the final suspension always in MCB. These cells were aliquoted into 1.5 ml microcentrifuge tubes for subsequent preincubations and pharmacological challenges. label experiments using 86 Double RbCl (Amersham) were accomplished by simultaneous loading of cells with~ 30 µCi of along the 3 H-serotonin. 86 Rb+ 35 Measurement of 3 H-Serotonin Release in Rat Mast Cells In a typical mast cell experiment 1.0 ml of 3Hserotonin packed cells were aliquoted into plastic microcentrifuge tubes. If preincubation was desired a small volume of the stock solution (i.e., lOOX CaC1 ) 2 first was added to the microcentrifuge tube. After the preincubation period small volumes of drug or hapten stock solutions were added to the tubes. shaken The tubes were then and placed in a 37°C shaker-water bath for the desired length of time. The tubes were then centrifuged at high speed in Eppendorf table top ultracentrifuges for three minutes. Aliquots of the supernate were transferred to scintillation vials. and 86 The 3 H-serotonin Rb were counted in scintillation fluid with a Beckman counter using standard methods. In a procedure to determine the maximum possible release of 3 H-serotonin, several cell samples were not centrifuged. were acidified with ~ These samples 25 µ£ of concentrated hydrochloric acid and placed in a steam bath for 10-20 minutes. Aliquots of the product were counted and the results served as the 100% potential release level for 3 serotonin in the rat peritoneal cell population. H- 36 P815 Mastocytoma: Origin and Maintenance The P815 mastocytoma was induced in a male DBA/2 mouse by repeated exposure to methylcholanthrene (Dunn and Potter, 1957). The distinguishing feature of the tumor cells were their high degree of granular metachromasia when stained by 0.5% ~ toluidine blue. The number of stained granules per cell was greatly reduced when compared to the normal mast cell. The tumor was first introduced to tissue culture by Lundak and Raidt (1973). The cell line was obtained from the Salk Institute of Biological Studies. The P815 mastocytoma cell line was maintained in incubators kept at 37°C and 5% co /100% H o. The medium 2 2 was Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% calf serum (GIBCO). Further additions were 1.2 mg/ml glutamine, 0.3 mM non-essential amino acids (GIBCO), 100 µg/ml streptomycin.and 100 units/ml penicillin G. Cell counts were determined with standard hemocytometers. Verapamil The verapamil used in this study was a generous gift from Knoll Pharmaceutical Company, Whippany, New Jersey. 37 RESULTS Results Establishing the General Nature of the Release of 3H-Serotonin from Mast Cells Four basic criteria were established to show that the release of 3 H-serotonin from mast cells caused by verapamil involved degranulation of mast cells; 1) dosedependent release of amines from the cell; 2) morphological changes similar to those caused by the "classical" degranulating agent, compound 48/80; 3) dependence of the process on metabolic energy; 4) demonstration that the drug did not lyse or kill the cell. The results establishing these criteria are described below. Verapamil causes a dose-dependent release of serotonin from rat mast cells (Figure 1). rat cells were loaded with 3 3 H- The H-serotonin in a method very similar to that described by Morrison et al (1974). They concluded that the release of 3 H-serotonin was exactly correlated with the release of histamine, an indication that both of these biogenic amines were released by the same mechanism upon degranulation of the mast cell. Morrison et al (1974) also showed that the 3 H-serotonin was taken up almost exclusively by the mast cells in a mixture of rat peritoneal cells and suggested that the 3 H-serotonin was bound in the granules 38 of the mast cell to the amine-protein-heparin complex described by Bergendorf f and Uvnas ( 1972). Mast cells exposed for one hour to high concentrations of verapamil (2 mg/ml) were observed under the light microscope to undergo a degranulation reaction similar to what is seen for mast cells exposed to the "classical" degranulation agent, compound 48/80. The degranulation of mast cells is dependent on cellular levels of ATP (Johanson, 1980). that release of 3 Figure 1 demonstrates H-serotonin from mast cells by verapamil was inhibited by preincubation of the cells in a mixture of the metabolic poisons, KCN and 2-deoxyglucose. As a positive control, the "classical" degranulating agent compound 48/80 was also shown to require metabolic energy for the degranulation of mast cells (Figure 2). Mast cells exposed for one hour to concentrations of verapamil that could induce maximal release of 3 in five minutes were found to exclude the trypan H-serotonin blue viability stain. Less than 2% of the mast cells showed staining by the dye indicating that the cells were viable and the drug was not releasing specific lysis. 3 H-serotonin through non- 39 Figure 1 The role of energy metabolism in the release of 3 H-serotonin from rat peritoneal cells challenged by verapamil. The metabolically inhibited cells (-o-o-) were preincubated in 10 mM 2-deoxyglucose and 0.3 mM KCN for one hour and 20 minutes at 25°c in glucose-free MCB. Normal cells (-•-•-) were preincubated in regular MCB in parallel with the metabolically inhibited cells. The challenge with verapamil was for one hour at 37°C. statistical error was estimated to be 10%. The 40 0 0 0 r-1 0 0 00 ,,....... r-1 E ............. M ~ '-"' c 0 .,..; ~ 0 0 ~ ro ~ ~ c © 0 c 0 u r-1 .,..; E ro ~ ro 0 0 ~ 0 0 N ~ © > 41 Figure 2 The role of energy metabolism in the release of 3 H-serotonin from rat peritoneal cells challenged with compound 48/80. The metabolically inhibited cells (-o-o-) were preincubated in 10 mM 2-deoxyglucose and 0.3 mM KCN for 40 minutes at 25°C in glucose-free MCB. Normal cells (-•-•-) were pre- incubated in regular MCB in parallel with the metabolically inhibited cells. The challenge with com- pound 48/80 was for one hour at 37oc. tical error was estimated to be 10%. The statis- 42 N M ,,...-.... M 00 E ............. N ~ ~ ........... ~ ~ N 0 ·l""'l ~ ro H ~ ~ 0 © 0 N 0 ~ 0 0 00 ............. © M 00 ~ ~ ~ ~ 0 N M 00 0 0 00 0 © 0 ~ E 0 u 43 86 Rb+- 3 H-Serotonin Double Label Experiments in Mast Cells Loading of 86 Rb+ and 3 H-serotonin into mast cells and then subsequently monitoring the release of these radioactive substances from the mast cells represents a novel experimental method (Figures 3 and 4). The results of the double label experiment indicate that compound 48/80 causes a dose-dependent increase in the initial rate (15 minute challenge) of 86 + Rb -efflux and release of 3 H-serotonin from rat peritoneal cells. (Figure 3) The results for rat peritoneal cells challenged with verapamil were somewhat different (Figure 4). Low concen- trations of verapamil cause a small decrease in the spon3 86 taneous release of H-serotonin and Rb+. Whereas high concentrations of verapamil caused an increase in the 3 86 + release of H-serotonin and Rb from the mast cells. The release of 86 Rb + is thought to be proportional + to the release of K from cells (Putney, 1979). However, 86 the exact relationship between K+-efflux and Rb+-efflux has not been established for the mast cell system. Compound 48/80 caused a concentration dependent increase in the rate of cells (Figure 5). 86 Rb+-efflux in P815 mastocytoma Unlike normal mast cells (Figure 2), relatively high concentrations of compound 48/80 were required to cause a significant increase in the rate of 86 Rb+-efflux from the P815 cells. Figure 5 showed 44 there was no difference between the dose-response curves 86 for Rb+-efflux in P815 cells challenged with compound 48/80 in the presence of extracellular calcium (2 mM Ca++) and in the absence of extracellular calcium (short preincubation in MCB containing 0.1 M EGTA)~ It has been shown that 86 Rb+-efflux is proportional to the concentra- tion of cytosolic Ca++ in other cell systems (Putney, 1979), accordingly we assume that the increase in 86 Rb+efflux in P815 cells challenged with compound 48/80 likewise reflects an increase in the concentration of calcium in the cytosolic pool. Figure 6 shows a dose-dependent increase in efflux in P815 cells challenged by verapamil. is very similar to the data from 86 86 Rb+- This result Rb+-efflux in P815 cells challenged with compound 48/80. *This short preincubation in 0.1 M EDTA was of short enough duration and low enough concentration to not significantly deplete the intracellular pools of calcium but insured there was complete removal of all extracellular calcium. In contrast, preincubation of mast cells in 2 mM EDTA for three hours caused mast cells to lose their ability to respond to compound 48/80 (Douglas and Ueda, 1973). 45 Figure 3 Double label experiment measuring the initial rates of release of 86 Rb + and 3 . H-seroton1n from rat peritoneal cells challenged with compound 48/80. 86 The cells were isolated and loaded with Rb+ and 3 H-serotonin by the methods described in Materials and Methods, Chapter II. challenge with at 37°C in MCB. The compound 48/80 was for 15 minutes The spontaneous release of 3 H- serotonin (---0---0---) was 795 ~ 70 cpm and the maximum possible release was 17,100 ~ 500 cpm. The spontaneous release of 86 Rb + (-•-•-•-•-) was 404 + 18 cpm and the maximum possible release was 795 + 25 cpm. The standard deviation for the blanks and the maximum possible release determine the ranges of the statistical error in this experiment. 480 ()... , ---- 'O <D U1 oj (!) 460 , --o----- ------ --------- ___ ..D 8000 'O (!) U1 oj (!) r-1 (!) ?-I 6000 s:: s:: •ri 0 r-1 +:> 0 <D P.:: ?-I © +,.0 fli P.:: ~ tO i 4000 440 00 sp.. C") sp.. C) C) 2000 420 400--------------------.L--------L---------__;L-______-L.._________J 0 5.0 4.0 3.0 2.0 1.0 0 Concentration Compound 48/80 (µg/ml) .r.=-- °' 47 Figure 4 Double label experiment measuring the initial rates of release of 86 Rb+ and 3 H-serotonin from rat peritoneal cells challenged with verapamil. The details of this experiment are described in Figure 3. The table below in- eludes standard deviations for the counts of the blank and maximum possible release. Spontaneous Release Maximum Possible Release 795 + 70 cpm 17,100 + 500 cpm 3 H-serotonin (---0---0---0---) 86Rb+ (-•-•-•-•-) 404 + 18 cpm 795 + 25 cpm 48 pes'B818B '' '' '' Ct) ~ L.(',) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CD u1uo+o.:ras-H 8 Wd'J 0 0 0 r-i C'\J 0 0 ~ '' '' ' '' '' ' 0 0 ' Ct) ' ' ''' '' '' '' ' '? 0 0 C'\J ' \ \ \ \ \ \ \ \ \ ~ 0 0 r-i I I I I ' l I 6, ------------'--~~~~~..&-~-----~~L-~~-~--_---:_~----~~---1~~--------'o 0 0 L.(',) 0 0 ~ 0 Ct) 0 49 Figure 5 The release of 86Rb+ from P815 Mastocytoma cells challenged by compound 48/80. The mastocytoma cells were suspended in a 2 µCi/ml 86RbC1 MCB for 37oc for two hours. 2 The cells were of washed five times in Ca-free MCB and divided in two. One aliquot was incubated for 30 minutes in 2 mM ca++ MCB (-•-•-) and the second aliquot was incubated 30 minutes in 0.1 mM EGTA MCB (--0---0---). The cells were aliquoted into small portions and challenged with compound 48/80 for 10 minutes. The maximum 86Rb+ count for the mastocytoma cells was 2320 cpm. mated error of each sample was 10%. The esti- 2500 100% '--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..., 0 2000 -- o---- - -- -- - - - ---------0-- "O Q) rn cd Q) M Q) p::: 1500 ,,O" ~ ,,," I • ~--------- ,, ,,. ,,' ,,(Y' + .n p::: Ln 0 tO 00 e~ 1000 C) 500 0 • , r-1"\ 1 ' AA , ' r::::f"\ ' <)("\("\ I <)h.{) ' '){){) Compound 48/80 concentration (µg/ml) V· I ')~{)) 500 51 Figure 6 The release of 86Rb+ from P815 mastocytoma cells by verapamil. The detailed procedure for challenging the mastocytoma cells with verapamil is described in Figure 5 . The cells were pre- incuba ted in EGTA (---0---0---) and 2 mM Ca++ (-•-•-•-) then challenged with verapamil for 10 minutes. 2500 100% - - ---- -- --------------0 2000 "d ()) en ro ()) r-i 1500 ()) ~ I + ..a 0:::: ;,/ I (.0 00 e 1000 ~ C) 500 0 50 100 150 200 250 300 Verapamil Concentration (µg/ml) 350 400 U1 N 53 DISCUSSION The Effect of Verapamil on Mast Cells The data in Figures 1 pamil caused the release of and 5 illustrate that vera3 H-serotonin from rat mast cells.This isthe first demonstration that verapamil could cause the release of biogenic amines from mast cells. The discovery that verapamil causes the release of 3 H-serotonin from mast cells is surprising considering the previously proposed mechanism of action of the drug and what is known about the mechanism of degranulation of mast cells. Fleckenstein (1977) proposed that vera- pamil was a member of the class of drugs that were specific "calcium-channel" antagonists. The release of histamine from mast cells caused by the classic degranulation agent compound 48/80 is thought to occur by a mechanism involving the release of calcium from intracellular pools into the cytosolic calcium pool (Ueda and Douglas, 1973). If the release of 3 H-serotonin from mast cells caused by verapamil is dependent on the release of calcium from membrane-bound intracellular pools and if the drug can function by blocking intracellular "calcium-channels" of these pools, then verapamil, itself, would not be expected to cause a release of 3 H- 54 serotonin from mast cells. The degranulation of mast cells caused by the interaction of antigen and membranebound IgE requires the influx of extracellular calcium (Hirata and Axelrod, 1980). If verapmil, functions by blocking "calcium-channels" and therefore inhibits the influx of extracellular calcium it would not be expected to cause the degranulation of mast cells. The surprising result that verapamil, itself, causes 3 the release of casts serious H-serotonin from mast cells, therefore, doubt on the proposition that the drug is acting strictly as a specific "calcium-channel" antagonist as described by Fleckenstein (1977). The measurement of 86 Rb+-efflux from mast cells was an attempt to define a role for cytosolic calcium in the mechanism of action of verapamil and compound 48/80. Figures 3 and 4 showed that the release of 3 H-serotonin from mast cells by compound 48/80 and high concentration of verapamil occurred with an increase in from the mast cells. 86 Rb+-efflux In a wide variety of cells and · · · th e efflux of 86 Rb+ ( an ana 1 ogue tissues an increase in of K+) was correlated with an increase in cytosolic Ca++ concentrations (Putney, 1979). If this correlation could be extended to the mast cell, the release of 3 H-serotonin by compound 48/80 and high concentrations of verapamil would feature an increase in the concentration of cytosolic 55 Ca ++ • If indeed the degranulation of the mast cell caused by compound 48/80 and high concentrations of verapamil features an increase in the concentration of cytosolic Ca++ and because the processes occur in the absence of extracellular calcium then the drugs could be mobilizing calcium from an intracellular pool into the cytosol. The data of Figure 4 show a dose-dependent effect of verapamil that may be due to the drug acting as a specific "calcium-channel" antagonist (Fleckenstein, 1977). Low concentrations of verapamil (< 50 µg/l) caused a small decrease in both the spontaneous release of SH-serotonin and the spontaneous efflux of 86 Rb + from mast cells. If the correlation described by Putney (1979) between 86 Rb+-efflux and the concentration of cytosolic Ca++ is 86 valid for mast cells, this decrease in Rb+-efflux caused by verapamil at low concentrations implies a decrease in the concentration of cytosolic Ca++. decrease in the spontaneous rate of 86 This slight Rb+-efflux is the only evidence that could be a consequence of the blocking of "calcium-channels" in mast cells. Other effects caused by high concentrations of verapamil, such as the increased release of SH-serotonin from mast cells, must occur while overriding the effects of the proposed blockage of "calcium-channels" in the mast cells. 56 Compound 48/80 (Figure 5) and verapamil (Figure 6) cause a dose-dependent increase in 86 Rb+-efflux from P815 mastocytoma cells. The P815 cells are thought to be similar to immature mast cells and contain fewer g~anules relative to the normal mature mast cell (Dunn and Potter, 1957). The P815 cells do not undergo the morphological changes associated with degranulation when exposed to the "classical" degranulation agent compound 48/80. Therefore, the results of Figures 5 and 6 seem to indicate that 86 the increase in Rb+-efflux caused by compound 48/80 and verapamil could occur in "mast cells" that are unable to degranulate. If one assumes 86 Rb+-efflux is correlated . h t h e concen t ra t.ion o f cytoso 1.ic Ca++ (P utney, 1979) , wit this result suggests that verapamil can, in fact, mobilize intracellular calcium into the cytosolic pool. In P815 cells, this early step in degranulation does not lead to degranulation itself because of a functional defect in the cells at some later step in the degranulation pathway. 57 Possible Indirect Effects of Verapamil on Blood Pressure Verapamil has been useful in controlling acute and chronic hypertension (Zsoter, 1980). We propose that a component of the hypotensive effect of verapamil is due to the release of vasoactive substances from mast cells and basophils. For example, histamine and the slow reacting substance of anaphylaxis (SRS-A) are vasoactive substances released from mast cells that could lower blood pressure. Histamine causes an increase in the leakage of fluids from the peripheral vasculature into the connective tissue and relaxation of the smooth muscles of the periperhal vasculature both of which can lower blood pressure (Middleton, 1980). Also, SRS-A causes an increase in peripheral vascular permeability (Lewis and Austen, 1977). Interestingly, Ross and Jorgensen (1967) found that the hypotensive effect of verapamil in cats was totally accounted for by changes in the peripheral circulation, a principal site of action for both histamine and SRS-A. Drug-induced hypotension caused by the release of vasoactive substances from mast cells is not without preceden~ for Baltzby et al (1949) found that compound -- 48/80, the "classical" agent of degranulation of mast cells, caused a large decrease in the blood pressure of dogs. Vasoactive substances released from mast cells 58 probably do not account for all of the hypotensive effect of verapamil as the ability of verapamil to cause the in vitro relaxation of smooth muscles of the vasculature (Rosenberger and Triggle, 1978) could also account for a component of the in vivo hypotensive effect of the drug. 59 REFERENCES Baltzby, R., Buck, J.S. deBeer, E.J. and Webb, F.J. (1949) J. Am. Chem. Soc. 71, 1301. Bergendorff, A. and Uvnas, B. (1972) Acta Physiol. Scand. 84, 320. Church, J. and Zsoter, T.T. (1980) Can. J. Physiol. Pharmacol. 58, 254. Clark, M. and Shapiro, D. (March 30, 1981) Newsweek 86. Cochrane, D.E. and Douglas, W.W. (1976) J. Physiol. 257, 433. Douglas, W.W. and Ueda, Y. (1973) J. Physiol. 224, 978. Dreifus, L.S., deAzevedo, I.M. and Watanabe, Y. (1975) Am. Heart J. 88, 95. Dunn, T.B. and Potter, M.J. (1957) J. Natl. Cane. Inst. 18, 587. Eto, S., Wood, J., Hutchins, M. and Fleischer, N. (1974) Amer. J. Physiol. 226, 1315. Evans, D.A. (1981) personal communication. Fleckenstein, A. (1964) Verb. Dtsch. Ges. Im. Med. 70, 81 Fleckenstein, A. (1977) Ann. Rev. Pharmacol. Toxicol. 17, 149. Fleckenstein, A. and Grun, G. (1969) Pflugers Arch. 307, R26. 60 Gardos, G. (1958) Acta Physiol. Acad. Sci. Hung. 14, 1. Golenhofen, K. and Hermstein, N. (1975) Blood Vessel 12, 21. Golenhofen, K. and Weston, W.H. (1976) in "Ionic Actions on Vascular Smooth Muscle" (E. Betz, ed.) Plenum Press New York, Haeusler, G. p. 21. (1971) Naunyn Schneiderberg's Arch. Pharmacol. 269, 446. Hirata, F. and Axelrod, J. (1980) Science 209, 1082. Ishizaka, T., Foreman, J.C., Sterk, A.R. and Ishizaka, K. (1979) Proc. Natl. Acad. Sci. USA 76, 5858. Johanson, T. (1979) Eur. J. Pharmacol. 58, 107. Johnson, T. (1980) Life Science 26, 61. Kohlhart, M., Bauer, B., Krause, H. and Fleckenstein, A. (1972) Pflugers Arch. 335, 309. Lew, V.L. and Ferreira, H.G. (1976) Nature 263, 336. Lewis, R.C. and Austen, K.F. (1977) Fed. Proc. 36, 2676. Ludak, R.L. and Raidt, D.T. (1973) Cell Irnmunol. Llalaisse, G. g, 60. W.J., Devis, G., Pipeleers, D.G. and Somers, (1976) Diabetologia 12, 77. Mascher, D. and Peper, K. (1969) Pflugers Arch. 307, 190. Middleton, E. (1980) J. Pharm. Sci. 69, 243. Mongar, J.L. and Schild, H.O. (1952) J. Physiol. 118, 461. Morrison, D.C., Roser, J.R., Henson, P.M. and Cochrane, C.G. (1974) J. Irnmunol. 112, 573. 61 Pinto, J.E.B. and Trifaro, J.M. (1976) British J. Pharmacol. 57, 127. Putney, J.W. Reuter, H. (1979) Pharm. Rev. 30, 209. (1965) Naunyn Schneiderberg's Arch. Exp. Pathol. Pharmakol. 251, 401. Reuter, H. (1967) J. Physiol. (Lond.) 192, 479. Rosenberger, L. and Triggle, D.J. (1978) "Calcium and Drug Action" (G.B. Weiss, ed.) Plenum Press, New York, p. 3. Ross, G. and Jorgensen, C.R. (1967) J. Pharm. Exp. Ther. 158, 504. Sandler, G., Clayton, G.A. and Thornicroft, S.G. (1968) Brit. Med. J. ~' 224. Schumann, H.J., Gorlitz, B.P. and Wagner, J. (1975) Naunyn Schmiederberg's Arch. Pharmacol. 289, 409. Shamroth, L., Krikler, D.M. and Garrett, G. Brit. Med. J. (1972) !, 660. Triggle, C.R., Grant, W.F. and Triggle, D.J. (1975) J. Pharmacol. Exp. Ther. 194, 182. Uvnas, B. and Thon, I.L. (1961) Exp. Cell Res. 23, 45. Whitman, R. (1968) Nature 219, 610. Zsoter, T.T. (1980) Amer. Heart J. 99, 806. 62 CHAPTER III A COMPARATIVE STUDY OF THE ACTIVATION OF MAST CELLS BY COMPOUND 48/80 AND VERAPAMIL 63 INTRODUCTION Crosslinking and Degranulation of Mast Cells; IgE Chapter II of this thesis examined two classes of drugs that caused stimulus-secretion coupling in mast cells. The bifunctionality of verapamil and the polymeric nature of compound 48/80 suggest that the drugs can act as crosslinking agents. This introduction briefly reviews the role of crosslinking and aggregation in the degranulation of mast cells. Many of the structures involved in the degranulation of mast cells by antigen and IgE are understood on a molecular basis. For example, a specific receptor for IgE on rat basophilic leukemic cells has been characterized as a glycoprotein of molecular weight 77,000 (Metzger, 1978). IgE binds reversibly to the receptor with a high affinity (Ka ~ 10 proximately 10 6 10 1 M- ) and there are ap- receptors per tumor cell (Metzger et al, 1976). IgE has the Y shape characteristic of immunoglobulins. The molecule is composed of two heavy and two light chains which form seven distinct globular domains. the Y is composed of two such domains, Each arm of (VH; VL and CHl; CL); the VH; VL domain contains the antigen combining site. The three remaining regions of the heavy chain (CH2, CH3, CH4) form three domains which together constitute the Fe 64 tail of the antibody; this tail contains the site which binds to the IgE receptor (Metzger, 1979). The primary structures of the E heavy chain of a human IgE-myeloma protein has been determined (Bennich and von Bahr-Lindstrom, 1974) as has the partial nucleotide sequence and the genomic location of the gene coding for the constant region of a mouse E heavy chain (Nishida et al, 1981). IgE binds with high affinity to its receptor in the absence of antigen. Such binding does not itself activate the mast cell nor will the binding of monomeric antigen to the cell-bound IgE activate the mast cell. For example, Siraganian et al (1975) demonstrated that bivalent antigen was the minimal unit required to degranulate mast cells. More- over, Ishizaka and Ishizaka (1969) found that · crosslinking IgE bound to mast cells with anti-IgE antibodies caused the mast cell to relase histamine. Monomeric Fab' fragments of the anti-IgE antibodies were ineffective in degranulating the mast cells. Furthermore, antibodies that bound directly to IgE-receptors were found to trigger the release of histamine from mast cells (Ishizaka and Ishizaka, 1978). Monomeric Fab' fragments of the anti-receptor antibody were ineffective in degranulating the mast cells. This finding demonstrated that crosslinking of the receptors for IgE, regardless of IgE and antigen, was sufficient to degranulate the mast cell. In the same system, crosslinking of 65 the IgE receptors with antibody caused an increase in Ca++ permeability in the mast cells (Ishizaka et al, 1979) and stimulated phospholipid methylation in the mast cells (Ishizaka et al, 1980). permeability to Ca ++ Phospholipid methylation and increased in the plasma membrane are early steps in the degranulation of mast cells caused by IgE (Hirata and Axelrod, 1980. Segal et al (1977) demonstrated that dimerized IgE, as opposed to monomeric IgE, was able to trigger the release of histamine from mast cells. The dimeric IgE was prepared by chemical crosslinking with a bifunctional reagent and did not require antigen for its activity. In summary, the evidence is overwhelming that the triggering of mast cells by IgE involves receptor aggregation via a crosslinking event. 66 Crosslinking and Degranulation of Mast Cells; NonAntibody Reagents A large variety of agents besides IgE are capable of releasing histamine from mast cells. Some of these agents clearly do not involve crosslinking in their mechanism. For example, A23187, a calcium ionophore activates mast cells by transporting calcium across the cell membrane (Cochrane and Douglas, 1974). Others, such as dextran with their extensive polymeric structure may trigger the release of histamine by the relatively non-specific aggregation of cell surface receptors (Baxter and Adamik, 1977). Compound 48/80 is one of a large number of polycationic molecules that cause the release of histamine from mast cells. Among thepolycationic agents that affect mast cells are polylysine, protamine, somatostatin, bee venom mellitin, polymixin B, and phylatoxins, bradykinin. The ana- C3a and C5a, have a large percentage of basic amino acids in their primary sequences (Hugli, 1975; Fernandez and Hugli, 1976) including a carboxyterminal arginine that is essential for activity (Dias da Silva and Lepow, 1967; Cochrane and Muller-Eberhard, 1968). 67 Sialic Acids and Neuraminidase Sialic acids are ubiquitous components of all vertebrate cell membranes (Gottschalk, 1972). charged sialic acids which The negatively occur as the terminal residues of complex heteropolysaccharides of glycoproteins are almost exclusively linked from carbon atom 2 of the sialic acid to either D-galastose (at positions 3 or 6) or to N-acetyl-Dgalactosamine (at position 3) or to other sialic acids (at position 8) (Tuppy and Gottschalk, 1972). In addition to glycoproteins, glycolipids have been found to have sialic acid components. For example, Hakomori and Saito (1969) found a glycosphingolipid on red cells that contained N-glycolylneuraminicacid and Kaufman et al (1968) isolated a similar glycosphingolipid from bird brains. On the red cell membrane, sialic acids account for 80% of the surface charge density at physiological pH as measured by microelectrophoresis (Herndier and Brooks, 1975). The high con- tent of sialic acid on plasma membranes and their negative charge at physiological pH make sialic acids likely candidates for binding with the polycationic agents that cause degranulation of mast cells (Baxter and Adamik, 1977). N-acetylneuraminic acid (a sialic acid) OH H 68 Neuraminidase is a specific enzyme which cleaves the terminal sialic acids off the heteropolysaccharide units of glycoproteins. The specificity of the enzyme is rela- tively independent of the preceding saccharide unit but is sensitive to the pattern of oxygen substitution on the sialic acid itself (Schauer, 1973). Esterification of the carboxyl group of neuraminic acid blocks the action of neuraminidase completely (Karkas and Chargaff, 1964). highly This specific action of neuraminidase insures that the treatment of cells with the enzyme removes only sialic acids and does not cause non-specific damage to the cell membrane. 69 RESULTS Molecular Weight Determination of Crosslinked Verapamil The G-15 column was used to separate the iodinated crosslinked verapamil from the unreacted iodine. This column was previously calibrated by tritiated sugars. The tetrasaccharide glucose glucose a(l + a(l + 4) glucose a(l + 3) 4) glucose isolated from Aspergillus niger was obtained via a generous gift by Dr. Nordin (Tung and Nordin, 1970). This tetrasaccharide was reduced with NaBT4 (Amersham) for 16 hours at 5°C. The reaction was terminated by cold NaBH4 ml anti the addition of glacial 3 acetic acid. Similarly H-glucose a(l + 4) glucitol and 3 H-glucitol were synthesized by borohydride reduction. The elution volumes (V) of these sugars on the G-15 column served as molecular weight markers for the crosslinked verapamil. A plot of V-V /V , where v is 0 0 0 the void volume, versus logarithm of molecular weight for the sugars and crosslinked verapamil is shown in Figure 7. A portion of the lyophilized pamil was resuspended in water. 125 1-crosslinked vera- Also dissolved in this solution were the molecular weight markers; reduced glutathione, vitamin . B , blue dextran, and bovine trypsin 12 inhibitor. The mixture was separated by gel filtration on a Sephadex G-25 column. The 125 1-crosslinked verapamil was monitored by gamma counting aliquots of the column 70 effluent. The column profiles of the molecular weight markers were determined by UV-visible spectromet!Y· A plot of V-Vo/Vo, were V is the elution volume and v is 0 the void volume, versus log of the molecular weight for the G-25 column is given in Figure 8. Extrapolat"ion from Figures 7 and 8 gives a molecular weight of 1290 ~ 170 for crosslinked verapamil on the G-15 column and 1260 + 180 for the G-25 column. The theoretical molecular weight for a monoiodinated dimer with a methylene bridge is 1173. The theoretical molecular weights of monoiodinated monomer and monoiodinated trimer with two methylene bridges are 570.5 and 1514, respectively. A dimeric structure for iodinated cross-linked verapamil is proposed since only one peak was found in the 125 rgamma count profile and there were no significant counts where a trimer or a monomer would theoretically occur in the profile. 71 Figure 7 Molecular weight determination of crosslinked verapamil gel filtration on a G-15 Sephadex column. The molecular weight standards were tritiated glucitol (182), glucose a(l+ 3) glucitol (344) and glucose a(l + 4) glucose a(l + 3) glucose a(l + 4) glucitol (668). Fig:ure 1.0 0.8 0 :> ........... ""-! N 0. 6 glucose dimer 0 :> I :> 0.4 0.2 ---- ..... _... ----------- 0 • 01 2.2 I I 2.4 ~--- ..... ..._. I ..... ._._ ._ - - - ----- ............. _-- .............. t I 2.6 I 2.8 log KW I _.., .... .-.. ....... _.... .... I 3.0 &I ~ l 3.2 73 Figure 8 Molecular weight determination of crosslinked verapamil: gel filtration of a G-25 Sephadex column. The molecular weight standards were blue 6 dextran (2 x 10 ), bovine tryspin inhibitor (6518), vitamin B (1355) and reduced glutathione (307). 12 Figure reduced glutathione 3.0 2.8 2.4 0 > ............. I...,....__.__._.-_,___,.,... _ _ .....,. ..... ~--- ..... - - - - - - - - - - 2.0 ....... .p. 0 >I > 1.6 1.2 0.8 bovine trypsin inhibitor 0.4_.l 2.0 I \ 2.4 \ J 2.8 I I I 3.2 log MW I \ 3.6 J I 3.8 75 NMR Characterization of Crosslinked Verapamil In order to elucidate the structure of the methylene bridged verapamil dimer, a 500.13 MHz proton nuclear magnetic resonance (nmr) study was undertaken on both the monomer and dimer in n2 o. A summary of the results follows. Figure 9 is the 500.13 MHz proton nmr spectrum for verapamil in n2 o. The assignments of the resonances were obtained via homonuclear decoupling experiments. One interesting feature is the asymmetry of the resonances for the isopropyl methyl groups (o ~ 0.8, 1.2 ppm). This is due to the fact that, because of the sterically hindered nature of the isopropyl group's position in the molecule, rotation of the group is sever~iy hindered. Thus, the chemi- cal shifts of the two methyl groups are affected differently by the ring currents of the adjacent phenyl group. Figure 10 shows the assigned spectrum and proposed structure for the methylene-bridged dimer. Most of the assignments were obtained using homonuclear decoupling. Confirmation of the existence of the bridge (o was obtained via a T 1 experiment. ~ 3.8 ppm) Because of the immobility of the -CH -bridge relative to the rapidly rotating -OCH 2 3 groups, the T for the -OCH 3 1 of the bridge should be shorter than that groups was observed. protons of the methylene bridge (o The signal assigned the ~ 3.8 ppm) had an inte- grated intensity of two protons per proposed dimer. 76 The asymmetric nature of the dimer was inferred from the loss of symmetry for the downf ield -CH to the isopropyl groups. If 3 groups attached the dimer were symmetric, this resonance would be a clean doublet; in fact, it is two overlapping doublets (which appear as a triplet). This in conjunction with the observation of two N-CH resonances (o 3 ~ 2.8 ppm), supports the proposed structure. The relative order of reactivity of 0-dimethoxybenzene to electrophilic substitution (para > ortho) and steric effects due to the bulky isopropyl and cyano groups (Evans, 1981, personal communication) were also considered in determining the position of the bridGing methylene. In order to detect the amount of terminal aromatic CH 0H groups within the sample, the nmr spectrum of the dimer was obtained in d 6 -DMSO. Two resonances at 4.7 ppm were found which could be 2 o ~ 4.4 and assigned to such groups. Their total concentration in the sample is less than 10%, as determined by comparing their integral with that for both the aromatic protons and the methylene bridge. the predominant species has the structure Thus, shown in Figure l OHi H~CO {) '~ '-cH~-CHz-N-CH~-CH; 0 OCH~ . HI 1 H CHC HI 9 J/ CHJ I C HJ HI OCHi OCH~ ...... ...... • a a d I L L L l t 5 ppm Figure 9. Proton magnetic resonance (500.13 I l O ppm ~JHz) spectrum and peak assignments of monomeric verapamil in D 0. 2 Q 02 OCH~ . H1 CO 3 i 1 OCH3 H CHk H 1 CO{)~--H~~Ho · 3 I HI HI c~ 3 CHc g c~ I 3 z,CHb_cHz-N-cH'-CH 1 2 2 2 0 H1 3 HI HI ~:c.H; 'cHz-CHz-N-cH~-cH;-Q c ---CH2 CH H b g cHj I ~ 3 H1 HI 3 . OCH' 3 "'co • l l I 5 ppm Figure 10. OCHi OCHi a l om; 3 a I HI Proton magnetic resonance (500.13 MHz) spectrum and peak assignments of crosslinked verapamil in n2 o. l O ppm 79 Figure 11 illustrates the relative ability of compound 48/80, crosslinked verapamil and verapamil to cause the 3 release of H-serotonin from mast cells. Several experiments were pooled and the mean concentrations of drug required to cause 50% of the maximum response was determined. The results for the release of 3 H-serotonin from mast cells are summarized below. Concentration Compound 48/80 (6) Crosslinked verapamil (3) Verapamil (3) at 50% Release 0.08 + 0.04 µg/ml 6.2 0.2 _µg/ml + + 20 180 µg/ml The effect of the treatment of mast cells with neuraminidase on the ability of compound 48/80, crosslinked verapamil and verapamil to release 3 H-serotonin from mast cells was determined in the experiment depicted in Figure lL The data show that the release of 3 H-serotonin from mast cells by compound 48/80 was inhibited by treatment of the cells with neuraminidase while in contrast neuraminidase treat- ment of the cells had an insignificant effect on the release of 3 H-serotonin from mast cells caused by verapamil and crosslinked verapamil. 80 Figure 11 The release of 3 H-serotonin from rat peritoneal cells challenged by compound 48/80, crosslinked verapamil and verapamil. The cells were isolated, loaded with 3 H-serotonin, and preincubated by the methods described in Materials and Methods, Chapter II. Sub- sequent experiments (Figure 16) have shown that the release of 3 H-serotonin by crosslinked verapamil was as high as 95 + 5%. The challenges by compound 48/80 (-•-•-•-•-) crosslinked verapamil (- • - • - •-) and verapamil (-!-4-!-!-) were for one hour at 37°C in MCB. was 12%. The spontaneous release of 3 H-serotonin (6) 81 100 s:: s:: •r-i 80 0 ..µ 0 ~ Q) U'J. I ~ (") 60 CH 0 Q) Cfl ~ Q) M Q) ~ 40 ~ 20 0 1.0 2.0 3.0 4.0 5.0 Log Concentration (ng/ml) 6.0 82 Figure 12 Effect of neuramindase on the release of 3 H-serotonin from rat peritoneal cells challenged with compound 48/80, crosslinked verapamil and verapamil. The cells were pre- incubated withneuraminidase exactly as described in Materials and Methods and challenged with the various drugs for one hour at 37°C. The values given for release of 3 H- serotonin (cpm) were calculated by subtracting a blank of 1050 cpm for control cells (-•-•-•-•-) and 1350 cpm for cells pretreated withneuraminidase (----0----0----0----). The molar concentrations of the drugs were calculated assuming that crosslinked verapamil is a dimer and compound 48/80 has a molecular weight by Morrison et al (1974). of 800 as determined The curves at the left are compound 48/80, the curves in the middle are crosslinked verapamil and the curves on the right are verapamil. The estimated error on the cpm of the released serotonin was 5%. 3 H- 6000 ...... 100% Release Level 5000 p (]) Cf1 I rn I (]) r-1 (]) I 4000 I I ~ I ~ I ·r-1 I ~ 0 .µ 0 H I I 3000 I ~ (}) U) I I '/ rt I ::c: ('I') 2000 1000 Cl-'.,.,,,. 0 ,,,.' o------ ~o------o--i.·o 0 compound 48/80 1.0 crosslinked verapamil log concentration (µM) 2.0 verapamil 3.0 00 w 84 Table III shows the results of two experiments examining the role of crosslinking of antigen in the release of 3 H-serotonin from mast cells by the IgE-14-205 protein. The crosslinking of ovalbumin by Woodward's reagents K and the relatively complex protocol of this experiment are described in detail in the Materials and Methods section. The addition of only crosslinked ovalbumin or only ovalbumin to the mast cells did not cause a significant release of SH-serotonin (not shown). The results of Table III are seen to indicate that crosslinked ovalbumin cause a greater release of 3 H-serotonin from IgE-treated mast cells than monomeric ovalbumin. Figure lSrepresents dose-response curves for the release of SH-serotonin from mast cells challenged with compounf 48/80 at various concentrations of extracellular calcium. The results show that increases in the concen- tration of extracellular calcium causes a decrease in the 3 ability of compound 48/80 to induce the release of H- serotonin from mast cells. This result agrees qualita- tively with the preliminary finding of Kagayama and Douglas (1974), that 110 mM Cac1 in the extracellular abolished the 2 degranulation of mast cells caused by compound 48/80. Figure 14 shows the effect of extracellular calcium concentration on the release of cells challenged with verapamil. 3 H-serotonin from mast The release of 3 H-serotonin 85 at the submaximal dose of verapamil (179 µg/ml) was relatively uneffected by the concentration of extracellular calcium. Figure 15 shows the effect of the concentration of 45 LaC1 on the uptake of ca into P815 mastocytoma cells. 3 At concentrations of LaC1 3 greater than approximately lmM, the La+++ caused a dose-dependent increase in the uptake of 45 ca into the mastocytoma cells. Figures 16 and examining the 17 illustrate timed experiments kinetics of the release of 3 H-serotonin from rat mast cells by compound 48/80, crosslinked verapamil and verapamil. Figure 16 shows that relatively high concentrations of verapamil and crosslinked verapamil cause the release of 3 H-serotonin from mast cells. This experiment illustrates that a component of the release of 3 H-serotonin by verapamil and crosslinked verapamil is fast (< 5 minutes). Similarly, the data of Figure 17 show that relatively low concentrations of these drugs could cause this same fast component (< 5 minutes) of release of 3 H-serotonin from the mast cells. 86 Table III Release and Retention of 3 H-Serotonin in Mast Chapter I II Cells Preincubated in IgE 14-205 Protein Then Challenged by Crosslinked Ovalbumin or Ovalbumin [IgE] [Ovalbumin] 0.50 µM 18.0 µM 0.50 µM 18.0 µM x 0.50 µM 0.14 µM 6.0 µM 0.14 µM 6.0 µM x 0.14 µM 0.14 µM 2.0 µM 0.14 µM 2.0 µM x 0.14 µM 0.27 µM 5.8 µM 0.27 µM 5.8 µM x 0.27 µM 1.08 µM 20.7-µM 1.08 µM 1.08 µM 20.7 µM 0.27 µM 0.27 µM 12.4 µM 0.27 µM 12.4 µM x x % Release % Retention 80 20 45 55 0 100 22 78 19 81 0 103 46 54 28 72 2 98 59 41 36 64 9 91 46 54 48 52 9 91 46 0 54 101 0 115 X refers to crosslinked ovalbumin 87 Figure 13 Effect of calcium concentration on 3 H-serotonin release from rat peritoneal cells challenged by compound 48/80. The rat peritoneal cells were isolated in regular MCB which contains no calcium. The cells were preincubated in various concentrations of CaC1 2 for 15 minutes at 37°C. The cells were then challenged by compound 48/80 for one hour at 37°C. The (-0-0-0-0-) curve represents cells preincubated in HCB containing no calcium. The (-•-•-•-•-) curves represent cells preincubated ++ in 2 mM Ca (upper) and 6 mM calcium (lower). The estimated error was 5%. 88 • 0 • 00 N m 0 00 0 ,,....._ r-1 E ......._ M ~ ~ ........... 0 0 00 ......._ 00 ~ © 0 ~ c ~ 0 ~ E 0 ~ u 0 ~ ~ .....,0 0 c ~ 0 rn ~ ~ c © c 0 u N 0 r-1 0 0 0 m 0 0 M 0 89 Figure 14 Effect of calcium concentration on 3 H-serotonin release from rat peritoneal cells challenged with verapamil. The rat peritoneal cells were isolated in regular MCB which contains no calcium. The cells were then preincubated in various concentrations of CaC1 2 for 10 minutes at 37°C. The cells were then challenged for one hour at 37°C wtih 510 µg/ml of verapamil (-•-•-•-) and 179 µg/ml of verapamil (-'-A-i-!-). was 5%. The estimated error 90 0 0 r-i 0 m 0 00 0 ~ ~ ~ E '-" 0 c 0 •ri © ~ ro ~ ~ c 0 ~ w 0 c 0 u E 0 ~ •ri ~ r-i 0 ro u 0 M 0 N 0 0 0 0 r-i 0 00 0 © 0 N 0 91 Figure 15 The effect of lanthanum concentration on calcium uptake in P815 mastocytoma cells. The P815 cells were aliquoted into MCB containing 2 mM ca++ and 45 ca at a specific activity of 0.58 µmole/10 6 cpm. After a short preincubation the P815 cells (1.3 x 10 6 cells per 1 ml sample) were exposed to various concentrations of LaC1 3 for 20 minutes at 37oc. The cells were then worked up on glass filters as described in Materials and Methods. The error in this experiment was large and was estimated from the standard deviation of the values for the blanks to be + 3 nmoles Ca/10 6 cells. 92 0 ~ ,,....... © M ~ ~ '-" ~ 0 ·r-l ~ ~ ro M ~ ~ ~ © 0 ~ 00 ~ 0 u E ~ ~ ro ~ ~ ~ ~ ~ ro ~ ~ 0 0 M ~ ~ 0 93 Figure 16 Rate of release of 3 H-serotonin from rat peritoneal cells challenged with high concentrations of verapamil and crosslinked verapamil. The cells were preincubated and challenged by the usual methods except that aliquots were sampled at various time intervals from large volumes (10 ml). Scintillation counting of the supernatants of the aliquots determined the release of 3 H-serotonin. The blank at 0 minutes was 4% of the total 3 Hserotonin release and at 91 minutes was 7% of the total 3 H-serotonin release. The concentration of verapamil was 880 µg/ml (-•-•-•-•-) and crosslinked verapamil 40 µg/ml (---0---0---). 94 9 0 crJ I I f t I I 0 00 I t '' I I 0 !:' I I ' 0 © -... r.n Q) .µ ;::$ 0 I..{) .,.,i::: E '--" Q) - .,.,E 0 ~ 0 (Y) 0 C'\l '\ '\ \ 0 \ l""""l b, ''' 0 0 l""""l " 0 0 00 0 E-1 95 Figure 17 Rate of release of 3H-serotonin from rat peritoneal cells challenged with crosslinked verapamil, verapamil, and compound 48/80. The cells were prein- cubated and challenged by the usual methods except that aliquots were sampled at various time intervals from large volumes (6 ml) of rat peritoneal cells in MCB. Scintillation counting of the supernatants of the aliquots determined the release of 3H-serotonin from the cells. The various drug concentrations were: compound 48/80 verapamil cross linked verapamil blank 0.16 µg/ml 330 µg/ml 5 µg/ml -·-·-·-·-·-·- -!:::.-!:::.-!:::.- -0-0-0- 96 70 60 ~ •M ~ 0 .;..> 50 0 ~ (J) Cl) I :i:: CV) ~ 0 40 (J) U1 ro Q) ,..-l (l) 0:: -eQ. 30 20 --- __. - - -tt' , ,, , ,, ,l::ir--- --- -- - -- A --~ 10 0 0 20 40 60 80 100 Time(minutes) 120 140 97 DISCUSSION Crosslinking and aggregation of IgE-receptors on the mast cell surface has been proposed as an obligatory event in the degranulation of mast cells (Metzger, 1979). A mechanism of action of verapamil and crosslinked verapamil might feature the drugs crosslinking cell surface structures in a manner analogous to the activation of mast cells by crosslinking IgE-receptors (Hirata and Axelrod, 1980) system. In fact, a chemically crosslinked deriva- tive of verapamil was 30 times more potent by weight at causing the release of 3 H-serotonin from rat mast cells relative to monomeric verapamil (Figure 11). We attempted to further examine the role of crosslinking and aggregation of cell surface structures by comparing the relative ability of chemically crosslinked ovalbumin versus monomeric ovalbumin to cause degranulation of mast cells in the presence of an IgE hybridoma protein that specifically binds to ovalbumin. Table 1 show The data of that ovalbumin crosslinked with Woodward's reagent K caused a greater release of 3 H-serotonin from rat mast cells preincubated in the IgE-14-205 protein than monomeric ovalbumin. This result does seem to agree with the general findings of Siraganian et al (1975) and others that chemically crosslinked antigen causes a greater IgE-mediated release of histamines than monomeric antigen. 98 Moreover,verapamil has a crude symmetry around the tertiary amino group making it a possible candidate for a bifunctional agent capable of crosslinking. The increased ability of crosslinked verapamil to cause the release of 3 H-serotonin from mast cells may be due to the avail- ability of more methoxyphenyl rings for binding to cellular target structures. The increased binding of a crosslinked molecule, relative to its monomeric counterpart, to another structure may be analogous to the so-called thermodynamic "chelate effect" for metal ions (Cotton and Wilkinson, 1972). The chelate effect refers to the enhanced stability of a metal complex system containing ring-forming ligands as compared to a system with similar monomeric ligands. The entropy for the formation of such chelates is significantly more favorable than that for the formation of an analogous complex from a metal ion and several ligands. In forming the chelate, the translational entropy of only one molecule is lost; in forming the multiliganded complex, the translational entropy of several molecules must be sacrificed. Besides simple thermodynamic considerations, the increased ability of crosslinked verapamil to cause release of 3 H-serotonin from mast cells relative to verapamil may be due to steric considerations in the crosslinking potential 99 of the drugs. For example, crosslinked verapamil may be able to span and consequently link sites that cannot be linked by monomeric verapamil. IgE binds to a specific glycoprotein on the mast cell surface (Metzger, 1979). Moreover, there is some circumstantial evidence that verapamil and D-600 can also bind to proteins. For example, Kaumann and Uchitel (1976) found significant differences in the (+) and (-) isomers of verapamil in their ability to inhibit potassium-induced contractures in frog skeletal muscle fibers. Furthermore, the (+) and (-) isomers of verapamil were shown to have different abilities to cause the decoupling of excitation from contraction in canine heart (Kaumann and Serur, 1975). The stereoselective pharmacology of (~)-verapamil suggests that the drug is binding to stereoselective sites on proteins. Discussion of Neuraminidase 3esults Experiments were designed t~ determine if verapamil interacts with the cell surface to cause degranulation of mast cells in a manner analogous to the binding of IgE to its receptor or if verapamil triggers degranulation by some intracellular mechanism of action. Rat peritoneal cells were treated with neuraminidase, an enzyme that specifically removes sialic acids from cell surface molecules, and then challenged with verapamil, crosslinked verapamil, and compound 48/80 100 (Figure 12). Pretreatment of the cells with neuraminidase abolished the ability of compound 48/80 to cause release of 3 H-serotonin from mast cells (Figure 12). Similarly, Baxter and Adamik (1977) had found that pretreatment with neuraminidase causes a significant decrease in the release of histamine from mast cells exposed to compound 48/80, protamine and polylysine. They also found that the neuraminidase pretreatment did not affect the ability of antigen to evoke IgE-mediated release of histamine from mast cells. Figure 12 shows that pretreatment of the cells with neuraminidase did not significantly affect the ability of verapamil or crosslinked verapamil to cause release of 3 H-serotonin from rat peritoneal cells. These results indicate that the exact sites of action of compound 48/80 and verapamil are different. Compound 48/80 causes degranulation of mast cells by binding to the sialic acids of cell surface molecules tive to hydrolysis by neuraminidase. that are sensi- In contrast, vera- pamil almost certainly does not require these neuraminidasesensi ti ve sialic acids to cause the release of from mast cells. 3 H-serotonin Conceivably, the verapamil and compound 48/80 could even by interacting with the same molecule. As a hypothetical example, the verapamil interacts with the protein portion of a glycoprotein and compound 48/80 binds to the saccharide groups of this same glycoprotein but there is not direct evidence for this hypothesis. 101 A simple way to explain why verapamil and crosslinked verapamil are not inhibited in their ability to cause the release of 3 H-serotonin from mast cells when the cells were pretreated with neuraminidase is to propose that these drugs act inside the cell to trigger the release of 3 H-serotonin. Other studies have inferred that verapamil not only blocks the so-called "slow channels" for Ca++ in the plasma membrane (Fleckenstein, 1977) but causes other effects by acting within the cell. For example, Church and Zsoter (1980) found verapamil inhibited the contraction of rabbit mesenteric vein caused by high concentrations of extracellular potassium. The drug did not alter the lanthanum-resistant uptake* of 45 ca by the vein during the verapamil-induced relaxation, implying that the effect was not due to the drug acting as a specific antagonist of the calcium channels of the plasma membrane but had an intracellular mechanism of action. Moreover, verapamil has been shown to inhibit the norepinephrine-induced development of tension in the isolated rat atrium (Church and Zsoter, 1980). Norepinephrine caused a *The 'l~nthanum-method" supposedly allows the direct measurement of 45ca influx by cell and tissues without interference due to binding of the 45ca to the cell surface and efflux of the label from the tissue (Van Breeman et al, 1973). However have found that La3+, itself, causes a dose-dependent uptake of 45ca into P815 mastocytoma cells (Figure 15) and similarly, Foreman and Mongar (1972) found that high concentrations of La3+ caused mast cells to release histamine, a ~rocess that can be caused by an influx of extracellular ca+ (Hirata and Axelrod, 1980). 102 rapid increase in tension that could be inhibited by verapamil. Church and Zsoter (1980) proposed that verapamil had an intracellular mechanism of action, supposedly by interfering with the distribution of calcium in the intracellular pools. However, this is not conclusive evidence since verapamil could act at the cell surface and exert its effect on the norepinephrine-induced tension via a mechanism involving a secondary messenger. Similarly, we have shown that verapamil can cause the release of 3 H-serotonin from mast cells in the absence of extracellular calcium; moreover the release was relatively insensitive to the concentration of extracellular calcium (Figure 13). These results are in contrast to those for the release of biogenic amines from mast cells challenged with compound 48/80; in this case release was significantly inhibited by the addition of extracellular calcium (Figure 14). These experiments suggest that the release of 3 H-serotonin from mast cells caused by verapamil is not due to an influx of extracellular calcium analogous to the mechanism of degranulation of mast cells by IgE-aatigen complexes (Hirata and Axelrod, 1980). Discussion of Possible Mechanisms Verapamil causes a wide variety of effects on mammalian myocardium, some of which cannot be explained by blockage of the so-called "slow calcium channels" (Bayer et al, 1975). For example, Bayer et al (1975) 103 found that the (~)-isomers of verapamil and D-600 have a quite specific inhibitory effect on the fast Na+ inward current in feline myocardium. The (+)-isomers also in- creased the threshold intensity of electrical stimuli needed to elicit conducted action potentials in the myocardium. Bayer et al (1975) also found that the (-)- isomers of verapamil and D-600 were more potent than the (+)-isomers in blocking the so-called specific calcium channels in the myocardium. This local anesthetic effect of verapamil and D-600 has also been shown in frog nerves where the drugs are l.6X times more active than procaine in blocking action potentials (Singh and Williams, 1972). A mechanism of action similar to the cationic anaesthetic effect of verapamil could provide a molecular rationale for the ability of verapamil to release 3 Hserotonin from mast cells. Seeman (1972) and Kwant and Seeman (1969) have shown that local anaesthetics such as phenothiazines displace membrane calcium from erthyrocyte ghosts and Sheetz and Singer (1974) suggested that cationic anaesthetics bind to the cytoplasmic side of the membrane bilayer which is relatively rich in the negatively charged phospholipids such as phosphatidylserine (Bretcher, 1972). The cationic drugs supposedly expand the cytoplasmic side of the bilayer and induce cup formations in erythrocytes. Furthermore, Frazier et al (1970) showed with microinjection techniques that the anaesthetic 104 action of several tertiary amine local anaesthetics was more effective on the cytoplasmic side of the nerve membrane. In accord with the above observations, verapamil may displace membrane-bound calcium from the cytoplasmic side of the plasma membrane and thereby trigger the release of granule-stored amines from mast cells. Unfortunately, the mechanism of action of cationic anaesthetics have not been resolved, but the anaesthetics do cause a variety of effects that could conceivably increase the cytosolic [Ca++] enough to activate mast cells. For example, positively charged anaesthetics displace loosely adsorbed Ca++ from reticulum membranes (Thorpe and Seeman, 1971) and reduce the entry of Ca ++ across mitochrondrial membranes (Tjioe et al, 1971). Furthermore, there is experimental evidence that high concentrations of verapamil affect the translocation of calcium across in vitro preparations of subcellular membrane fractions. For example, high concentrations of verapamil have been shown to inhibit calcium uptake by membrane and endoplasmic reticulum fractions from rabbit aorta (Thorens and Haeusler, : 1979). Moreover, Crankshaw et al (1977) found that D-600 (520 µg/ml) and verapamil (490 µg/ml) significantly reduced Ca ++ accumu- lations in subcellular fractions of the plasma membrane, endoplasmic reticulum, and the mitochrondrial membrane of rat myometrium. Lastly, Balzer (1972) found a similar 105 effect for verapamil in an in vitro membrane fraction of the sarcoplasmic reticulum of skeletal muscle. Perhaps, verapamil causes an increase in the concentration of cytosolic Ca++ by inhibiting the calcium-ATPase pumps of membranes defining the cytosolic pool. This event may trigger the release of histamine by mast cells challenged by verapamil. An unanswered question is whether the in vitro inhibition of calcium-ATPase pumps by verapamil in subcellular membrane fractions is relevant to the intact mast cell. Verapamil could possibly interact with the ubiquitous ++ intracellular Ca -receptor, calmodulin, and cause the degranulation of mast cells. Calmodulin has been implicated in a wide variety of calcium-dependent effects such as activation of a phosphodiesterase (Cheung, 1970) an adenyl cyclase (Bronstron et al, 1975) a phospholipase A2 in platelets (Wong and Cheung, 1979) and ATPase-dependent calcium pumps of erythrocyte membranes (Levine and Weiss, 1980). Some or all of these effects could be postulated to affect degranulation of mast cells depending on whether verapamil potentiates or counteracts the action of calmodulin. However, such speculation is pointless until the existence of sufficient calmodulin has been demonstrated in the mast cell. 106 Kinetics of the Release of SH-Serotonin from Mast Cells The kinetics of the release of SH-serotonin by verapamil and crosslinked verapamil show interesting features (Figures 16 and 17). At concentrations of verapamil and crosslinked verapamil that do not cause maximal release of SH-serotonin, the pattern of release appears to be biphasic. For release of SH-serotonin caused by verapamil or crosslinked verapamil, there appears to be a fast release (< 5 minutes) followed by a lag time then a slower release (Figure 17). A possible explanation of this biphasic kinetics is that the drugs first cause a release of SH-serotonin by interacting with the cell surface, in a manner analogous to compound 48/80, then penetrate the membrane and interact with intracellular targets to bring about the slow release. In accord with this possibility, the crosslinked verapamil showed a larger release of SH-serotonin in the·fast phase then did monomeric verapamil; nevertheless crosslinked verapamil showed a lower from the mast cells. overall release of SH-serotonin This increased ability of cross- linked verapamil to cause the fast release of SH-serotonin from mast cells could be due to its ability to aggregate cell surface molecules as a crosslinking agent in a manner analogous to the polymeric compound 48/80. In contrast, monomeric verapamil could be more effective in the slow 107 phase of release of 3 H-serotonin simply because higher concentrations of the drug are penetrating the membrane and interacting with intracellular targets. 108 REFERENCES Balzer, H. (1972) Naunyn-Schmeiderberg's Arch. Pharmacol. 274, 256. Baxter, J.H. and Adamik, R. (1977) Biochem. Pharm. 27, 487. Bayer, R., Kalusche, D., Kaufmann, R. and Mamhold, R. (1975) Naunyn Schmeiderberg's Arch. Pharmacol. 290,81. Bennich, H. and von Bahr-Lindstrom, H. (1974) "Progress in Immunology," (ed. L. Brent and J. Hollborrow) North Holland-Amsterdam, Vol. 1, p. 49. Bretcher, M.S. (1972) J. Mol. Biol. 71, 523. Bronstron, C.O., Huang, Y.C., Breckenbridge, B.N. and Wolf, D.J. (1975) Proc. Natl. Acad. Sci. USA 72, 64. Cheung, W.Y. (1970) Biochem.Biophys. Res. Commun. 33, 533. Church, J. and Zsoter, T.T. (1980) Can. J. Physiol. Pharmacol. 58, 254. Cochrane, C.G. and Douglas, W.W. (1974) Proc. Natl. Acad. Sci. USA 71, 408. Cochrane, C.G. and Muller-Eberhard, H.J. (1968) J. Exp. Med. 127, 371. Cotton, F.A. and Wilkinson, G. Chemistry" 3rd ed. (1972) "Advanced Organic (John Wiley and Sons) p. 650. Crankshaw, D.J., Janis, R.A. and David, E.E. (1977) Can. J. Physiol. Pharmacol. 55, 1028. Dias da Silva, W. and Lepow, I.H. (1967) J. Exp. Med. 125, 921. Fernandez, H.N. and Hugli, T.E. (1976) J. Immunol. 117, 1688. Fleckenstein, A. (1977) Ann. Rev. Pharmacol. Toxicol. 17, 149. Foreman, J.C. and Mongar, J.L. (1972) Nature 240, 255. 109 Frazier, D.T., Narahashi, T. and Yamada, M. (1970) J. Pharm. Exp. Ther. 171, 45. Hakomori, S. and Saito, T. (1969) Biochemistry 12, 5082. Herndier, B. and Brooks, D.E. (1975) unpublished results. Hirata, F. and Axelrod, J. (1980) Science 209, 1082. Hugli, T.E. (1975) J. Biol. Chem. 250, 8293. Gottschalk, A. (1972) "Glycoproteins. Their Composition, Structure, and Function" Elseiver, New York. Ishizaka, K. and Ishizaka, T. (1969) J. Imrnunol. 103, 508. Ishizaka, K. and Ishizaka, T. (1978) J. Imrnunol. 120, 800. Ishizaka, T., Foreman, J.C., Sterk, A.R. and Ishizaka, K. (1979) Proc. Natl. Acad. Sci. USA 76, 5858. Ishizaka, T., Hirata, F., Ishizaka, K. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 1903. Kagayama, M. and Douglas, W.W. (1974) J. Cell Biol. 62, 519. Kaumann, A.J. and Serur, J.R. (1975) Naunyn Schmeider- berg's Arch. Pharmacol. Kaumann, A.J. and Uchitel (1976) Naunyn-Schmiederberg's Arch. Pharmacol. 292, 21. 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Siraganian, R.P., Hook, W.A. and Levine, B.P. (1975) Imrnunochemistry 12, 149. Thorens, S. and Haeusler, G. (1979) Eur. J. Pharmacol. 54, 79. Thorpe, W.R. and Seeman, P. (1971) J. Pharmacol. Exp. Ther. 179, 324. Tjioe, S., Hasigaard, N. and Bianchi, C.P. (1971) J. Neurochem. 18, 2171. 111 a Tung, K.K. and Nordin, J.H. (1970) Analytical Biochem. 38, 164. Tuppy, H. and Gottschalk, A. (1972) in "Glycoproteins" (Gottschalk, A., ed.) Elsevier, North Holland, p. 403. Van Breeman, C., Farinai, B.R., Casteels, R., Gerba, F., Wuytack, F. and Deth, R. (1973) Phil. Trans. R. Soc. Lond. B 265, 57. Wong, P. and Cheung, W.Y. Commun. 90, 473. (1979) Biochem. Biophys. Res. lllb CHAPTER IV A STUDY OF CONTRACTURE OF SKELETAL MUSCLE CAUSED BY COMPOUND 48/80 112 INTRODUCTION Muscle Contracture As A Model for Excitation-Contraction Coupling A muscle contracture has been defined as a decrease in the length of a muscle or an increase in muscle tension produced without muscle action potential (Gasser, 1930). In contrast, muscle twitches require a muscle action potential and are designated as contractions. A difficulty in studying excitation-contraction coupling in skeletal muscle is separating the complex ionic events involved in the generation of the action potential from possible ionic or biochemical events associated with the actual coupling of the action potential with the release of calcium from the sarcoplasmic reticulum. Muscle contractures, because they do not depend on muscle action potentials, offer a simpler system for study. However, use of muscle contractures as a basis for inferences on excitation-contraction coupling should involve great caution because of the possible non-physiological nature of the experiment. However, in the past contracture systems have provided useful models for excitation-contraction coupling. For example, there is the so-called potassium contracture which features the de- 113 polarization of the muscle membrane by replacement of Na+ by K+ in the extracellular medium. Frank (1964) demonstrated that the removal of extracellular caicium abolishes the potassium contracture but does not affect the depolarization of the muscle membrane. This result suggested that the extracellular calcium pool was the source of "trigger-Ca++" in the potassium-contracture. A more thorough analysis of the kinetics of the removal of calcium in potassium-contracture suggests that the rate of exchange of "trigger-Ca++" was intermediate between the rate of exchange of extracellular calcium, which was rapid, and the rate of exchange of intracellular calcium of the sarcoplasmic reticulum, which was shown to be slow (Frank, 1979). Similarly, muscle contractions can be abolished in buffer without calcium, but only after Ca++ has been removed for approximately ten minutes. This is much longer than the few seconds required for Ca++ to diffuse out of the t-tubular system and shorter than the estimated four hours required to significantly deplete the pool of calcium in the sarcoplasmic reticulum (Frank, 1979). The parallel finding for the potassium-contracture of skeletal muscle and the contractions of skeletal muscle caused by an action potential seem to imply that the source of "trigger-Ca++" is the same for both systems and is likely to be membrane-bound calcium. 114 Caffeine causes a contracture in skeletal muscle at concentrations greater than 3.0 mM (Axelsson and The.sloff, 1958). This contracture does not require extracel~ular calcium and does not cause depolarization of the sarcolemma. Caffeine is believed to act directly on the sarcoplasmic reticulum and to cause calcium release from that organelle (Ebashi, 1976). The molecular mechanism of this release is not known. This contracture system has limited utility for understanding EC coupling between the t-tubules and the sarcoplasmic reticulum but may be of use in under~ standing the function of the sarcoplasmic reticulum (Hasselbach, 1980). This study involves the development of an experimental model of excitation-contraction coupling. We have found that compound 48/80, a promoter of stimulus-secretion coupling in mast cells causes a contracture in muscle. The possibility of a mechanistic link between stimulussecretion coupling in mast cells and excitation-contraction coupling in skeletal muscle was investigated using the compound 48/80 to induce muscle contracture. 115 Protein Turnover in Muscle Continuous protein turnover is a feature of muscle and many other tissues and cells (Goldberg and St. John, 1977). Protein degradation. as well as protein synthesis is precisely regulated in the normal cell. Protein breakdown may function to remove abnormal proteins, to degrade enzymes or structures not needed in the immediate function of the cell, and to provide amino acids for other tissues under nutritional stress. The rates of protein synthesis and degradation in tissues can be changed by extracellular signals such as hormones and by the activity of the tissue. Protein turnover ultimately can determine the growth, stability or atrophy of tissue. There is evidence that protein turnover in normal and pathological muscles is sensitive to the distribution of calcium in the intracellular pools. Kameyama and Etlinger (1979) had found that protein turnover in rat soleus muscles could be affected by treatment of the muscles with the Ca-ionophore, A23187. Pennington (1978) proposed that muscular dystrophy, a disease characterized by abnormalities in protein turnover, might also exhibit abnormalities in cellular calcium metabolism. 116 Chapter II of this thesis presents evidence that, in mast cells, compound 48/80 can mobilize intracellular stores of calcium leading to degranulation of the cell. In the normal excitation-contraction cycle of skeletal muscle the muscle action potential triggers a series of events which lead to the mobilization of calcium from the intracellular organelle, the sarcoplasmic reticulum (Hasselbach, 1980). Since compound 48/80 causes a con- tracture in frog skeletal muscle, we were interested in a possible relation between protein turnover and the mobilization of intracellular calcium in frog skeletal muscle challenged with compound 48/80. 117 MATERIALS AND METHODS Isolation of Frog Skeletal Muscles The muscles were isolated from frogs of the genus Rana pipiens (Carolina Biological). The frogs were ·double pithed and pinned down. The skin was cut away from the leg and the exposed muscles were kept wet by oxygenated frog Ringer's solution. All muscles were removed by a technique such that the tendons of origin and insertion could be easily cut with no damage to the muscle membrane. Fine pointed forceps and corneal scissors proved indispensible to this task. The muscles removed, with the ranges in masses, were: the sartorius (80-400 mg), semitendinosus (70-300 mg), semimenbranosus (280-1200 mg), gracilis major (240-950 mg), gastrocnemius (420-1940 mg), and a tightly bound complex of the peronius, tibialis articus and tibialis posticus (220-800 mg). This "shin muscle" complex was not separated for fear of damaging the muscle membrane. When more than one frog was used in an experiment care was taken to match their sizes. 118 Denervation of the Sartorius Muscle Intact frogs were set in 10% ethanol until they ceased to show any movement reflexes. A small incision was made over the sartorius muscle. (~ 2 cm) The other leg was left intact to later serve as a control. With fine point forceps and corneal scissors the sartorius muscle was separated from the gracilis major muscle to expose the sartorius nerve. damaging the muscles. Care was taken to avoid The nerve was cut causing a con- traction/twitch of the sartorius. The skin was closed with wound clips and after a week the sartorius muscles were isolated and submitted to pharmacological challenge. 119 Treatment of Frog Muscles with Neuraminidase Frog skeletal muscles were treated with neuraminidase derived from Vibrio cholerae (Calbiochem-Behring) .. Typical experiments exposed the muscle to 0.25 units/ml in 0.7 ml of PAB for 75-90 minutes at 25°c. The muscles were then transferred to another PAB aliquot and subjected to the various pharmacological challenges. One unit of neuraminidase activity is defined as the amount of enzyme that will release 1 µmole of N-acetylneuraminic acid from human acid a -glycoprotein per minute at 37°C in 0.05 M 1 sodium acetate buffer, pH 5.5 containing 5 mM calcium chloride. 120 Measurement of the Efflux of 86 Rb+ from Frog Mu~~le MuseJ.cs were preincubated in 10 µCi/ml of 86 RbCl in PAB at 25°C for 1.5 hours. from the muscles by The excess 86 Rb+ was ~ashed dipping them in a large volume of PAB then incubating the muscles in a 5 ml volume of the buffer. Thewashwas then repeated. The rationale for this procedure was to remove loosely associated 86 Rb+ from the muscle. After this preparation, the muscles were put into plastic tubes containing PAB and challenged with the various pharmacological agents. Aliquots of the extramuscular buff er were sampled and analyzed by the standard methods of scintillation counting. 121 Protein Degradation Measurements All experiments involving protein degradation ~ere performed in protein anabolism buffer (PAB). PAB is a balanced buffer specifically designed to maintain protein synthesis. The amphibium PAB was modified from the basic frog Ringer buffer with adaptation from a system (Kameyama and Etlinger, 1979). mammalian muscle PAB consisted of 116 mM NaCl, 2.5 mM KCl, 2 mM MgC1 2 , 10 mM Tris(hydroxymethyl)aminomethane, 10 mM glucose, 1.8 mM CaCl2, 1.0 mM isoleucine, 1.0 mM leucine, 1.0 mM valine and 10 units/£ insulin. The PAB was titrated to pH 7.4 with concentrated hydrochloric acid. The isolated frog muscle was incubated in PAB at 25°c. After a specific drug challenge the muscle was placed in PAB containing 10% trichloroacetic acid (TCA). The original PAB was also made 10% in TCA. The TCA served three purposes: to precipitate any exogenous protein released by the muscle; to precipitate the drug 48/80 to prevent interference with the subsequent tyrosine assay and to precipitate the protein of the intact muscle so that the muscle could easily be broken up to release internal pools of tyrosine. The muscles were generally left overnight at 5°C before being broken up by manual manipulation with a glass stirring rod. 122 Each muscle of a given experiment was manipulated for approximately the same time. The precipitated muscle debris was pelleted by centrifugation and the supernatant was analyzed. It was found that the muscles could be broken up very easily by the addition of an aliquot of saturated sodium carbonate solution to the TCA precipitated muscle. This approach was tried in several experi- ments and was believed not to the assay. The supernatant of the original reaction volume (containing released tyrosine) and the supernatant of the precipitated muscle (internal tyrosine pool) were analyzed by the spectrofluorimetric method of Waalkes and Udenfriend (1957). The assay consisted of deriva- tizing tyrosine with l-nitroso-2-napthol and analyzing the extracted product spectrofluorimetrically (activation 460 mµ; measurement 570 mµ). The instrument used was a Perkin-Elmer MPF-4 fluorescence spectrometer. kindly loaned by Dr. M. A. Raftery. In most experiments the total protein degradation was cited as the sum of the released tyrosine and the internal tyrosine pool. 123 Origin and Maintenance of L8 Myoblast Cell Line The L8 cell line was a gift of Dr. H. Blau of Stanford University (Blau and Epstein, 1979). The line was or- ginally derived from newborn rat skeletal (thigh) muscles (Yaffe, 1968). These cells retain the ability to fuse to form multi-nuclear syncytia (myotubules). Upon formation of the myotubules the L8 cells cease division and DNA synthesis and synthesize a number of proteins of myotubules including myosin and the muscle isozymes of creatine kinase. The L8 myoblasts were grown at 37°C in 5% co /100% H 0 2 2 in Waymouths media supplemented with 10% fetal calf serum. The ~reconf inement monolayers were passed by first washing the cells with Ca and Mg - free Dulbecco's phosphate buffered saline and then digesting the monolayer for three minutes at 37oc with 0.125% trypsin. 1:10 to 1:20. The cells were split 124 Protein Degradation in L8 Cells The cells were grown from low density to approximately 5 x 10 5 cells per plate. The growth medium was washed off the monolayer with several rinses of PBS. 1.5 µ£ of PAB, adjusted with NaCl for mammalian cells, was added to the plates. The cells were challenged with 48/80 for one hour. After this treatment 0.25 ml of 30% TCA was added to 1 ml of the supernatant to precipitate protein and drug. One ml of 30% TCA was then added to the remaining 0.5 ml of PAB. The monolayer of precipitated cells was mechanically detached from the tissue culture plate by a rubber policeman. aliquots were centrifuged to remove Both the TCA precipitate and the supernatant analyzed for tyrosine. The total of the two aliquots represented the total free tyrosine in the L8 cells. Differences in free tyrosine levels indicated protein degradation by the assumptions of Kameyama and Etlinger (1979). 125 45 Measurement of Ca Uptake In a typical muscle calcium exchange experiment, muscles 45 were exposed to cacl in the presence of 25°c PAB. The 2 45 ca was 1 µCi/ml in a 2 ml volume. The specific activity of the 45 ca was 5 x 10 1.8 mM CaC1 . 2 4 cpm/µmole Ca at a concentration of After the designated time period for the challenge the muscles are serially washed in 5 ml PAB. After four washes 45 (~ 15, 15, 30, 30 minutes) the external ca counts have stabilized at a level original specific acitivity. had stabilized Because the wash counts at such a low value, it was assumed that all "loosely" bound surface the muscle. 45 Ca had been removed from The muscles were then digested in 3 N nitric acid on a steam bath for two hours. aliquot of the supernatant was taken. solution. Muscle debris, if any after the digestion, was centrifuged and an was present neutralized of~ 0. 4% of the with The lation counter. 45 an aqueous The aliquots were sodium hydroxide ca was then counted on a Beckman scintil- 126 Assay of Protein Synthesis in Frog Skeletal Muscle The muscles were preincubated in oxygenated protein anabolism buffer (PAB) at 2s 0 c for 20 minutes. had been made 0.6 µCi/ml with and 0.5 mM with cold tyrosine. in this medium for ~ 14 C-tyrosine (Amersham) The muscle was challenged 1 hour in presence of a drug or in the absence of drug as a control. treated The PAB Then the muscle was with 30% trichloroacetic acid (TCA) at s 0 c for at least 12 hrs. The muscle-TCA tube was spun at 2,000g for 10 minutes. The supernatant was collected and extracted three times with diethyl ether to remove the TCA which can interfere with the scintillation counting. of 14 An aliquot was counted to determine the amount c-tyrosine present. Another aliquot was taken to determine the amount of tyrosine present in the cytoplasmic pools. The tyrosine was measured by the Waalkes and Udenfriend (1957) method described elsewhere in this study. The TCA precipitate (the bulk of the muscle) was washed then solubilized in 6N NaOH for several hours in a steambath. An aliquot of this mixture was neutralized and counted. This count represented the amount of tyrosine incorporated into the muscle protein. 14 c- Total tyrosine incorporation was determined by dividing the specific activity of the cytoplasmic pool of tyrosine by the total 14 c-tyrosine incorporated into the muscle protein. 127 Protein synthesis was expressed as pmoles tyrosine incorporated in 1 mg of muscle per hr. 128 RESULTS General Isolated frog muscles undergo a readily observable contracture when exposed to the drugs; compound 48/80 and verapamil. Typical concentrations that would cause a readily observable contracture in the muscle were: 500 µg/ml-2 mg/ml for compound 48/80 and 1.3 mg/ml of verapamil. The contractures were characterized by a significant change of length in the muscle the sartorius) and the development of of plasticity. (~ 40% for rigor or loss The general description of the contracture caused by compound 48/80 was very similar to the caffeineinduced contracture/rigor described by Schwartz et al (1978). The contractures were often scored on a relative basis. A slight change in the morphology of a muscle relative to a untreated control muscle was given a value of one plus (+). A large or maximum contracture was given a value of four pluses (++++). If no difference was observed between the untreated control muscle and the treated muscle, the treated muscle was scored as "no contracture". The scoring system though semi- quantitative, is reproducible. For example, no contrac- tures were ever observed on the muscles exhaustively pretreated with neuraminidase. Control muscles for the neuraminidase experiment treated with compound 48/80 129 (between 1 mg/ml and 2 mg/ml) always elicited a strong contracture response. Our focus was on the ionic events associated with contracture and with protein turnover during the contracture. The experiments were usually designed to get the maximum contracture response such that differences in ionic flux or protein turnover between treated muscles and control muscles would be significant for a small number of samples. Muscle contracture caused by compound 48/80 was tested for its dependence on a nerve-muscle interaction De- nervated frogsartorii in which the nerve had been allowed to atrophy were observed to undergo the same contracture in the presence of compound 48/80 as a sartorius muscle not surgically altered. The denervatedsartorii showed the same characteristic compound 48/80-dependent increase in 45 ca-uptake (2-3 fold) relative to control as the nonsurgically altered sartorius muscle. A preliminary experiment indicated that treatment of the sartorius muscle with EDTA (2 mM for 20 minutes) could diminish, but did not abolish, the contracture caused by 48/80. Frog skeletal muscles bathed in PAB with no Ca++ and challenged with compound 48/80 showed no observable difference in contracture or rate of protein degradation relative to muscles incubated in the normal 1.8 mM Ca++ PAB. 130 Neuraminidase and Skeletal Muscle Neuraminidase treatment of frog skeletal muscles prevented the observable contracture caused by compound 48/80. Similarly, the release of 3 H-serotonin from mast cells was inhibited by pretreatment of the cells with neuraminidase (see Chapter III, Figure 12). 131 Efflux of 86 Rb+ From Skeletal Muscle The results of Figures 18 and 19 show the effects of verapamil and compound 48/80 on the efflux of 86 Rb+ from frog skeletal muscles. Figure 18 illustrates the dose-dependent increase in 86 + the initial rate of efflux of Rb from frog skeletal muscle caused by compound 48/80. This result was simi- lar to that seen in Chapter II for P815 cells challenged with compound 48/80. dependence in the 86 (Figure 5) Figure 19 shows the + Rb -efflux in the frog sartorius muscle as a function of concentration of 48/80 and as a function of the time during which the muscle was exposed to the agent. rate of 86 At all doses of compound 48/80 the Rb+-efflux was fairly constant during the 62 minutes in which samples were taken. Figure 20 shows the effect of verapamil on 86 Rb+- efflux in frog skeletal muscle challenged with verapamil. The verapamil at low concentrations (< 400 µg/ml) caused 86 + an inhibition of the spontaneous release of Rb from the muscle while at higher concentrations the drug causes an increase in the 86 + Rb -efflux from the muscle. This result is very similar to that in Chapter II obtained 86 for 86 Rb+-efflux from rat mast cells loaded with Rb+3H-serotonin and challenged by verapamil. 132 Figure Initial rates of 86 18 Rb+-efflux from frog skeletal muscle challenged with compound 48/80. The 6 muscles were preincubated in 10 µCi/ml of ~ RbCl in PAB at 25°C for 1.5 hours. The muscles were prepared for pharmacological challenge by sequential incubation in two 5 ml volumes of PAB. Prior to each 10 minute incubation the muscles were dipped in a large volume of PAB. After prepara- tion, the muscles were challenged with compound 48/80 for 10 minutes. Aliquots of the extramuscular buff er were sampled and counted by the usual methods of scintillation counting. in cpm/mg per min. The rates are expressed The muscles used were the semimembranosus C-• - •- • - ) and the sartorius (-•-•-•-•-•-). The estimated error was + 5%. 133 0 N 00 ~ r--l ,,.-..... r--l E ........... ~ ~ E "-" r--l 0 00 N r--l ........... ~ ~ ~ 0 r--l 0 c 0 ·r-i ~ ~ ~ ~ 00 0 c © 0 c 0 u ~ 0 ~ 0 N 0 0 0 r--l 00 0 134 Figure Efflux of 86 19 Rb+ from a frog sartorius muscle challenged with compound 48/80 for different periods of time. The details of this experiment are described in Figure 18. The curves represent a direct count of an aliquot of the buffer containing the sartorius divided by the muscle mass. The aliquots were sampled at 10 minutes (lowermost curve), 37 minutes (middle curve) and 62 minutes (uppermost curve). for this experiment was The estimated error 50 cpm/mg. 135 0 0 0 C\I 0 0 00 r-1 !"""-. r-1 E 0 0 m r-1 ............. b.O ;1 '-" 0 00 ............. 0 0 ~ r-1 'O ~ 00 c ::I 0 0 0 C\I r-1 ~ E 0 u c 0 •r-1 0 0 0 -!..l r-1 -!..l ~ ~ c Q) t) c 0 0 00 0 0 m 0 0 ~ 0 0 C\I 0 0 0 m 0 0 u 136 Figure 20 Initial rates of 86 Rb+-efflux from frog skeletal muscle challenged with verapamil. Details of the experimental procedure are described in Figure 18. Semitendinosus muscle (---0---0---0---) and muscles of the shin complex (-•-•-•-) were challenged by the drug for 10 minutes at 25°C. 86 Rb+-efflux was expressed in units of cpm/mg per min. The estimated error was 5%. 137 0 N m r-f ,,....... r-f ~ r-f N r-f s M s '-" .......... r-f ·r-i sro ~ ro H © > 0 \ r-f ~ 0 ~ \ 0 \ ·r-i \ ~ \ 00 \ 0 \ \ ro H ~ ~ © 0 \ ~ \ © \ 0 N 0 0 0 0 0 © (!') N 0 0 0 u 138 Protein Degradation in Skeletal Muscle Compound 48/80 (2 mg/ml) caused a large increase in the rate of protein degradation in frog skeletal muscles relative to controls (Figure 21). Protein degradation was estimated to be a function of release of free tyrosine into the medium and into the internal pools of the muscle. This amino acid was chosen because it is not metabolized and does not itself alter the rates of protein synthesis or degradation (Kameyama and Etlinger, 1979, Fulks et al, 1975). significant This effect was relatively fast because differences in the tyrosine levels released into the extracellular pool could be detected in the first 15 minutes after challenge by the drug. The approxi- mately 2-fold increase in net protein degradation was correlated with the characteristic contracture of the muscle caused by compound 48/80. 139 Figure 21 shows the effect of compound 48/80 on protein degradation in frog skeletal muscle. The re- sults are pooled for four quartets of muscles; the sartorius, the semitendinosus, the gracilis major and the so-called shin complex. The statistical analysis using the t-test for paired observations (Sokal and Rohlf, 1969) is summarized in the following Table: control vs 48/80 (2 mg/ml) significant (P < 0.05) control vs neuraminidase not significant neuraminidase vs neuraminidase then 48/80 not significant The results of a preliminary experiment on protein degradation were very similar to Figure 12 and are summarized in the following Table: Relative Degradation Rate Treatment control (normalized) 100% compound 48/80 (2 mg/ml) 296% neuraminidase 139% neuraminidase then compound 48/80 108% In all experiments involving protein degradation only the muscles challenged with 6ompound 48/80 and not pretreated with neuraminidase showed a contracture. Figure22 shows that compound 48/80 caused a dose- dependent increase in protein degradation in LS myoblast cells. 140 Figure 21 Protein degradation in frog skeletal muscle : effect of neuraminidase and compound 48/80. This histogram summarizes the pooled results derived from four quartets of muscles in one controlled experiment. The muscles used were the sartorius, semitendinosus, gracilis major and the shin complex. The control values were normalized within the quartets and a ratio determined for values of the muscles treated with neuraminidase and/or 48/80 (2 mg/ml). c~mpound The error bars on the treated muscles represent the standard deviation of these ratios of treatments to controls. The standard deviation of the control values before normalization was 78 + 12 pmoles Tyr/mg per hr. % Tyrosine Release Relative to Control 0 J.-J J.-J 0 0 0 I I I I 0 Normalized Controls CJl Pretreatment Neuraminidase + 48/80 0 tv w CJl 0 0 0 I I I . 48/80 Pretreatment Neuraminidase tv CJl 0 .. I . ~ ~ ~ .. Ir .. .. 142 Figure 22 Effect of compound 48/80: L8 myoblast cells. protein degradation in The L8 myoblast cells were challenged with compound 48/80 for one hour at 37°C. Total protein degradation was measured as the sum of the tyrosine released into the extracellular buffer and the free tyrosine in the intracellular pools. The units were nmoles Tyr/10 6 cells. The details of the method for measuring protein degradation in tissue culture cells are described in Materials and Methods, Chapter IV). 143 3.0 2.5 ,....... U) r--i r-i 0,) u © 0 r-i ...._ U) 0,) 2.0 r-i 0 ed ...._ Q) U) ~ 0,) r--i 1.5 0,) ~ Q) d ·r-! U) 0 M ~ ~ 1.0 0.5 0 0.5 1.0 1.5 2.0 2.5 Concentration compound 48/80 (mg/ml) 3. 144 Uptake of 45 ca in Skeletal Muscles Compound 48/80, at concentrations sufficient to cause an observable contracture, also caused a significant increase in 45 Ca-uptake into frog skeletal muscle. In four distinct experiments involving seven muscle pairs, compound 48/80 (2 mg/ml) caused a (2.2 ! 0.5)-fold increase 45 over controls in initial ca-uptake. Neuraminidase pretreatment of the muscles did not abolish this effect. For example, three muscle pairs, pretreated with neuraminidase then challenged with compound 48/80 (2 mg/ml) showed a (2.2 ! 0.6)-fold increase over muscles only treated with neuraminidase in initial 45 ca-uptake. Figure 23 shows that neuraminidase-treated muscles have a higher rate of muscles. 45 Ca-uptake relative to non-treated The results from another•pair of muscles were pooled with the two pairs of muscles in Figure 21 for a test of statistical significance. Using a t-test for paired comparisons (Sokal and Rohlf, 1969) the difference between 45 ca-uptake in muscles treated with neuraminidase and control muscles was found to be statistically significant (P < 0.05). The measured 1145 ca-uptake" represents the amount of label that has been taken up during the exposure of the muscle to the 45 ca and then retained by the muscle until completion of a workup consisting of four washes over a 145 period of approximately 1.5 hours. The purpose of these washings is to exchange membrane-bound calcium. Since some intracellular 45 45 ca with cold ca could exchange with calcium in the extracellular medium during the workups, the measured 45 ca retained in the muscle - need not represent total uptake of 45 ca during the pharmacological challenge but instead represents the difference between the 45 ca gained during exposure of the muscle to 45 ca during the incubation with drug and the subsequent loss of the 45 ca during the workup of the muscle. Indeed, calcium exchange does occur in frog sartorius muscle when exposed to frog Ringer's solution. For example, Cosmos and Harris (1961) found that in muscle incubation in frog Ringer's there was a steady net inflow of Ca++; yet there was some exchange of label indicating an efflux of 45 ca out of the muscle. Assuming that a gram of muscle equals a milliliter of myoplasm, the increase in 45 ca-uptake caused by compound 48/80 translates to a rate of increase in myoplasmic [Ca++] of rv 3 µM per minute if the "influx" of calcium calculated from 45 ca-uptake was totally directed into the myoplasm with no uptake by other intracellular calcium pools. Figure 23 shows that verapamil inhibits the uptake of 45 ca caused by compound 48/80. pamil, alone, Interestingly, vera- causes a contracture and seems moreover to enhance the contracture caused by compound 48/80. 146 Figure 23 Calcium uptake in frog skeletal muscle: of neuramindase _and compound 48/80. effect Figure shows the results of a representative experiment derived from two quartets of frog skeletal muscle. The muscles were incubated in PAB at 37oc with 45 ca at a specific activity of 0.724 pmole/cpm. The challenge by 48/80 was for 12 minutes. The neuramindase pretreatment was ~ 85 minutes at 0.25 units/ml. The key observation was that only the muscles challenged by compound 48/80 (2 mg/ml) showed the characteristic contracture. The control muscles, muscles pretreated with neuramindase, and muscles pretreated with neuramindase and subsequently challenged by compound 48/80 were uniformly flaccid. Calcium Uptake (pmoles/mg per minute) 0 Control f-1 t.J VJ ii::.. CJ'! (j) 0 0 0 . 0 . 0 0 . .. I ' I ' . ...:] 00 0 0 • I I I I I-' .i::-- . " I- ~ Compound 48/80 Neuraminidase Neuraminidase then Compound 48/80 . I .. .. . ' 148 Figure 24 Effect of verapamil and compound 48/80 on calcium uptake in frog skeletal muscle. represents The histogram an experiment involving a quartet of frog semimembranosus muscles. The challenge by the various drugs was for five minutes with the verapamil and compound 48/80 being added simultaneously to the one muscle. The control muscles did not show any observable contracture. Muscles treated only with verapamil or compound 48/80 showed an observable contracture. The muscle treated with both drugs simultaneously was observed to have a "stronger" contracture than muscle treated with only compound 48/80 or only verapamil. The estimated error was + 3 pmoles/mg. Calcium Uptake (pmoles/mg) 0 Control tv t--1 01 0 01 0 01 0 I I I I I I I tv w . 01 f-1 ..i::-. \0 Verapamil (2 mM) 48/80 (2 mg/ml) Verapamil (2mM)+ 48/80 (2 mg/ml) w t--1 I 150 Protein Synthesis in Skeletal Muscle Figure 25 shows the effect of compound 48/80 on the rate of protein synthesis in frog skeletal muscle. In these experiments only muscles challenged with compound 48/80 and not pretreated with neuraminidase showed an observable contracture. A statistical analysis of the results of two experiments examining protein synthesis in frog skeletal muscle indicated no significant difference in the rate of protein synthesis between any two of the experimental treatments. The statistical test used was a t-test comparison for paired values (Sokal and Rohlf, 1973). 151 Figure 25 Protein synthesis in frog skeletal muscles: effect of neuraminidaseand compound 48/80. This histogram summarizes the results from five quartets of muscles in two separate experiments. The control muscles were normalized to the 100% level of tyrosine incorporation. Examples of the incorporation of tyrosine into control muscles are: semitendinosus 7.0 pmole Tyr/mg per hr sartorius gracilis major 7.8 pmole Tyr/mg per hr 18.5 pmole Tyr/mg per hr semimembranosus 15.9 pmole Tyr/mg per fir The incorporation of Tyr into muscles pretreated with neuroaminidase and/or challenged with 2 mg/ml compound 48/80 was divided by the control values. A standard deviation of these ratios for each group was calculated and is represented by the error bars on the histogram. % Tyrosine Incorporation Relative to Control 0 ~ ~ m ._.., oo f-1 ~ o 0 0 0 0 0 0 I ··~~~~~~~~~~~.-~~~~--.-~~~~--:ii------------------ Normalized muscles Compound 48/80 .• ' t--i l..11 N Pretreatment Neuraminidase Pretreatment .. Neuramindase then Compound 48/80L-~~~~~~~~~~~~~~~~~~~--------1 I ' 153 DISCUSSION Model for the Contracture of Frog Skeletal Muscle Caused by Compound 48/80 In order to simplify the following, the discussion begins with the presentation of a hypothetical model for the mechanism by which pharmacological agents such as compound 48/80 interact with muscles and thereby cause effects such as contracture. Subsequently, the results obtained in these studies, and of others, will be discussed in terms of this postulate. The model features two, unconnected processes which can be initiated by agents such as compound 48/80; (i) release of Ca ++ from the sarcoplasmic reticulum into the myoplasm which causes the muscle to contract. (ii) an independent increase in the rate of Ca++ influx across the cell membrane. (This influx need not result in a net increase in [Ca++] in the myoplasm as the incoming calcium may be taken up by the sarcoplasmic reticulum and/or pumped back out of the cell as rapidly as it enters.) The mechanistic pathway of the contractures caused by compound 48/80 is initiated by the binding of the drug to a surface molecule situated on the membrane of the t-tubule; this surf ace molecule includes sialic acid 154 residues which must be intact for interaction with compound 48/80. The interaction of compound 48/80 with a surface molecule in the t-tubules transmits a "signal" to a region of the membrane of the sarcoplasmic reticulum which is essentially in contact with the membrane of the t-tubule; this causes a sharp increase in the calcium permeability across the membrane of the sarcoplasmic reticulum such that large amounts of calcium flow out of this organelle into the surrounding myoplasm. (The nature of the "signal" is not known but could be an important component of the coupling of excitation to contraction in skeletal muscle. Chapter I). This increase in myoplasmic [Ca++] in turn activates the contractile apparatus causing the muscle to contract. Moreover, the increase in myoplasmic [Ca++] activates calcium-dependent protein degradation in the muscle and also causes an increase in K+ permeability of the outer membranes of the muscle. In a separate process, compound 48/80 causes an influx of extracellular Ca++ across those regions of the muscle membrane not in direct contact with the terminal cisternae of the SR. This entering Ca++ can be rapidly taken up by the intracellular calcium pools, mainly the sarcoplasmic reticulum. As noted earlier, the sarcoplasmic reticulum has the ability to remove very rapidly calcium from the myoplasm by virtue of the high rate and capacity of its ATP-dependent Ca++ pumps; indeed, such removal of 155 calcium from the myoplasm by the sarcoplasmic reticulum leads in normal circumstances to the rapid relaxation of a contracted muscle. If compound 48/80 does not interact with the t-tubular "trigger" structures but can still cause the influx of ++ ++ extracellular Ca across the muscle membrane, Ca pumping capacity of the SR may well be sufficient to pre. [Ca++] f rom reaching the threshold vent the myoplasm1c required for contractures. Thus one may observe an uptake of Ca++ without concomitant contraction. However, when compound 48/80 also activates the "trigger" structures causing release of Ca++ from the SR and also allows the concentration of myoplasmic [Ca++] to reach the threshold required for contraction. (The amount of calcium released in this way from the sarcoplasmic reticulum greatly ++ exceeds the capacity of any ATP-dependent Ca pumps to remove Ca ++ from the myoplasm.) Figure 26 is a schematic diagram of skeletal muscle illustrating the structures of skeletal muscle relevant to the proposed model. 156 Figure 26 - Schematic of Skeletal Muscle sarcolemma Function propagation of initial action potential t-tubule excitation-contraction coupling terminal cisternae release of Ca ++ Structure longitudinal reticulum reuptake of Ca "trigger" structure stimulus-contracture coupling ? excitation-contraction coupling ? sarcolemma t-tubule / l "trigger" structures ++ terminal cisternae longitudinal reticulum myoplasm contractile apparatus T actin filament T myosin filament 157 Evidence Relevant to Location of "Trigger" Structures in t-Tubules The contracture of frog skeletal muscle caused by compound 48/80 is inhibited by pretreatment of the muscle with neuraminidase (Figures 2land 25). Neuraminidase does not penetrate the plasma membrane (Gottschalk, 1972), thus the target molecule for compound 48/80 that "triggers" the contracture should reside on the surface of . the muscle and its activity should depend on sialic acid residues susceptible to removal by neuraminidase. In this regard, Dorrscheit-Kafer (1977) found that pretreatment of frog sartorii with neuraminidase caused the threshold membrane potential for contraction to be raised and found that increases in extracellular Ca++, an ion that preferentially binds to sialic acids over other physiological ions (Jaques et al, raised the contraction threshold. 1977),also This finding indicated that a cell surface structure which required sialic acids for maximal activity was involved in excitationcontraction coupling in skeletal muscles. This molecule and the neuraminidase-sensitive "trigger" molecule of our model may well be identical. 158 The model proposes that the interaction of compound 48/80 occurs with the "trigger" structure located on the surface of the t-tubules. In this regard, the study of glycerol-treated muscles demonstrated that the t-tubules were essential for excitation-contraction coupling. Micrographs indicate that glycerol treatment causes a reorganization of the t-tubules in such a way that they no longer appear as the intermediate element of the triad of the SR, t-tubule and the sarcolernma and seem physically pinched off from the sarcolernma (Howell and 1967). Jenlon Muscles treated in this way with glycerol quickly loose their twitch response and their ability to contract in 35 mM K+ Ringers but still respond to caffeine, a direct activator of the SR. These observations indicate that the coupling between the muscle potential action and the SR has been disrupted by the glycerol treatment, Morphologically, the t-tubules lie is close proximity to the terminal cisternae of the SR and approximately 80% of the surf ace area of the t-tubules about the terminal cisternae (Peachey, 1965). Compound 48/80 likely causes the activation of Ca++ release from the sarcoplasmic reticulum by a process which depends on receptors which contain sialic acid residues and which are localized in the t-tubules. 159 Similarly, normal excitation-contraction depends on a coupling process localized in the t-tubules and dependent on sialic acid susceptible to neuraminidase (DorrsheidtKafer, 1977). These similarities lead to the suggestion in the model that the cell surface molecules involved in these two processes are the same. Incidentally, both compound 48/80 (MW 800) and V. Cholerae neuraminidase (MW 90,000) should be able to enter the lumen of the t-tubules as even larger molecules such as ferritin (MW 460,000) and peroxidase (MW 200,000) can easily enter these regions (Constantin, 1975). 160 Evidence for an Increase in Myoplasmic [Ca++] A central feature of the model for contracture caused by compound 48/80 is that in normal muscle the drug triggers a process that leads to an increase in myoplasmic [Ca++]. Likewise, the normal excitation-contraction cycle of skeletal muscle features a transient increase in myoplasmic [Ca++] from 0.1 µM to approximately 2 µM (Caputo, 197m with the source of this increase being the sarcoplasmic reticulum (Hasselbach, 1980). Therefore, we will examine the evidence that compound 48/80 does indeed cause an increase in myoplasmic [Ca++]. 1) 86 Rb+-Efflux Compound 48/80 causes a dose-dependent increae in the efflux of 86 Rb+ from frog skeletal muscle (Figure l~. In non-excilatory cells, K+-efflux (Rb+ is an analogue of K+) is correlated with an increase in the cytosolic concentration of Ca++. This increase in the cytosolic [Ca++] is believed to increase the permeability of K+ across the plasma membrane which causes the K+ to flow out of the cell down it concentration gradient (Putney, 1979). If one extends this hypothesis to skeletal muscle, the increase in the 86 Rb+-efflux caused by compound 48/80 suggests an increase in the myoplasmic [Ca++]. 161 2) Protein Degradation Further evidence that contracture of frog skeletal muscle caused by compound 48/80 features an increase in myoplasmic [Ca++] was derived from studies of degradation in the muscles. protein We will briefly discuss the results and than will review the findings of others that show the rate of intracellular proton degradation reflects cytosolic [Ca++]. Figure 21 shows a significant increase in the rate of protein degradation in muscles challenged with compound 48/80 relative to control muscles. This increase in the rate of protein degradation correlates with the observable contracture of frog skeletal muscle caused by compound 48/80. In contrast, there was no significant increase in the rate of protein degradation in muscles pretreated with neuraminidase and subsequently challenged with compound 48/80 relative to control muscles pretreated with neuraminidase but then not challenged with compound 48/80 (Figure 21). Pretreatment of the muscle with neuraminidase also abolishes the contracture caused by compound 48/80 further implying that increase in the rate of protein degradation may correlate with contracture of the muscle. Further evidence along these lines is provided by our finding that a significant dose-dependent increase in the rate of protein degradation 162 was seen in L8 myoblast cells treated with compound 48/80 (Figure 22). We suggest that during contracture the relative increase in the rate of protein degradation was due to . . t h e concentration . . [C a ++] . an increase in of myoplasmic This correlation has been previously proposed by others. Kameyama and Etlinger (1979) showed that the rate of protein degradation in rat soleus muscles in the presence of ++ extracellular Ca was increased when the muscles were exposed to the calcium ionophore, A23187 relative to controls in the same medium but not treated with the ionophore. Cochrane and Douglas (1973) demonstrated that mast cells in the presence of extracellular Ca++, and treated with A23187 underwent degranulation, a process likewise thought to reflect on an increase in cytosolic ++ [Ca ] (Chapter II, Theoharides and Douglas, 1978). Therefore, if A23187 treatment of mast cells and rat soleus muscle effects intracellular [Ca++] in the same manner, then rat soleus muscles should experience an increase in myoplasmic [Ca++] on treatment with A23187 and such an increase in [Ca++] would lead to an increase in protein degradation. 163 There is circumstantial biochemical evidence showing that protein degradation in skeletal muscle depends on the intracellular concentrations of calcium. For example, Reddy et al (1975) and Kar and Pearson (1978) have isolated neutral proteases that are activated by Ca++ and hydrolyze a variety of muscle proteins. More- over, Dayton et al (1976) have purified a Ca++_activated protease, possibly involved inmyofubrillarprotein turnover, from porcine skeletal muscle. 3) Summary In summary, the increase in protein degradation which correlates with contracture caused by compound 48/80 (Figure 21) and the increase in the efflux of 86 Rb+ (Figure 18) which depends on the dose of compound 48/80 suggest that an increase in the concentration ++ of myoplasmic [Ca ] may be the central feature of the mechanism of contracture in frog skeletal muscle caused by challenges with compound 48/80. 164 Possible Source of Ca++ for Contracture In our proposed model for contracture caused by compound 48/80, there are two independent processes that could effect the concentration of myoplasmic [Ca++]: an increase in the rate of Ca++ influx across the cell membrane and/or the net release of Ca ++ from the SR. Experimental evidence suggests that extracellular calcium is not sufficient to cause contracture of frog skeletal muscle challenged with compound 48/80. Accordingly, the sarcoplasmic reticulum seems the most likely source of calcium required for contraction. 1) Extracellular Calcium Evidence that extracellular calcium is not the source includes the following observation. Compound 48/80 could cause a contracture of isolated frog skeletal muscle incubated in physiological buffer containing no calcium. This contracture was similar to a normal contracture casued by the drug in the presence of the regular 1.8 mM Ca ++ physiological buffer. This observed independence of the contracture on extracellular calcium is similar to the lack of a requirement for extracellular calcium in the degranulation of mast cells caused by compound 48/80 (Uvnas and Thon, 1961),we have also discussed (Chapter III) 165 a) Measurement of 45 Ca-uptake - The uptake of extracellular calcium by muscles challenged with compound 48/80was increased relative to control muscles; however this increase was not correlated with a contracture of the muscle (Figure 23). Specifically challenge with compound 48/80 of muscles pretreated with neuraminidase causes the same relative increase in 45 ca uptake as does challenge with compound 48/80 of muscles not treated with the enzyme; these muscles however did not undergo a contacture. The model asserts that compound 48/80 causes an increase in the influx across the sarcolemma of extracellular calcium; this process leads to an increased uptake of 45 Ca without, necessarily an 1ncrease.1nmyop · . 1 asm1c . [Ca++], because the ATP-dependent pumps of the SR take up the 45 ca upon entry into the myoplasm and prevent its exchange with the extracellular medium. When the SR is not activated by compound 48/80 because of neuraminidase pretreatment of the muscle, the influx of extracellular calcium caused by compound 48/80 is not sufficient to activate the contractile apparatus because the SR takes up the entering Ca++ sufficiently rapidly to not increase the myoplasmic [Ca++] enough to cause the muscle to contract. 166 b) Effect of Verapamil - Verapamil caused a decrease 45 in the uptake of ca in frog skeletal muscles simultaneously challenged with compound 48/80 relative to muscles only treated with 48/80 but does not inhibit the contracture (Figure 24). In fact, the two drugs where added simultaneously to a muscle seen to cause a "stronger" contracture than either did alone. Verapamil blocks the influx of calcium across the plasma membrane in a variety of preparations of smooth and cardiac muscle (Fleckenstein, 1977). If this mechanism of action could be extended to skeletal muscle, then the results of Figure 24 are readily explained; the drug inhibited the increase in uptake of 45 ca caused by compound 48/80 by specifically blocking the influx of extracellular calcium in the muscle. of the Since this blockage by verapamil influx of extracellular calcium does not inhibit contracture caused by compound 48/80, we conclude that an increase in uptake of extracellular calcium does not correlate with contracture of the muscle. According to our model, this suggests that verapamil blocks calcium influx across the sarcolemma but does not interfere with the operation of the receptors in the t-tubules which on binding of compound 48/80 mediate the contracture. 167 Interestingly, verapamil, alone, of frog skeletal muscle. causes a contracture This is a surprising result for verapamil has been defined as a specific blocker of calcium channels of the plasma membrane of a number of cells and tissues (Fleckenstein, 1977) but the contraction of muscle depends on the mobilization of Ca++ from the SR into the myoplasmic pool (Hasselbach, 1980) not calcium influx across the sarcolemma. Indeed, verapamil may be acting as a blocker of calcium channels when the drug at low concentrations causes a decrease in the spontaneous efflux of 86 Rb+ from frog muscles (Figure 20). decrease in 86 This Rb+-efflux could be reflective of a decrease in myoplasmic [Ca++] (see Chapter II, Putney (1979)) . caused by verapamil blocking the spontaneous flux of Ca++ into the myoplasm from membrane bound pools and/or the extracellular medium. However, at higher concentrations of verapamil this effect due to the blockage of calcium channels is overcome and there is an increase in myoplasmic ·[ca++] which leads to observed increases in 86 Rb+-efflux and to the observed contracture. In this case, the mechanism of contracture of skeletal muscle caused by verapamil is unknown but may be related to the mechanism of degranulation of mast cells caused by the drug (Chapter III) in verapamil is proposed to act only after entry into the cell. 168 c) Summary - In summary, we have examined several lines of experimental evidence and made an analogy to mast cells to propose that in skeletal muscle the increase in calcium influx from the extracellular environment caused by compound 48/80 is neither a sufficient condition nor a necessary condition for contracture. Accordingly, the extracellular pool does not seem to be a likely ++ source of Ca that causes the increase in myoplasmic [ca ++] and su b sequent 1 y causes contracture. The mechanism of the increased uptake of 45 Ca caused by compound 48/80 probably depends on a membrane component which interacts with compound 48/80 to increase the rate of influx of calcium into the muscle. component of the sarolemma is not The function of this affected by treat- ment of the muscle with neuraminidase and the influx of 45 ca is taken up by an intracellular calcium pool, presumably, the sarcoplasmic reticulum. 2) Sarcoplasmic Reticulum Possible indirect evidence that the sarcoplasmic reticulum is the source of Ca++ required for the contracture of muscle caused by compound 48/80 is that contracture caused by high concentrations of caffeine is generally similar in appearance ie shortened muscle and loss of plasticity (Schwarz et at, 1978). Moreover, caffeine contracture is caused by the direct interaction of the ++ . drug with the SR causing the release of Ca into the myoplasm (Weber and Herz, 1968). 169 Conclusion and Further Tests of the Model The contracture of skeletal muscle challenged with compound 48/80 correlates with an increase in the rate of protein degradation and the rate of 86 Rb + -efflux in the muscle; these observations suggest an increase in myoplasmic [Ca++]. Compound 48/80 also caused an increase in the uptake of 45 Ca, however this increase does not correlate with contracture. Pretreatment with neuraminidase of the muscles abolished the contracture indicating that compound 48/80 interacts with a "trigger" structure that contains sialic acid residues essential for activity, such pretreatment does not however effect the increase in rate of influx of Ca++ in response to compound 48/80. The model proposes that the "trigger" structure is located on the t-tubular membrane proximate to the terminal cisternae of the sarcoplasmic reticulum. Thus a test of the model would involve treatment of the muscle with glycerol causing a separation of the t-tubules from the sarcolemma and subsequent challenge with compound 48/80. If the contracture does not occur under these conditions the site of the ''trigger'' structure would likely be in the t-tubules. As a positive control, caffeine should still be able to cause a contracture under these conditions otherwise the glycerol treatment would 170 also be affecting the function of the SR. glycerol-~reatedmuscles (Note, are still activated by caffeine, Howell and Jenlon, 1967). The increase in 86 Rb+-efflux caused by compound 48/80 implies that there was also an increase in myoplasmic ++ [Ca ]. The question is whether the increase in myoplasmic [Ca++] was directly the result of the interaction of compound 48/80 with the "trigger" structure proposed in the model. If pretreatment of the muscle with neuraminidase lowers the increase of 86 Rb+-efflux caused by 48/80 then one would conclude that the "trigger" structure sensitive to neuraminidase is indeed important . . [Ca++] . in controlling myoplasmic If, however, 86 Rb + - efflux caused by compound 48/80 is not affected by neuraminidase then the model is not valid unless other factors besides the release of calcium from the SR into the myoplasm can influence the K+( 86 Rb+) channels 86 Rb+-efflux. For example, could "sense" the increased influx of Ca++ across the muscle membrane seen in muscles pretreated with neuraminidase and challenged with compound 48/80. Furthermore, neuraminidase itself causes an increase in 45 Ca uptake in skeletal muscle (Figure 23 and similarly in cultured heart cells (Langer et al, 1976) Again, this increased influx of Ca++ across the muscle . . [Ca++] l membrane ( as d istinct from increased myop 1 asmic 171 caused by neuraminidase could be "sensed" by the K+ ( 86 Rb+) channels thus causing an increase in 86 Rb+- efflux such that the experiment would not reveal any change in myoplasmic [Ca++] caused by compound 48/80. The existence of a highly specialized organelle, such as the sarcoplasmic reticulum, for the generation and removal of transient increases in cytosolic [Ca++] has not been demonstrated in mast cells, nevertheless compound 48/80 causes the degranulation of mast cells presumably by mobilizing an intracellular pool of calcium (see Chapter II). An experiment examining the effect of neuraminidase treatment on uptake of 45 ca into mast cells challenged with compound 48/80 could show a result similar to skeletal muscle (Figure23); neuraminidase treatment of mast cells inhibits degranulation but does not change the increase in challenge with compound 48/80. 45 ca-uptake caused by Using our model for skeletal muscle, such a result would indicate that the activation of an intracellular calcium pool in the mast cell was dependent on the close proximity of the pool to neuraminidase-sensitive "trigger" structures of the plasma membrane and the increase in 45 ca-uptake was due to ATP-dependent calcium pumps on an intracellular pool rapidly taking up the increase in the influx of extracellular Ca++ and preventing the cytosolic [Ca++] from reaching the 172 threshold for degranulation. This finding would suggest that the functional internal structure .. of the mast cell . is complex and similar to skeletal muscle. However, influx of extracellular Ca++ caused by crosslinking of receptors to IgE is sufficient and necessary to cause degranulation of mast cells (Hirata and Axelrod, 1980) indicating that this, possibly large, influx of Ca++ ++ is not prevented from increasing cytosolic \ [Ca ]to the threshold for degranulation by bein~ caken up by an intracellular calcium pool analagous to the SR in skeletal muscle. 173 Parallels Between Pharmacological Challenge of Rat Mast Cells and Skeletal Muscle. A Summary Table IV lists the findings of this thesis on rat mast cells and frog skeletal muscle that are similar for both tissues. These similar findings indicate that certain steps of the mechanistic pathway of siimulus-secretion coupling in mast cells might be analogous to steps in the coupling of pharmacological stimulus to contracture or even to excitation-contraction coupling in skeletal muscle. Such steps include the binding of compound 48/80 to receptors of the cell surface which have sialic acid residues. This in turn causes the mobilization of calcium from intracellular pools into the cytosol consequently activating the function characteristic of the tissue; degranulation of mast cells and activation of the contractile apparatus in skeletal muscle. Interestingly, mast cells also contain a "contractile apparatus'' composed of actin microfilaments, that have been proposed upon activation to eject the granules from exocytotic pits at the cell surface (Cochrane and Douglas, 1974). 174 Table IV, Chapter IV - Summary of Similar Findings on Rat Mast Cells and Frog Skeletal Muscle of this Thesis Similar Findings 1. Implication compound 48/80 causes . . increase in cytoso l"ic [Ca++] an increase in 86 Rb+-efflux 2. no absolute requirement for extracellular Ca 3. not required for activation verapamil inhibits mobilization inhibit spontaneous of Ca 86 Rb+ ++ at those concentra- tions high doses of verapamil verapamil overcomes the caused increase re- inhibition of mobilization of lease of 5. ca ++ into . . t h e tissue low doses of verapamil release of 4. ++ . f lux o f in 86 Rb+ . an d acti- Ca++ and itself causes mobiliza- vat ion tion of Ca++ processes of activation the target of compound 48/80 are neuraminidase- contains essential sialic sensitive acid. 175 REFERENCES Axelsson, J. and Thesleff, S. (1958) Acta Physiol. Scand. 44. 55. Blau, H. and Epstein, C. (1979) Cell 17, 95. Caputo, C. (1978) Ann. Rev. Biophys. Bioeng. 2, 63. Cochrane, D.E. and Douglas, W.W. (1974) Proc. Natl. Acad. Sci. USA 71, 408. Constantin, L.L. (1975) Prog. Biophys. Mol. Biol. 29, 197. Cosmos, E. and Harris, E.J. 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