Pharmaceutical Sciences, Faculty of
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UBCV
Mahey, Rajesh
Doctor of Philosophy - PhD
University of British Columbia
The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells.
CALCIUM TRANSPORT AND ATP HYDROLYTIC ACTIVITIES IN GUINEA-PIG PANCREATIC ACINAR PLASMA MEMBRANES by RAJESH MAHEY B.Sc. (Hon.), Sunderland Polytechnic, England, 1983 M.Sc, University of British Columbia, Canada, 1987 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE FACULTY OF GRADUATE STUDIES THE FACULTY OF PHARMACEUTICAL SCIENCES Division of Pharmacology and Toxicology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1991 © Rajesh Mahey, 1991 ln presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology. Faculty of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada ' Date April 30, 1991 DE-6 (2/88) A B S T R A C T The aim of the present investigation was to determine whether a plasma membrane high affinity C a 2 + - A T P a s e plays an integral role i n the maintenance of cytoplasmic free C a 2 + in pancreatic acinar cells. To achieve this, the C a 2 + -transport and C a 2 + - A T P a s e activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain a n ATP-dependent high affinity C a 2 + - p u m p and a high affinity C a 2 + -dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity C a 2 + - A T P a s e activity as well as the Ca 2 + - transport were found to be dependent on M g 2 + , whereas the low affinity ATPase activity appeared to be inhibited by M g 2 + . The high affinity ATPase activity was 7-fold greater in magnitude than the Ca 2 + - transport . Whereas the Ca 2 + - transport was very specific for A T P as a substrate, the high affinity C a 2 + - A T P a s e showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca 2 + - transport and the high affinity Ca 2 + -dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca 2 + - transport and C a 2 + - A T P a s e activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca 2 + - transport . These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca 2 + -dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes C a 2 + - A T P a s e confirmed the presence of endogenous calmodulin. ii Abstract The catalytic subunit of cyclic AMP-dependent protein kinase stimulated C a 2 + transport, suggesting that cyclic A M P may have a role in the regulation of Ca 2 + -pump-mediated C a 2 + efflux from pancreatic acini. Ca 2 + -dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. H A 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the C a 2 + -transport and Ca 2 + -dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated C a 2 + transport, it may be concluded that H A 1004 is ineffective i n blocking the actions of c A M P -dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C , the phorbol ester T P A and the diacylglycerol derivative, S A - D G , failed to stimulate the Ca 2 + -uptake activity. However, these agents produced stimulation of the Ca 2 + -dependent ATPase activity i n the presence of phosphatidylserine. C G P 41 251, a potent and selective inhibitor of protein kinase C , did not inhibit the Ca 2 + - transport or Ca 2 + -dependent ATPase activities. These observations suggest that protein kinase C may not be involved i n the regulation of the plasma membrane C a 2 + -pump i n guinea-pig pancreatic acinar cells. These results also point to another difference between Ca 2 + - transport and the C a 2 + - A T P a s e activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca 2 + -uptake , suggesting that I P 3 - and/or IP 4 -mediated C a 2 + releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity C a 2 + - A T P a s e is not the biochemical expression of plasma membrane Ca 2 + - transport in panreatic acini. Our results imply a role for iii Abstract calmodulin and cAMP-dependent protein kinase, but not protein kinase C , in the regulation of C a 2 + efflux from pancreatic acinar cells. iv T A B L E O F C O N T E N T S Abstract ii Table of Contents v List of Tables viii List of Figures '. ix List of abbreviations xii Acknowledgements xv Dedication xvi Introduction 1 Morphology and Function of the Exocrine Pancreas 1 Morphology 1 The Enzyme Secretory Process .3 Composition of Pancreatic Acinar Cell Secretion 4 Regulation of Enzyme Secretion 6 Stimulus-secretion coupling 8 Role of Calcium 9 Role of Inositol Phosphates 12 Involvement of Protein Kinase C 15 Other Intracellular Messengers of Pancreatic Enzyme Secretion . .18 Interactions Among Intracellular Messengers 19 Intracellular C a 2 + Oscillations and Ca 2 + - induced C a 2 + release . . . 19 Effect of Calcium on Exocytosis 22 Calcium Homeostasis 23 The Plasma Membrane Calcium Pump 26 Regulation of the Plasma Membrane Calcium Pump 32 Calmodulin 32 Limited Proteolysis 34 Other Regulators 34 Objective 36 Materials and Methods 38 Materials 38 v Table of Contents a) . Radiochemicals: 38 b) . Reagents: 38 Methods 41 Preparation of Pancreatic Acini 41 Preparation of Plasma Membranes from Pancreatic Acini 42 Purification of C a 2 + - A T P a s e from Pancreatic Acinar Plasma Membranes 42 Measurement of Calcium Uptake Activity 44 Assay of C a 2 + - A T P a s e Activity 45 Assay of Na + /K+-ATPase Activity 47 Determination of Endogenous Calmodulin 47 Phosphorylation of Guinea-pig Acinar Plasma Membranes .48 Polyacrylamide Gel Electrophoresis and Autoradiography 49 Sodium Dodecyl Sulphate Polyacrylamide gels 49 Acid gels 49 Staining, Destaining and Drying 50 Autoradiography .51 Protein Assay 52 Determination of Free Calcium Concentration 52 Statistical Analysis 53 Results 54 The Plasma Membrane Preparation 54 Optimization of Assay Conditions 57 Characterization of C a 2 + Transport 62 Effects of C a 2 + and Calmodulin 62 Effect of M g 2 + 62 Substrate Specificity .65 Characterization of C a 2 + - A T P a s e Activity 65 Effect of M g 2 + 65 Effect of C a 2 + , Calmodulin and K + 70 Formation of the Phosphorylated Intermediate of the Calcium Pump 70 Substrate Specificity 74 Regulation of Ca 2 + - transport and C a 2 + - A T P a s e by Protein Kinases and Inositol Phosphates 76 Regulation by Protein Kinase A 76 vi Table of Contents Regulation by CaM 76 Determination of Endogenous CaM 80 Removal of Endogenous CaM 88 Regulation by Protein Kinase C 88 Regulation by Inositol Phosphates 90 Solubilization and Purification of Ca2+-ATPase 90 Experiments with Plasma Membranes from Human Pancreas 97 Discussion 102 Basic Characterization 103 Regulation of Ca2+-ATPase and Ca2+-transport 108 Is the High Affinity Ca2+-ATPase the Ca2+-Pump? 115 Purification of the Ca2+-Pump 116 Studies with the Human Pancreatic Acinar Plasma Membranes 117 Original Contributions to the Literature 117 Summary and Conclusions 118 Bibliography 120 vii LIST OF TABLES Table I. Regulators of pancreatic exocrine secretion and their intracellular messengers 7 Table II. A comparison of the specific activities (SA) at different stages of the guinea-pig pancreatic acinar plasma membrane preparation 56 Table III. Effect of protein kinase inhibitors on 4 5 C a 2 + uptake in plasma membrane vesicles .78 Table IV. Effect of inhibitors CGS 9343B and CGP 41 251 on Ca 2 +-dependent ATPase activity at different Ca 2 + concentrations 86 Table V. Effect of Protein kinase C, SA-DG and TPA on Ca2+-dependent ATPase activity of guinea-pig pancreatic acinar plasma membranes. . . .92 Table VI. Summary of the effects of different potential regulators on the Ca2+-uptake and Ca2+-ATPase activities of guinea-pig pancreatic acinar plasma membrane preparations 109 viii L I S T O F F I G U R E S Figure 1. Diagramatic representation of a pancreatic acinar cell 2 Figure 2. Scheme of the possible routes followed by secretory proteins in pancreatic acinar cells 5 Figure 3. Reaction cycle of the human red cell Ca2+-pump 28 Figure 4. Flow-chart for the preparation of guinea-pig pancreatic acinar plasma membranes 43 Figure 5. Recovery of protein, Ca2+-ATPase activity and Na+/K+-ATPase activity in different centrifugation fractions of guinea-pig pancreatic acinar plasma membrane preparations 55 Figure 6. Effect of varying protein concentration on 45Ca2+-uptake in guinea-pig pancreatic acinar plasma membranes 58 Figure 7. Effect of varying protein concentration on Ca2+-dependent ATPase activity in guinea-pig pancreatic acinar plasma membranes. . . .59 Figure 8. Time-course of Ca2+-ATPase activity 60 Figure 9. Effect of freeze-thawing on Ca2+-dependent, Mg2+-dependent and total ATPase activity 61 Figure 10. Ca 2 + activation of 4 5 C a 2 + uptake by guinea-pig pancreatic acinar plasma membrane vesicles 63 Figure 11 Effect of Mg 2 + on 45Ca2+-uptake in guinea-pig pancreatic acinar plasma membrane vesicles 64 Figure 12. Effect of CDTA on 4 5 C a 2 + uptake in guinea-pig pancreatic acinar plasma membrane vesicles 66 Figure 13. 45Ca2+-uptake in the presence of different nucleotide substrates in guinea-pig pancreatic acinar plasma membrane vesicles 67 Figure 14. Effect of Mg 2 + on Basal ATPase activity 68 Figure 15. Effect of calcium on Ca2+-dependent ATPase activity in the absence and presence of various magnesium concentrations 69 Figure 16. Effect of Ca 2 + on Ca2+-dependent ATPase activity in the absence or presence of 6 |ig/ml calmodulin 71 ix List of Figures Figure 17. Effect of calcium on Ca 2 + -dependent ATPase activity i n the absence and presence of various potassium concentrations 72 Figure 18. Autoradiogram of phosphorylated proteins i n guinea-pig pancreatic acinar plasma membranes. 73 Figure 19. Substrate specificity of Ca 2 + -dependent and Mg 2 + -dependent hydrolytic activities in guinea-pig pancreatic acinar plasma membranes 75 Figure 20. Effect of the catalytic subunit of protein kinase A on C a 2 + activation of 4 5 C a 2 + uptake 77 Figure 21. Effect of the catalytic subunit of protein kinase A and the inhibitor H A 1004 on Ca 2 + -dependent ATPase activity 79 Figure 22. Effect of trifluoperazine on 4 5 C a 2 + uptake in guinea-pig pancreatic acinar plasma membrane vesicles .81 Figure 23. Effect of compound 48/80 on 4 5 C a 2 + uptake i n guinea-pig pancreatic acinar plasma membrane vesicles .82 Figure 24. Effect of trifluoperazine on Ca 2 + -dependent ATPase activity at different C a 2 + free concentrations 83 Figure 25. Effect of compound 48/80 on Ca 2 + -dependent ATPase activity at different C a 2 + free concentrations 84 Figure 26. Effect of compound 48/80 on total ATPase activity at different C a 2 + free concentrations and i n the presence of 0.5 m M M g 2 + 85 Figure 27. Stimulation of the erythrocyte C a 2 + - A T P a s e indicator activity by exogenous C a M 87 Figure 28. Protein profile (absorbance at 280 mm) of fractions eluted from a Sephadex G-75 chromatography column after applying solubilized guinea-pig pancreatic acinar plasma membranes 89 Figure 29. Effect of Protein kinase C or 12-O-tetradecanoyl phorbol-13-acetate on 4 5 C a 2 + uptake by guinea-pig pancreatic acinar plasma membrane vesicles 91 Figure 30. Effect of l-stearoyl-2-arachidonoyl-s/i-glycerol on 4 5 C a 2 + uptake. 93 Figure 31. Effect of IP4 and I P 3 on 4 5 C a 2 + - u p t a k e i n guinea-pig pancreatic acinar plasma membranes 94 x List of Figures Figure 32. ATPase activity and absorbance at 280 nm of fractions collected from CaM-agarose column following loading and washing 96 Figure 33. ATPase activity and absorbance at 280 nm of fractions eluted from the CaM-agarose affinity column 98 Figure 34. Enrichment of Ca2+-dependent ATPase and Na+/K+-ATPase activities in human pancreatic acinar plasma membranes 99 Figure 35. Effect of CaM and TFP on Ca2+-dependent ATPase in human pancreatic acinar plasma membranes 101 xi LIST OF ABBREVIATIONS A C h acetylcholine A D P adenosine 5'-diphosphate A P ammonium persulphate A T P adenosine 5'-triphosphate ATPase adenosine 5'-triphosphatase B A P T A bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid ° C degrees celcius C-subunit catalytic subunit of cAMP-dependent protein kinase C a M calmodulin C a M - P K calmodulin-dependent protein kinase c A M P adenosine 3',5'-cyclic monophosphate C C h carbachol C C K cholecystokinin-pancreozymin C C K - 8 CCK-octapeptide C D T A trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetate c G M P guanosine 3',5'-cyclic monophosphate C i curie c IP 3 cyclic l:2,4,5-trisphosphate C T P cytosine 5'-triphosphate D G diacylglycerol ~Ei high affinity state of the enzyme E 2 low affinity state of the enzyme E D T A ethylenediaminetetraacetic acid E G T A ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid E R endoplasmic reticulum xii List of abbreviations G T P guanosine 5'-triphosphate H E P E S N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] I P 3 inositol 1,4,5-trisphosphate I P 4 inositol 1,3,4,5-tetrakisphosphate ITP inosine 5'-triphosphate K c a concentration of free C a2 + at half-maximal response k D a kilodaltons K R B Kreb's Ringer bicarbonate L i D S l i thium dodecylsulphate M molar micro m A milliamperes min minutes Mops 3-(N-morpholino)propanesulfonic acid M r relative molecular mass N A D P H p-nicotinamide adenine dinucleotide phosphate (reduced) P A phosphatidic acid P C phosphatidylcholine PI phosphatidylinositol P i inorganic phosphate P I P 2 phosphatidylinositol 4,5-bisphosphate P K A cAMP-dependent protein kinase (protein kinase A) P K C protein kinase C P M S F phenylmethylsulfonyl fluoride p N P P p-nitrophenyl phosphate PS pho sphatidyl serine R E R rough endoplasmic reticulum xiii List of abbreviations r p m revolutions per minute S . E . M . standard error of mean S A - D G l-stearoyl-2-arachidonoyl-sn-glycerol S D S sodium dodecylsulphate S D S - P A G E sodium dodecylsulphate polyacrylamide gel electrophoresis T C A trichloroacetic acid T E M E D tetraethyl-methylenediamine T F P trifluoperazine T P A 12-O-tetradecanoyl phorbol-13-acetate Tris tris(hydroxy)aminomethane v/v volume per unit volume V I P vasoactive intestinal polypeptide v v max maximal velocity w/v weight per unit volume w/w weight per unit weight xg unit gravitational force xiv ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my co-supervisors, Professor Sidney Katz and Dr . Michael Bridges for their advice, encouragement and guidance throughout the study. The constructive criticism and suggestions by the members of my research committee, Dr . Jack Diamond, Dr . Ray Pederson and Dr . Derek Applegarth are gratefully acknowledged. I wish to thank the chair of my research committee, Dr . James Axelson for being very helpful during the study. I would like to thank the members of Dr . Katz's laboratory for being very helpful and cooperative throughout my study. I wish to thank my friends and colleagues i n the Faculty of Pharmaceutical Sciences, especially Sue Panesar and Bruce Allen, for making my stay an enjoyable experience. I am very thankful to Dr . Paul Keown and Dr . M a r k Meloche for providing the human pancreatic tissue obtained through the Pacific Organ Retrieval for Transplantation program. Finally, I would like to gratefully acknowledge the continuous financial support by the Canadian Cystic Fibrosis Foundation throughout my studies. xv D E D I C A T I O N To m y pa ren ts for t he i r con t inuous encouragement xvi INTRODUCTION MORPHOLOGY AND FUNCTION OF THE EXOCRINE PANCREAS Morphology The pancreas is a solid glandular organ, structurally and functionally associated with the upper part of gastrointestinal tract (Harris et al., 1979). It has both endocrine and exocrine functions. The exocrine pancreas comprises the bulk of the organ. For example, 82% of pancreatic volume in guinea-pigs is exocrine tissue (Bolender, 1974). Major structural components of the exocrine pancreas consist of acinar cells and the ductal cells. The acinar cells are organized into acini, which are small groups of cells arranged around a common luminal space. The acini are responsible for the synthesis and secretion of digestive enzymes. Ductal cells, on the other hand, are responsible for fluid and electrolyte secretions (Grossman and Ivy, 1946; Harper and Scratcherd, 1979). The acinar cells are polarized cells consisting of basal, lateral and apical membrane surfaces (Fig. 1). Only the basal membrane is exposed to the circulation and contains receptors for secretagogues, while the enzymes are released at the apical membrane. The total cell volume is composed of 54% cytoplasmic matrix, 22% rough endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules and 0.7% condensing vacuoles. The total membrane surface area consists of 60% RER, 21% mitochondria, 9.9% Golgi complex, 4.8% plasma membrane, 2.6% zymogen granules, 1.8% plasma membrane vesicles and 0.4% condensing vacuoles (Bolender, 1974). RER occupies the intranuclear and paranuclear regions of the cell, whereas the apical pole of the cell contains the Golgi complex and zymogen granules (Ekholm and 1 Introduction Figure 1. Diagramatic representation of a pancreatic acinar cell. In general, features of the resting cell are represented. However, the sequence of events (a-d) illustrated in the apical zone of the cell represent features of the stimulated cell, a = caveolus, b = coated vesicle, c = endocytotic vesicle, d = multivesicular body, (from Case, 1978). 2 Introduction Edlund, 1959; Ekho lm et al., 1962a; Kern , 1986). Zymogen granules are the secretory units and are concentrated beneath the luminal plasma membrane to be released during stimulation. In contrast to the acinar cell, the ductal cell cytoplasm contain relatively few mitochondria and small amounts of Golgi complex and R E R (Dixon, 1979; Ekho lm et al., 1962b). The ductal cells collectively form the duct system which is subdivided into three branches, the intralobular, interlobular and main pancreatic ducts (Bencosme and Lechago, 1969; Kern , 1986). These ducts perform the collecting and draining function of the pancreatic secretions. The intralobular ducts are located within the acinar lobules and drain the pancreatic juice secreted by the acini. F lu id from these ducts is drained into the interlobular ducts. These i n turn drain into the main pancreatic duct which empties into the duodenum. The Enzyme Secretory Process Acinar cell secretion consists of a series of events including the synthesis of secretory product, its transport though intracellular compartments and its eventual release into the extracellular space. Most of our knowledge of the secretory pathway comes from the original studies by Jamieson and Palade (Jamieson and Palade, 1967, 1971a, 1971b, 1977, Palade, 1975). Their results can be summarized as follows: Secretory proteins are synthesized on R E R and translocated into its cisternae. F r o m here, the proteins are transported to the Golgi apparatus i n small vesicles which are assumed to function as shuttling vesicles between the transitional elements of the R E R and Golgi elements. After various modifications of the protein mixture within the Golgi compartments, the products are packaged within membranes derived from Golgi saccules. The proteins i n the resulting condensing vacuoles are then progressively 3 Introduction concentrated to form the mature zymogen granules. These granules are stored just inside the apical membrane. An appropriate stimulation then leads to the fusion of zymogen granules with the apical plasma membrane and extrusion of their contents into the acinar lumen by a complex process of exocytosis (Fig. 2). This process is not well understood, but it appears to involve a specific and as yet unknown "recognition step" between the granule membrane and the apical plasma membrane, followed by the transient fusion of these membranes. After mediating the discharge process, the membrane material is recycled (Herzog and Reggio, 1980). Composition of Pancreatic Acinar Cell Secretion The human pancreas synthesizes and secretes more, as well as a wider range of proteins per gram of tissue than any other organ (Rinderknecht, 1986). However, because human tissue is, for the most part unavailable for study, much of our knowledge of pancreatic secretory proteins has been gained from studies in experimental animals. Most of the early methods for separation of pancreatic secretory proteins involved column chromatography. However, in 1975, Scheele used two-dimensional electrophoresis to separate the mixture of proteins from guinea-pig exocrine pancreatic secretion (Scheele, 1975). This method yields higher resolution from smaller quantities of protein than the chromatographic separation methods. The secreted enzymes include serine proteases (such as trypsin, chymotrypsin and elastase), exopeptidases (including carboxypeptidases A and B), phospholipase A 2 , lipase, colipase, nonspecific carboxylesterase, amylase, pancreatic ribonuclease and pancreatic deoxyribonuclease (Rinderknecht, 1986). A number of these enzymes are 4 Introduction SECRETORY PATHWAYS IN PANCREATIC ACINAR CELL CONSTITUTIVE PARAGRANULAR REGULATED Figure 2. Scheme of the possible routes followed by secretory proteins i n pancreatic acinar cells. The different organelles involved are shown. RER-rough endoplasmic reticulum, T V -transition vesicles, CV-condensing vacuoles, IG-immature granules, MG-mature granules, ZG-zymogen granules, C . (as in CIS-C.)-cisternae. (from Beaudoin and Grondin, 1991). 5 Introduction secreted as inactive zymogens that are activated by trypsin. Trypsin itself is converted to the active form by enterokinase secreted by the duodenal mucosa. Regulation of Enzyme Secretion Secretion of digestive enzymes from the exocrine pancreas is controlled by a number of peptide hormones and neurotransmitters present i n the pancreatic ganglia (see Table I). However, the actual mechanism(s) involved i n stimulus-secretion coupling are only now beginning to be understood. Cholecystokinin-pancreozymin ( C C K ) and the neurotransmitter acetylcholine (ACh) are known to participate i n the physiological mechanisms for enzyme secretion. Evidence for the involvement of these secretagogues in enzyme secretion has come from both in vivo and in vitro studies. The muscarinic cholinergic agents A C h , carbachol (CCh) and bethanechol can stimulate amylase secretion from pancreas (Argent et al., 1973; Case and Clausen, 1973; Will iams and Chandler, 1975; Williams et al., 1976). Using radiolabelled antagonists, [ 3 H] quinuclidinyl benzilate and [ 3 H] N-methyl scopolamine, muscarinic cholinergic receptors have been demonstrated to be present on pancreatic acinar cells (Dehaye et al., 1984; Larose et al., 1981; N g et al., 1979). The other physiological stimulant, C C K , can stimulate amylase release two-fold in isolated pancreatic acinar cells (Christophe et al., 1976) and up to 20-fold in the perfused rat pancreas (Kondo and Schulz, 1976b; Williams, 1984). One possible explanation for this difference i n amylase secretion is that exocytosis may require the presence of a specialized domain within the apical plasma membrane which is absent i n isolated cells (Williams, 1984). The naturally occurring peptide analogues of C C K , gastrin and caerulein, were also 6 Introduction Table I. Regulators of pancreatic exocrine secretion and their intracellular messengers, (from Williams and Hootman, 1986). Regulator Intracellular messenger Actions ACb, CCK Bombesin, Substance P VIP, Secretin C a 2 + C a 2 + Cyclic AMP Insulin, epidermal growth Unknown factor Digestive enzyme secretion; Cl"-rich pancreatic juice; digestive enzyme synthesis; trophic effects Digestive enzyme secretion Stimulation or potentiation of digestive enzyme secretion; pancreatic juice secretion Potentiation of digestive enzyme secretion Somatostatin Unknown Inhibition of secretion 7 Introduction shown to increase enzyme secretion (Deschodt-Lanckman et al., 1976; M a y et al., 1978). Radiolabeled C C K ( 1 2 5 I - C C K ) and other analogues have been used to demonstrate C C K binding sites on pancreatic acinar cells (Jensen et al., 1980; Sankaran et al., 1980, 1982). H i g h affinity binding sites for bombesin and related compounds have also been demonstrated on pancreatic acinar cells (Jensen et al., 1978). These compounds act on receptor sites distinct from the C C K receptors to evoke enzyme secretion (Jensen et al., 1978; Petersen and Philpott, 1979). Substance P and its analogues have also been shown to evoke enzyme release by acting on specific receptors (Jensen and Gardner, 1979, 1981). Secretin and vasoactive intestinal polypeptide (VIP), while mainly involved i n fluid and electrolyte secretion by the exocrine pancreas (Case et al., 1980; Folsch and Creutzfeldt, 1977; Komarov et al., 1939; Said and Mutt, 1977; Sewell and Young, 1975), also regulate enzyme secretion i n some species. These compounds, along with the peptide histidine isoleucine occupy specific receptors linked to the adenylate cyclase second messenger system leading to increased enzyme secretion i n guinea-pig and rat, but not i n cat, dog and mouse (Jensen and Gardner, 1981; Jensen et al., 1981, 1983; Robberecht et al., 1977). The physiological relevance of insulin receptors on the surface of pancreatic acini (Korc et al., 1978) is not certain. Insulin may be a potentiator of enzyme release, rather than a stimulant (Williams et al., 1981). STIMULUS-SECRETION COUPLING As described above, the acinar cells are polarized cells. Therefore, the need for an intracellular messenger may be more pronounced in these cells than others since the physiological signal has to be carried from the stimulation sites at the basal membrane to the the enzyme releasing sites at the apical 8 Introduction membrane. The role of intracellular messengers has been extensively studied i n the pancreatic acinar cell. Some of the accumulated knowledge is discussed below. Role of Calcium Enzyme secretion has been postulated to be triggered by increased cytosolic free C a 2 + . The involvement of cytosolic free C a 2 + i n the signalling process during pancreatic secretion has been extensively reviewed (Schulz, 1980; Sung and Williams, 1988; Williams, 1980). Most secretagogues discussed above have been shown to increase intracellular free C a 2 + . It is now established that this increase occurs as a result of release from intracellular stores as well as an increased permeability of the plasma membrane to C a 2 + . The resting levels of intracellular free C a 2 + i n pancreatic acinar cells have been reported to be 90-105 n M in mouse (Ochs et al., 1985; Powers et al., 1985), 100-160 n M i n guinea-pig (Pandol et al., 1985a) and 120-125 n M i n rat (Bruzzone et al. , 1986; Mual lem et al., 1988b). This concentration increases to over 1 L I M within seconds i n response to stimulation by secretagogues (Muallem et al., 1988b; Ochs et al., 1985; Pandol et al., 1985a; Powers et al., 1985). This increase, however, is transient (Muallem et al., 1988c; Ochs et al., 1985; Pandol et al., 1985b; Streb and Schulz, 1983). The requirement of C a 2 + for enzyme secretion was demonstrated in early experiments using Ca 2 + - f ree buffers and ethylene glycol bis(fi-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-containing media. These studies showed that while initial enzyme secretion was independent of extracellular C a 2 + , sustained release could only be maintained i n its presence (Argent et al., 1973; Case and Clausen, 1973; Elmer l et al., 1974; Williams, 1980). More recently, it has been shown that C C h failed to produce full stimulation of 9 Introduction enzyme release i n the presence of bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) , an intracellular C a 2 + chelator (Dormer, 1984). Furthermore, amylase release from pancreatic acini suspended i n C a 2 + -containing media could be initiated with the C a 2 + ionophore A23187 (Elmerl et al., 1974; Ponnappa and Williams, 1980). A good correlation between an increase i n free C a 2 + and enzyme release was shown i n isolated pancreatic acini (Dormer, 1984; Pandol et al., 1985a; Powers et al., 1985); antagonists of the secretagogues inhibited both amylase release and the rise i n cytosolic free C a 2 + (Powers et al., 1985). The threshold concentration of intracellular free C a 2 + required for amylase release i n the mouse pancreatic acini was calculated to be 280 n M (Ochs et al., 1985). A stoichiometric relationship appears to exist between changes in intracellular free C a 2 + and amylase release (Ochs et al., 1985). It was reported that 1.2% of total amylase is released over a 30 minute period for each 100 n M increase in C a 2 + . Extensive research has been carried out to determine the source of the increased intracellular C a 2 + . A n early study demonstrated that i n superfused pancreatic fragments, amylase release i n response to short pulses of A C h stimulation at half-hour intervals was not affected by exposure to a Ca 2 + - free solution, even when E G T A was present (Petersen and Ueda, 1977). This study and others have shown that C a 2 + may be released from an intracellular store. Secretagogues have been shown to increase both release of intracellular C a 2 + and its influx. The initial effect of secretagogues appears to be the release of C a 2 + from intracellular stores (Pandol et al., 1987). C C h caused a rapid rise in intracellular free C a 2 + i n the presence or absence of extracellular C a 2 + i n mouse (Muallem et al., 1988c) and guinea-pig pancreas (Pandol et al., 1985a, 1987). Similar results were obtained with C C K i n guinea-pig pancreas (Pandol et al., 10 Introduction 1985a). After stimulation with C C h , the C a 2 + store appears to be depleted or desensitized to C C K (Pandol et al., 1987) indicating that both secretagogues release C a 2 + from a common intracellular store (Powers et al., 1985; Schulz, 1980) . The release of sequestered C a 2 + has also been proposed to be the cause of an initial increase i n C a 2 + efflux (Dormer et al., 1981) as observed i n many studies using 4 5 C a 2 + prelabelled acini (Case and Clausen, 1973; Heisler, 1974; Kondo and Schulz, 1976a; Matthews et al., 1973). Ear ly evidence suggested mitochondria to be the intracellular C a 2 + store (Clemente and Meldolesi, 1975; Wakasugi et al., 1982). However, more recent evidence has shown that C a 2 + released during stimulation originates from the endoplasmic reticulum (ER) (evidence discussed in the next subsection). A recent report has suggested two separate non-mitochondrial intracellular pools (TheVenod et al., 1989). In addition to their action in initiating the release of C a 2 + from intracellular C a 2 + stores, secretagogues have been reported to increase plasma membrane permeability to C a 2 + i n pancreatic acinar cells (Wakasugi et al., 1981) . Activation of rat pancreatic acini by C C K was shown to result i n a 7-fold increased permeability of the plasma membrane to C a 2 + (Muallem et al., 1988b). Direct uptake of 4 5 C a 2 + into dispersed acini can be demonstrated with both secretagogues and the C a 2 + ionophore A23187 (Kondo and Schulz, 1976b). The influx of extracellular C a 2 + appears to be more important i n sustained release (Ochs et al., 1985; Schulz, 1980). As demonstrated in guinea-pig pancreatic acinar cells, the sustained phase of amylase release is unaffected by CCh-induced depletion of intracellular C a 2 + stores (Pandol et al., 1985a). However, using rapid time resolution, C a 2 + influx was shown to occur before internal release i n some cells (Blackmore, 1988; Sage and Rink, 1987). It has also been suggested that low physiological concentrations of agonists cause C a 2 + 11 Introduction influx, while high concentrations produce C a 2 + release (Exton, 1988). Another study showed that the rate of rise i n intracellular C a 2 + i n response to C C h was dependent on extracellular C a 2 + , while the onset and peak amplitude were unaffected (Ochs et al., 1985). This indicates that, although not directly dependent on it, the rise i n intracellular C a 2 + may be regulated by external C a 2 + . Role of Inositol Phosphates The physiological significance of agonist-stimulated breakdown of Phosphatidylinositol (PI) was first suggested by Michell (1975). The second messenger involved in the release of C a 2 + from intracellular stores is inositol-1,4,5-trisphosphate (IP3). The receptor-mediated activation of phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) (Putney et al., 1983) to produce IP3 (Berridge and Irvine, 1984; Doughney et al., 1987; Pandol et al., 1985b). Hydrolysis of PIP2 to diacylglycerol (DG) and I P 3 has now been established as a part of the signal transduction mechanism i n the control of a variety of cellular processes such as secretion, metabolism, phototransduction and cell proliferation (Berridge and Irvine, 1984). Whereas I P 3 is water soluble and is released into the cytosol to mobilize intracellular C a 2 + , D G operates within plasma membranes to activate protein kinase C (PKC). Over the past few years, evidence has also accumulated indicating the production of I P 3 from PIP2 during receptor-mediated stimulation of amylase release from pancreatic acinar cells. Secretagogues such as C C h and caerulein have been demonstrated to cause PIP2 breakdown in the exocrine pancreas (Putney et al., 1983). Following agonist stimulation, the increase in I P 3 is rapid (Powers et al., 1985). In other experiments, it was shown that CCK-octapeptide (CCK-8) stimulated PI and P I P 2 breakdown i n guinea-pig pancreatic acini i n the absence of both 12 Introduction extracellular and intracellular C a 2 + (Pandol et al., 1985b). This suggests that the phosphoinositide breakdown is independent of extracellular C a 2 + and mobilization of intracellular C a 2 + stores. The hydrolysis of phosphoinositides is not accelerated by A23187. In fact, PI turnover was shown to precede C a 2 + release (Putney et al., 1983). Furthermore, production of IP3 is noted within 5 seconds of stimulation by C C h , which is rapid enough to cause the release of sufficient C a 2 + from R E R to give rise to the observed increase i n cytosolic free C a 2 + (Doughney et al., 1987). Another study showed that the increase i n intracellular free C a 2 + is transient, whereas PI breakdown continues after 5 minutes (Pandol et al., 1985b). There is evidence to suggest that IP3 causes the release of C a 2 + from intracellular stores, possibly E R or calciosomes. I P 3 caused C a 2 + release from nonmitochondrial stores i n permeabilized insulin-secreting cells (Biden et al., 1984). In other tissues, such as permeabilized smooth muscle cells, IP3 has been shown to mimic the effects of receptor-linked agonist on both contraction and the mobilization of intracellular C a 2 + (Bitar et al., 1986). Streb and colleagues were the first investigators to show a direct release of C a 2 + from nonmitochondrial intracellular stores by IP3, using permeabilized rat acinar cells (Streb et al., 1983). Another study demonstrated that C C h and I P 3 act on the same pool of releasable C a 2 + (Streb and Schulz, 1983). The nonmitochondrial store was the same C a 2 + store which is released by A C h . Us ing rat pancreatic acinar cells, this I P 3 releasable intracellular C a 2 + store was later demonstrated to be the E R (Streb and Schulz, 1983; Streb et al., 1984). Other reports have since confirmed these findings (Brown et al. , 1987; Mual lem et al., 1987; Richardson and Dormer, 1984). IP 3 -induced translocation of C a 2 + across the E R membrane was shown to be through an ion channel rather than a carrier (Joseph and Williamson, 1986). 13 Introduction This r P 3 -activated C a 2 + channel is blocked by heparin (Ferris et al., 1989). The C a 2 + efflux across E R membranes was balanced by the movement of monovalent cations and anions in permeabilized hepatocytes (Joseph and Williamson, 1986). A n I P 3 receptor has recently been solubilized, purified and characterized from rat cerebellum (Supattapone et al., 1988b). This protein has a molecular mass of 260,000 daltons and a for I P 3 of 0.1 u M . The binding of I P 3 to the receptor was reversibly inhibited by 300 n M C a 2 + i n the particulate fraction, but not affected by up to 1.5 m M C a 2 + i n purified receptor preparations (Supattapone et al., 1988b). The I P 3 receptor has been localized to the E R (Ross et al., 1989), reconstituted (Ferris et al., 1989, 1990) and sequenced (Furuichi et al., 1989; Maeda et al., 1990). A possible feedback inhibition mechanism may operate for C a 2 + release i n pancreatic acinar cells: IP 3-mediated C a 2 + release has been shown to be inhibited by C a 2 + itself i n permeabilized A K 4 2 J cells, a pancreatic acinar cell line (Willems et al., 1990; Zhao and Muallem, 1990). This inhibition was explained by a possible loosely attached cytosolic factor which interacts with the IP 3-sensitive C a 2 + channel in a Ca 2 + -dependent manner to modulate the IP 3 -induced C a 2 + release. L P 3 has also been suggested to regulate C a 2 + movements across the plasma membrane. It stimulated C a 2 + influx into T-lymphocytes (Kuno and Gardner, 1987) and rat mast cells (Penner et al., 1988). I P 3 also inhibited 4 5 C a 2 + efflux from rat brain synaptosomes (Fraser and Sarnacki, 1990). However, i n other studies, IP 3 -induced C a 2 + release from subcellular fractions of rat pancreatic acini did not correlate with plasma membrane markers (Streb et al., 1984). Cyclic l:2,4,5-trisphosphate (cIP 3), produced during agonist stimulation, has also been shown to release sequestered C a 2 + with the same potency as I P 3 14 Introduction (Irvine et al., 1986; Wilson et al., 1985). However, the turnover rate of cIP 3 is considerably slower than that of IP 3 itself. Therefore, the cIP 3 formed probably would not directly influence either C a 2 + release or C a 2 + entry. However, since LP 3 is short-lived, cLP3 may take over the role of IP 3 such that the rapid metabolism of the latter is no longer of consequence (Hughes et al., 1988). Inositol 1,3,4,5-tetrakisphosphate (IP4) has also been shown to rapidly increase in pancreatic acinar cells during stimulation and has been suggested to increase C a 2 + entry into these cells (Muallem, 1989). There is direct evidence for the role of IP 4 in the stimulation of C a 2 + influx in sea urchin oocytes (Berridge, 1987) . IP4 may initiate C a 2 + signals by opening voltage-sensitive C a 2 + channels in the plasma membrane (Petersen, 1989). Binding sites for IP 4 have been demonstrated in many cells including the adrenal cortex (Enyedi and Williams, 1988) . Other reports have suggested that IP 4 is not involved in the regulation of extracellular C a 2 + entry (Hill and Boynton, 1990). In perfused lacrimal glands, it was shown that the actions of ACh on C a 2 + entry could only be mimicked in the presence of both IP 3 and IP 4 and not by either agent alone (Morris et al., 1987). IP 4 was recently postulated to be involved in replenishing the IP 3-sensitive C a 2 + pool, possibly via interaction with the Ca 2 +-ATPase of ER or calciosomes rather than the plasma membrane (Hill and Boynton, 1990). This might be explained by an interesting model proposed by Irvine (1990) in which the IP 4 receptor is located between ER and the plasma membrane when the two membranes are in close apposition. Involvement of Protein Kinase C Evidence has suggested that a rise in intracellular C a 2 + by itself is not sufficient to stimulate and maintain secretion from isolated acini (Sung and Williams, 1988). For example, the C a 2 + ionophore ionomycin produced only one-15 Introduction third the level of amylase release compared to C C h in mouse pancreatic acini (Ochs et al. , 1985), and C C h was shown to produce a 3-fold higher effect than maximal C a 2 + concentrations in permeabilized cells (Kimura et al., 1986). Furthermore, a divergence between Quin-2 fluorescence changes and amylase secretion has been observed at supramaximal agonist concentrations (Ochs et al., 1985; Powers et al., 1985). These data suggest that an additional event occurs at higher agonist concentrations. This additional stimulatory pathway may involve P K C . A n endogenous P K C activity has been identified i n the pancreas (Burnham and Williams, 1984; Wrenn et al., 1981). D G , the other product of PIP2 hydrolysis and the natural activator of P K C (Nishizuka, 1984, 1988), stimulated amylase secretion, but produced no change in intracellular C a 2 + i n guinea-pig pancreatic acini (Pandol et al., 1985a). The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of P K C , has also been shown to stimulate amylase secretion from guinea-pig pancreatic acini without affecting the intracellular free C a 2 + concentration (Ansah et al., 1986; Pandol et al., 1985a). In addition, synthetic 1,2 D G and other phorbol esters were shown to stimulate amylase release from pancreatic acini (Gunther, 1981; Merritt and Rubin, 1985). Since the rise in D G is slow and prolonged compared to IP3 (Exton, 1987), P K C has been proposed to affect the sustained release of enzymes rather than the initial secretion (Sung and Williams, 1988). Furthermore, P K C itself is phosphorylated and therefore can remain active after D G and intracellular C a 2 + levels have decreased (Exton, 1987). It has been postulated that the initial and sustained phases of secretion are mediated by two separate intracellular pathways, the initial phase being controlled by IP3 and the sustained phase by D G (Pandol et al., 1985a). 16 Introduction P K C may also have a negative feedback effect over secretagogue-induced C a 2 + transients (Nishizuka, 1988). Preincubation of guinea-pig pancreatic acini with T P A produced a time- and dose-dependent inhibition ( I C 5 0 = 30 nM) of the CCh-induced increase i n intracellular free C a 2 + , reaching maximal inhibition within 3 minutes (Ansah et al., 1986). T P A was also shown to produce a marked decrease i n CCK-8-evoked pancreatic secretory response i n the anesthetised rat (Francis et al., 1990). This response was blocked by polymyxin B , an inhibitor of P K C . In human platelets, P K C was reported to cause initial secretion and aggregation, followed by negative feedback on receptor-mediated mobilization of intracellular C a 2 + and hydrolysis of PIP2 (Zavoico et al., 1985). The evidence provided by these studies suggests that P K C may have a direct regulatory role over intracellular free C a 2 + . In other studies, P K C was shown to act synergistically with the C a 2 + ionophore (Merritt and Rubin, 1985). It has been postulated that complete activation of amylase release requires stimulation of both Ca 2 + -dependent and P K C pathways (Merritt and Rubin, 1985; Pandol et al., 1985a). It has recently been suggested that phosphatidylcholine (PC) breakdown may be more important than PI hydrolysis i n the regulation of P K C and perhaps other cell functions. The generation of D G from P C is quantitatively greater than from P L P 2 and may be a major factor in the regulation of P K C (Exton, 1988). Another report has suggested two separate pathways for D G formation (Cockcroft et al., 1985). According to this hypothesis, in an early phase, D G is formed from P I P 2 and i n a later phase, from phosphatidic acid (PA) which is formed from PI via phospholipase D . 17 Introduction Other Intracellular Messengers of Pancreatic Enzyme Secretion Secretin and V I P are believed to stimulate amylase release via cyclic adenosine 3',5'-monophosphate (cAMP) (Gardner and Jensen, 1981). Both agonists have been shown to increase c A M P levels between 8- and 30-fold i n dispersed acinar cells or i n intact acini from the guinea-pig pancreas (Korman et al., 1980; Robberecht et al., 1976). A much larger increase can be observed i n the presence of a cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthene (Gardner et al., 1982). Furthermore, somatostatin, which inhibits amylase release in vivo, inhibited the increase i n c A M P by V I P and secretin in guinea-pig pancreatic acini (Esteve et al., 1983). These observations indicate that, at least i n guinea-pig pancreas, c A M P plays a role in stimulus-secretion coupling. In other species, such as mouse, cAMP-mediated secretagogues do not affect amylase release by themselves, but can potentiate the release by Ca 2 + -mediated secretagogues (Burnham et al., 1988). Another cyclic nucleotide, cyclic guanosine 3',5'-monophosphate (cGMP), is increased in response to Ca 2 + -re leas ing secretagogues and A23187 (Christophe et al., 1976, Berridge, 1984). However, elevations of intracellular c G M P by addition of 8-bromo or dibutyryl c G M P produced no effect on C a 2 + fluxes or amylase release (Gunther and Jamieson, 1979). Therefore, the increase i n c G M P levels may be secondary to increased C a 2 + via stimulation of guanylate cyclase. A potential mediator role for c G M P in stimulus-secretion coupling is not clear. P A , which is formed during secretagogue-mediated phospholipid breakdown has been postulated to act as an endogenous ionophore to mediate the inward movement of C a 2 + that occurs during occupation of the surface membrane receptors (Putney et al., 1980). 18 Introduction Interactions Among Intracellular Messengers The C a 2 + , P K C and cAMP-dependent protein kinase (PKA) systems appear to interact with one another to influence enzyme secretion. P K C and C a 2 + ionophore were shown to produce synergistic stimulation of amylase release i n pancreatic acini (Kimura et al., 1986; Merritt and Rubin, 1985; Pandol et al., 1985a). P K A produced a stoichiometric phosphorylation of IP 3 -binding protein i n rat cerebellum to cause a substantial decrease in the potency of L P 3 i n releasing C a 2 + (Supattapone et al., 1988a). Calmodulin-dependent protein kinase (CaM-PK) and P K C were found to be ineffective in phosphorylating this protein. O n the other hand, i n permeabilized rat pancreas, c A M P and analogues stimulated Ca 2 + - induced secretion (Kimura et al., 1986). Furthermore, V I P , secretin and 8-bromo c A M P were shown to potentiate the effects of Ca 2 + -mediat ing agents i n the pancreas (Burnham et al., 1984; Collen et al., 1982). T P A has been shown to enhance the cAMP-mediated secretion (Kimura et al., 1986). This effect may be due to a PKC-induced phosphorylation of adenylate cyclase, leading to the potentiation of its activity (Yoshimasa et al., 1987). Adenylate cyclase activity can also be stimulated by C a M - P K (Klee and Newton, 1985). O n the contrary, c A M P was shown to markedly inhibit C a 2 + mobilization and secretion in platelets (Nozawa, 1987). This effect may have been due to the stimulation of C a 2 + uptake into intracellular stores (Nozawa, 1987). Intracellular C a 2 + Oscillations and Ca 2 + - induced C a 2 + release Recent developments have shown that instead of a continuous flow out of an open channel, C a 2 + may be released in 'quantal' form i n response to secretagogue stimulation (Irvine, 1990, Mual lem et al., 1989). This was probably best illustrated by an elaborate study performed in Xenopus oocytes using 19 Introduction confocal fluorescence Ca 2 + monitoring to measure localized Ca 2 + release by flash photolysis of caged IP3 (Parker and Ivorra, 1990). This study demonstrated an abrupt onset of Ca 2 + release in an all or none manner with increasing LP3 liberation. The quantal release of Ca 2 + may explain the Ca 2 + oscillations observed in single cell preparations. Yule and Gallacher (1988) noticed that the treatment of single pancreatic acinar cells with low concentrations of ACh produced oscillations of intracellular Ca 2 + , detected by changes in fura-2 fluorescence. While these oscillations could be initiated in Ca2+-free medium, they could only be sustained in Ca 2 + containing medium. The frequency of these oscillations did not depend on the ACh concentration (Yule and Gallacher, 1988). The oscillations were more prominent at lower agonist doses (Stuenkel et al., 1989). On the other hand, low concentrations of CCK were shown to produce a small sustained release of Ca 2 + , and Ca 2 + oscillations were only observed at high CCK concentrations (Tsunoda et al., 1990). It has recently been suggested that Ca 2 + oscillations result from a small localized release of IP3 (Zhao et al., 1990). If so, this may explain the results of Dormer and colleagues who demonstrated that inositol phosphate formation was less sensitive to CCh than was the stimulation of amylase release (Doughney et al., 1987). It is likely that these authors were unable to measure such a minute local release of IP3. Ca 2 + oscillations appear to die away during agonist application making way to a more sustained elevation of Ca 2 + (Parker and Ivorra, 1990). This phenomenon probably reflects diffusion of Ca 2 + across the cell and not IP3 accumulation, since a recent study showed that a constant IP3 infusion caused a steady release of Ca 2 + resulting in repetitive Ca 2 + spikes (Wakui et al., 1990). In fact, a constant supply of IP3 appears to be necessary for Ca 2 + oscillations. It is now established that activation of receptors linked to IP3 20 Introduction formation generally evoke oscillating cytoplasmic C a 2 + signals with submaximal agonist concentrations (Goldbeter et al., 1990). Although the mechanism for intracellular free C a 2 + oscillations i n non-excitable cells is not clear, several suggestions have been made. It is possible that more than one mechanism exists (Zhao and Mual lem, 1990). Studies with A R 4 2 J pancreatic cells indicate that C a 2 + oscillations result from a combination of stimulation of C a 2 + release by IP3 and inhibition of C a 2 + induced C a 2 + release (Zhao et al., 1990). Since C a 2 + oscillations can be induced by I P 3 , its agonists or C a 2 + itself, it was concluded that C a 2 + - i n d u c e d C a 2 + release is responsible for the oscillations (Osipchuk et al., 1990; Wakui and Petersen, 1990). The C a 2 + oscillations are not due to fluctuations in the levels of I P 3 (Wakui et al., 1989). A lack of inhibition of Ca 2 + - induced C a 2 + release by heparin indicates that it is not dependent on I P 3 or the IP 3-sensitive channel (Wakui et al., 1990). According to a recent study in single mouse oocytes, the role of LP 3 -induced C a 2 + release may be to gradually raise C a 2 + levels to the point where Ca 2 + - induced C a 2 + release is triggered (Peres, 1990). The P K C pathway does not appear to be crucial for C a 2 + oscillations (Zhao et al., 1990), but it may have a negative feedback influence (Tsunoda et al., 1990). T P A was shown to inhibit C a 2 + oscillations triggered by a C C K analogue i n A R 4 2 J cells (Zhao et al., 1990). It was proposed that the inhibitory regulation is due to the phosphorylation of a target protein by P K C . c A M P was also shown to negatively regulate the C a 2 + oscillations. The reduced amplitude and increased frequency of C a 2 + oscillations caused by c A M P (Zhao et al., 1990) may be due to the phosphorylation of the I P 3 receptor (Supattapone et al., 1988a). 21 Introduction Effect of Calcium on Exocytosis Very little is known about the mechanism by which C a 2 + brings about secretion i n acinar and other secretory cells. Calmodulin (CaM) has been suggested as a regulatory factor in the secretagogue activation of intracellular C a 2 + release i n pancreatic acinar cells (Chien and Warren, 1988). In other studies using an 1 2 5 I - C a M gel overlay technique, C a M was shown to bind to a 230,000 daltons protein of possible cytoskeletal origin (Ansah et al., 1984). Therefore, it was suggested that C a M regulates the secretory process by interacting with the cytoskeleton (Ansah et al., 1984). The molecular mechanism underlying the actions of C a 2 + during exocytosis has been proposed to involve specific or multifunctional C a M - P K (Cohn et al., 1987; Exton, 1987). To this end, a multifunctional C a M - P K of 51,000 daltons has been identified and purified from rat pancreas (Cohn et al., 1987; Gorelick et al., 1983). M a n y secretory processes are thought to involve CaM-dependent phosphorylation of cytosolic and membrane proteins (Exton, 1987). However, the C a 2 + - C a M complex may affect this phosphorylation without the kinase, and furthermore, C a 2 + may affect it without C a M . C a M - P K is known to modulate neurotransmitter release in the brain by acting on synapsin I which results in an altered interaction between synaptic vesicles and the plasma membrane (Llinas et al., 1985). A similar mechanism may be involved in pancreatic acinar cells. It is postulated that Ca 2 + -dependent phosphorylation of a yet-unknown specific regulatory protein is involved i n secretion. Phosphorylation of a ribosomal S6 protein was shown to be stimulated by C C h and C C K i n rat pancreas (Freedman and Jamieson, 1983). However, dephosphorylation of two proteins of 21,000 and 20,500 daltons appears to correlate better than phosphorylation with the onset of secretion (Burnham and Williams, 1982). Dephosphorylation of these proteins could be 22 Introduction prevented by atropine over a similar time period as the atropine-induced inhibition of amylase release. A Ca 2 + -act ivated, CaM-dependent protein phosphatase identified and characterized i n mouse pancreatic acinar cytosol (Burnham, 1985) may be responsible for this dephosphorylation. In addition, C a 2 + may be responsible for the fusion of zymogen granules with the apical plasma membrane (Milutinovic et al., 1977). CALCIUM HOMEOSTASIS A precise regulation of cytosolic free C a 2 + is critical for this ion to serve as a physiological signal i n the secretory process of pancreatic acinar cells. Extracellular C a 2 + concentration is 10,000-fold higher than intracellular levels, and this leads to a continuous leakage of C a 2 + into the cell down its electrochemical gradient. In addition, the cytosolic C a 2 + rises very rapidly during stimulation. Therefore, to terminate the stimulus and to maintain an appropriate resting free C a 2 + level, the cell must have mechanism(s) to continuously sequester and extrude excess C a 2 + . In the pancreas, the endoplasmic reticulum (Richardson and Dormer, 1984; Streb and Schulz, 1983), mitochondria (Wakasugi et al., 1982) and plasma membrane (Bayerdorffer et al., 1985a, b; Kribben et al., 1983; Schulz et al., 1986a) have all been ascribed roles i n C a 2 + regulation. Although mitochondria and E R have both been suggested as organelles involved i n the intracellular sequestration of C a 2 + (Streb and Schulz, 1983; Wakasugi et al., 1982), recent evidence (Muallem et al., 1987; Schulz et al., 1986b) indicates that the E R may play the more important role. A n A T P -dependent Ca 2 + -uptake with a K ^ a of 1.1 | i M was demonstrated i n mouse pancreatic microsomes (Ponnappa et al., 1981). However, in more recent studies of rat pancreatic acinar E R , much higher affinities for C a 2 + have been reported 23 Introduction for the C a 2 + - p u m p and the C a 2 + - A T P a s e activities (Brown et al., 1987; Mual lem et al., 1987). A number of studies have shown that the E R C a 2 + - p u m p is activated during stimulation of the pancreas by secretagogues. E R isolated from secretagogue-stimulated pancreatic acini from rat and mouse showed increased C a 2 + uptake (Ponnappa et al., 1981; Richardson and Dormer, 1984). In addition, E R isolated from CCh-stimulated rat pancreatic acini showed decreased C a 2 + content and increased C a 2 + - A T P a s e activity (Brown et al., 1987). Both of these effects were prevented after blockade with atropine. More recently, Mual lem and colleagues demonstrated that secretagogues stimulated the C a 2 + - p u m p of an agonist-sensitive C a 2 + pool (believed to be the E R ) i n permeabilized pancreatic acinar cells (Muallem et al., 1987, 1988d); the apparent affinity of this C a 2 + - p u m p for C a 2 + was increased 3-fold by secretagogue stimulation, while the maximal velocity was raised 2-fold (Muallem et al., 1987). The E R C a 2 + - p u m p was shown to regulate free C a 2 + down to 0.4 u M (Streb and Schulz, 1983). F r o m the evidence available to them, Dormer and colleagues suggested that the E R C a 2 + - p u m p was more sensitive to free C a 2 + than the plasma membrane extrusion systems (Dormer et al., 1987). It is possible that one component of the nonmitochondrial C a 2 + pool is the recently described 'calciosomes' (Volpe et al., 1988). These structures are similar to E R and are distributed throughout the cytoplasm and may be the targets for I P 3 . Since the capacity of intracellular organelles to sequester C a 2 + is limited, extrusion of the cation from the cell is a necessity. It is here that the plasma membrane plays a very important role in the maintenance of intracellular free C a 2 + levels (Carafoli et al., 1986). In many mammalian cell types, two plasma membrane Ca 2 + -extrus ion mechanisms have been identified; a C a 2 + -transporting ATPase pump and a Na + /Ca 2 + -exchanger . 24 Introduction The Na + /Ca 2 + -exchanger utilizes a N a + gradient established by the N a + / K + - A T P a s e pump to drive its exchange of N a + for intracellular C a 2 + (Schatzmann, 1985). It is generally regarded as a low affinity system (Slaughter et al., 1987, Rengasamy et al., 1987). The precise role of this system i n C a 2 + homeostasis of non-excitable cells remains unclear. Bayerdorffer and colleagues (Bayerddrffer et al., 1985b) showed that unlike other tissues, the N a + / C a 2 + -exchanger i n rat pancreatic acinar cells appears to be a high-affinity C a 2 + extrusion system. A high affinity Na + /Ca 2 + -exchanger has also been reported in human small intestine basolateral membranes (Kikuchi et al., 1988). A more recent study of the Na + /Ca 2 + -exchanger i n dispersed pancreatic acini did not determine its affinity for C a 2 + (Muallem et al., 1988a). This system may work side-by-side with the C a 2 + - A T P a s e to extrude C a 2 + from the cell. However, despite its high affinity, the C a 2 + extrusion rate of the exchanger appears to be very low at approximately 0.12 nmoles/mg/min (Bayerdorffer et al., 1985b). In addition, some reports have indicated that the Na + /Ca 2 + - exchanger is not significantly stimulated during secretagogue-activation i n pancreatic acini (Muallem et al., 1988a, b). Therefore, the exchanger may play only a minor role i n C a 2 + homeostasis, with most, i f not all, C a 2 + efflux from pancreatic acinar cells being mediated by the plasma membrane C a 2 + - p u m p (Muallem et al., 1988a). The plasma membrane C a 2 + - p u m p may be more important in regulation and maintenance of low intracellular free C a 2 + levels i n pancreatic acinar cells, but its activity i n isolated plasma membrane preparations has not been sufficiently well characterized to fully assess this possibility. In rat pancreatic acini, the net C a 2 + efflux is thought to be regulated by the plasma membrane C a 2 + - p u m p (Muallem et al., 1988a). The rate of C a 2 + efflux i n pancreatic acinar cells is increased during stimulation by secretagogues (Case and Clausen, 1973; 25 Introduction Heisler, 1974; Kondo and Schulz, 1976a; Matthews et al., 1973; Muallem et al., 1988b). It has also been suggested that the pancreatic acinar plasma membrane Ca2+-pump can regulate intracellular Ca 2 + concentrations to the same or a lower level than that achieved by the ER (Schulz et al., 1986b). The properties of plasma membrane Ca2+-pump are discussed below. The Plasma Membrane Calcium Pump The active transport of Ca 2 + is an ATP-requiring process and is believed to be carried out by a Ca2+-ATPase enzyme (Sarkadi and Tosteson, 1979; Schatzmann, 1975). The plasma membrane Ca2+-ATPase has been well characterized in the erythrocyte (reviewed by Carafoli et al., 1986; Schatzmann, 1986) and in cardiac muscle (Caroni and Carafoli, 1981a; Caroni et al., 1983), and has been linked to a Ca2+-translocating function. The erythrocyte Ca 2 + -ATPase is considered the prototype, and many of the properties discussed here were first described for this enzyme. A Mg2+-dependent Ca2+-stimulated ATPase in the plasma membranes of human erythrocytes was first demonstrated by Dunham and Glynn (1961). Subsequent to this, a Ca2+-transport activity was reported in these membranes (Schatzmann, 1966). The two activities were subsequently shown to be closely coupled (Schatzmann and Vincenzi, 1969). The Ca2+-ATPase displays a high affinity for Ca 2 + (KCa = 0.5 uM) and can hydrolyze ATP at a rate of 150-500 nmoles/mg/sec (Carafoli et al., 1986). Protons have been suggested as the counter ions for the erythrocyte Ca2+-pump (Niggli et al., 1982). The K^ a for cardiac sarcolemmal Ca2+-ATPase is 0.4 uM (Caroni et al., 1983). In the absence of CaM, the purified Ca2+-ATPases appear to have much lower affinities for Ca 2 + than the enzymes in situ (Kc a = 10 |iM for erythrocyte and 20 fiM for sarcolemmal) (Caroni et al., 1983; Niggli et al., 1981). 26 Introduction The erythrocyte enzyme demonstrates a high substrate specificity for ATP (Sarkadi et al., 1979). Although Ca-ATP and free ATP have been suggested as the physiological substrate of the enzyme, Mg-ATP generally appears to be the true substrate (Enyedi et al., 1982; Penniston, 1982). The number of C a 2 + ions pumped per mole of ATP hydrolyzed has been the subject of much controversy. While it appears that the stoichiometry of the pump can vary between 1:1 and 2:1 (Ca 2 +:ATP) depending upon the concentration of the effectors used in the assay system (Akyempon and Roufogalis, 1982; Larsen et al., 1981; Sarkadi, 1980), a 1:1 ratio is generally thought to reflect the physiological situation (Carafoli et al., 1986; Larsen et al., 1978). The molecular weight of the monomelic erythrocyte plasma membrane Ca2 +-pump determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is approximately 138,000 daltons (Carafoli et al., 1986). However, the enzyme is believed to be present within the membrane in an oligomeric form. A dimeric arrangement has been suggested by azidocalmodulin-binding experiments (Hinds et al., 1982). Molecular weight determinations by a radiation-inactivation study revealed that the functional form of the Ca2+-pumping ATPase within the native erythrocyte membrane is a dimer of 290,000 daltons (Minocherhomjee et al., 1983). A recent publication described the isolation and sequencing of cDNAs coding for a plasma membrane Ca2+-pump from a human teratoma library (Verma et al., 1988). Comparison of the cloned sequence with the sequences from the purified erythrocyte Ca2 +-ATPase showed 86% identical residues. The calculated molecular weight of the translated clone is very similar to the observed M r for erythrocyte Ca2 +-ATPase. Contrary to expectations, the primary sequence showed little resemblence to another extensively studied and cloned C a 2 + transporter, the sarcoplasmic reticulum Ca2 +-pump (Brand! et al., 27 Introduction 1986; MacLennan et al., 1985; Verma et al., 1988). A close resemblence between the two proteins is only observed i n one hydrophobic region including residues 885-905 (Verma et al., 1988). Other resemblences between the two C a 2 + transporters are i n the regions that are generally conserved i n ion transporters such as the A T P binding site i n the ATPases. Controlled trypsin proteolytic digestion of the purified erythrocyte C a 2 + -ATPase results i n a number of small successive fragments of 90, 85, 81 and 76 kilodaltons (kDa) (Benaim et al., 1984; Zurini et al., 1984). A l l of these fragments retain the Ca 2 + - s t imulated ATPase activity, while the 90, 85 and 81 k D a fragments can also transport C a 2 + following reconstitution into liposomes (Benaim et al. , 1984, 1986; Zurini et al., 1984). The proteolysis of the 85 k D a fragment to the 81 k D a cleavage product results i n a loss of the C a M binding site with a concommitant persistent stimulation of C a 2 + - A T P a s e activity. The high affinity Ca 2 + - transport ing ATPase of erythrocyte membranes forms a Ca 2 + -dependent phosphorylated intermediate which is acid-stable, but very labile i n the presence of hydroxylamine and alkalis (Katz and Blostein, 1975). This phosphorylated intermediate has a molecular weight of 135,000-145,000 daltons (Katz and Blostein, 1975; K n a u f et al. , 1974; Wolff et al., 1977) and forms part of the reaction cycle of the enzyme. The reaction sequence proposed by Schatzmann (1985) is shown i n Figure 3. The transport cycle begins with the binding of C a 2 + and A T P to the enzyme, resulting i n the formation of a high affinity Ca 2 + -dependent phosphoenzyme, E i C a P (Katz and Blostein, 1975; Niggli et al., 1979b; Rega and Garrahan, 1975; Richards et al., 1978). The formation of this intermediate does not require M g 2 + (Rega and Garrahan, 1975), but is accelerated by the presence of this ion (Enyedi et al., 1980; Rega and Garrahan, 1978; Schatzman and Burgin, 1978). In the next step, E ] C a P is converted to a more reactive intermediate E 2 C a P 28 Introduction which has a lower affinity for Ca 2 + and is hydrolyzed rapidly to release Pj in the presence of Mg 2 + (Garrhan and Rega, 1978; Katz and Blostein, 1975). During this step, Ca 2 + is thought to be transported to the extracellular surface of the cell (Sarkadi, 1980). The next step is the hydrolysis of the phosphorylated intermediate to E 2 and the dissociation of Ca 2 + (Rega and Garrahan, 1978). The final step in the cycle is the conversion of the E 2 state back to the Ej^ state. Caj ADP ATP + E T ^ A rv E^a-P Mg2+?* *Mg2+ E (Mg)-ATP 2 V II V E2Ca~P P Ca, H,0 o "2 III Figure 3. Reaction cycle of the human red cell Ca2+-pump. In the normal mode the cycle turns in a clockwise direction. Ca^ represents ionized cytosolic Ca 2 + , Ca0 represents ionized extracellular Ca 2 + and P represents inorganic phosphate. E1 and E 2 are two conformational forms of the protein. Requirements of the reactions are indicated by asterisks, (from Schatzmann, 1985). A high affinity Ca2+-ATPase has been demonstrated in rat pancreatic acinar cell plasma membranes (Al-Mutairy and Dormer, 1985; Ansah et al., 1984, Hurley et al., 1984). This activity shows characteristics similar to the Ca2+-transporting ATPase of erythrocytes. It has a K Q & of 0.65-1.7 |iM (Al-Mutairy and Dormer, 1985; Ansah et al., 1984; Dormer and Al-Mutairy, 1987), shows an apparent requirement for Mg 2 + , forms a Ca2+-dependent 29 Introduction phosphorylated intermediate and is stimulated by C a M and acidic phospholipids (Ansah et al., 1984). In a recent study, a high affinity Mg 2 + -dependent, C a 2 + -ATPase activity was localized to the cytoplasmic surface of the plasma membrane i n rat pancreatic acinar cells (Ochs et al., 1988). In addition, a C a 2 + -transport activity has been observed i n rat (Bayerdorffer et al., 1985a) and cat (Kribben et al., 1983) pancreatic acinar plasma membranes by one laboratory. The rat C a 2 + - p u m p was found to be electrogenic, had a K Q & of 0.9 | i M and was specific for A T P (Bayerdorffer et al., 1985a). However, the results from most studies of pancreatic acinar plasma membranes demonstrate a low affinity, non-specific C a 2 + ( o r M g 2 + ) - A T P a s e activity, i.e. an enzyme which can only be stimulated with high concentrations of either C a 2 + or M g 2 + , and can utilize either di- or tri-phosphates as substrates (Forget and Heisler, 1976; H a m l y n and Senior, 1983; Lambert and Christophe, 1978; LeBel et al., 1980; Mart in and Senior, 1980). Similar low affinity, C a 2 + ( o r Mg 2 + ) -st imulated, di-/tri-phosphatase activities have been reported i n a number of other tissue types such as rat stomach smooth muscle (Kwan and Kostka, 1984), corpus luteum (Minami and Penniston, 1987; V e r m a and Penniston, 1981) rat adipocytes (Pershadsingh and McDonald, 1980), cardiac muscle (Anand-Srivastava et al., 1982), liver (Lotersztajn et al., 1981, 1982), rat osteosarcoma (Murray et al., 1983) and rat kidney cortex (Parkinson and Redde, 1971). This enzyme appears to be insensitive to monovalent cations, ouabain, ruthenium red, vanadate and the mitochondrial inhibitors sodium azide and oligomycin. The low affinity, non-specific C a 2 + ( o r M g 2 + ) - A T P a s e has been suggested to be an ecto-enzyme (Ansah et al., 1984; H a m l y n and Senior, 1983). The ecto-ATPase of pancreatic acinar cells (also referred to as a diphosphohydrolase) has been characterized and purified from the pig (LeBel et al., 1980). The purified 30 Introduction diphosphohydrolase can hydrolyze both di- and triphosphates with a pH optimum between 8 and 9. It is insensitive to oligomycin, ouabain and ruthenium red. The function of such ecto-enzymes is uncertain. Some reports suggest that in pancreas, it may be an enzyme located within the zymogen granule membrane which is inverted during exocytosis (Harper and Scratcherd, 1979); as such, it may be involved in the storage of zymogen granules and the process of exocytosis (LeBel and Beattie, 1985). Other functions for the ecto-ATPase have been postulated in other tissues. It was suggested that the ecto-ATPase of the mammary gland (13762 mammary-adenocarcinoma ascites), with similar properties to the pancreatic enzyme, may serve to control extracellular ATP concentrations (Carraway et al., 1980). An ecto-ATPase purified from rat heart sarcolemma is thought to play a role in the production of adenosine which increases coronary blood flow and the oxygen supply to the heart (Tuana and Dhalla, 1988). In mast cells, an ecto-ATPase was shown to be associated with Ca 2 + influx (Chakravarty, 1987). It is possible that two separate activities are present in the pancreas, a Ca2+(or Mg2+)-ATPase and a distinct high affinity Ca2+-ATPase that is responsible for Ca 2 + transport. In addition to the low affinity Ca2+(or Mg2+)-ATPase, a high affinity Ca2+-ATPase activity has been demonstrated in some of the tissues mentioned above (Iwasa et al., 1982; Kikuchi et al., 1988; Lotersztajn et al., 1981, 1982; Minami and Penniston, 1987; Verma and Penniston, 1981). The high affinity Ca2+-ATPase of liver plasma membranes has been suggested to be the Ca2+-pumping protein (Lotersztajn et al., 1981; Pavoine et al., 1987). However, this activity differs from the erythrocyte prototype in a number of ways: it is not stimulated by CaM (Iwasa et al., 1982; Lotersztajn et al., 1981); it shows a higher Ca2+-affinity (K a^ = 13-87 nM); it has no requirement for external Mg 2 + (Lin and Fain, 1984; Lotersztajn et al., 1981); it has a broader 31 Introduction nucleotide specificity; it has lower sensitivity to vanadate (Lin and Fa in , 1984; Lotersztajn et al., 1981, 1982) and a stoichiometry of 0.3 ( C a 2 + : A T P ) for C a 2 + transport by the reconstituted purified ATPase (Pavoine et al., 1987). Regulation of the Plasma Membrane Calcium Pump Calmodulin Calmodulin, as it was termed by Cheung (Cheung et al., 1978), is a ubiquitous protein (Cheung, 1980). It is a heat stable, acidic, Ca 2 + -b ind ing , globular protein with a molecular weight of 16,723 daltons (Klee and Vanaman, 1982). The highest concentration of C a M i n mammalian tissues is found i n the brain (Dedman et al., 1977; Watterson et al., 1976). C a M regulates a variety of cellular processes and stimulates a number of enzymes (Scharff, 1981). The first observation of the effects of C a M on C a 2 + - A T P a s e was made by Bond and Clough (1973), who reported that a non-hemoglobin protein present in the hemolysate of human erythrocyte membranes increased the C a 2 + - A T P a s e activity of isolated erythrocyte membranes. C a M has been demonstrated in and isolated from erythrocytes (Gopinath and Vincenzi, 1977; Jarrett and Penniston, 1977). The stimulatory effects of C a M on C a 2 + transport and C a 2 + - A T P a s e in the plasma membrane vesicles from these cells are well established (Hinds et al., 1978; Larsen and Vincenzi, 1979; Penniston, 1983; Sarkadi, 1980; Schatzmann, 1982, 1985). This stimulation results i n a 30-fold increase i n the affinity for C a 2 + and 3-4 fold increase in maximum velocity of the pump (Foder and Scharff, 1981; Roufogalis and Mauldin, 1980; Scharff and Foder, 1978, 1982; Smallwood et a l , 1988). C a M generally requires C a 2 + for its actions. Saturation of C a M with C a 2 + results i n a conformational change (Bromstrom and Wolff, 1981) which exposes 32 Introduction hydrophobic groups which i n turn appear to be involved i n the binding of C a M to the effector molecules (Tanaka and Hidaka, 1980). The suggested mechanism of C a M stimulation of C a 2 + - A T P a s e is the potentiation of both phosphorylation (Allen et al., 1987; Enyedi et al., 1980; Mual lem and Karl ish , 1980; Rega and Garrahan, 1980) and dephosphorylation (Allen et al., 1987; Jeffery et al., 1981; Rega and Garrahan, 1980) of the Ca 2 + -dependent phosphoprotein intermediate, resulting in the increased turnover of the transport cycle. C a M has also been isolated and purified to homogeneity from whole homogenates of dog pancreas (Bartelt et al., 1986). In addition, rat pancreatic acinar plasma membrane C a 2 + - A T P a s e can be stimulated by C a M (Ansah et al., 1984; Dormer and Al-Mutairy, 1987) and inhibited by micromolar concentrations of the C a M antagonists trifluoperazine (TFP) and chlorpromazine (Ansah et al., 1984). Furthermore, the 1 2 5 I - C a M gel overlay technique demonstrated Ca 2 + -dependent binding of C a M to a 133,000 daltons protein which may be the C a 2 + - A T P a s e (Ansah et al., 1984). It should be noted, however, that the CaM-induced stimulation of C a 2 + - A T P a s e s is not universal. The liver and corpus luteum C a 2 + - A T P a s e s are not stimulated by exogenous C a M , even after washing the plasma membranes with E G T A to remove any endogenous C a M (Lotersztajn et al., 1981; Verma and Penniston, 1981); either C a M i n these plasma membranes is very tightly bound or these enzymes are insensitive to it. Oligomerization can stimulate purified C a 2 + - A T P a s e i n the absence of C a M (Kosk-Kosicka et al., 1990). Since C a M inhibits oligomerization (Vorherr et al., 1991, Kosk-Kosicka et al., 1990), it was suggested that the CaM-binding domain may be involved i n this process. Furthermore, a synthetic peptide C a M -binding domain appeared to bind the C a 2 + - A T P a s e (Enyedi et al., 1989; Vorherr 33 Introduction et al., 1991). These data implicate CaM-binding domain in the stimulation of the C a 2 + - p u m p . Limited Proteolysis Controlled tryptic digestion can also stimulate the high affinity C a 2 + -ATPase activity. Digestion of C a 2 + - A T P a s e to a fragment of 81,000 daltons results in stimulation of the enzyme to a similar extent as that observed with C a M treatment of the native enzyme (Benaim et al., 1984; Zurini et al. , 1984). This cleavage also results i n a loss of the C a M binding site. Therefore, it appears that the C a M binding domain exerts some type of inhibition on the C a 2 + - A T P a s e and that the masking of this domain by CaM-binding or oligomerization or its proteolytic cleavage removes this inhibition from the enzyme. Calpain, a Ca 2 + -dependent endogenous protease (Pontremoli and Melloni, 1986) also stimulates C a 2 + - A T P a s e by proteolytic fragmentation (Wang et al., 1989). It too results i n a loss of C a M stimulation, but'yields different proteolytic fragments (Wang et al., 1988, 1989). Other Regulators Regulation of C a 2 + - A T P a s e by other agents has not been characterized to the same extent as the C a M and proteolytic regulation. The erythrocyte and cardiac sarcolemmal C a 2 + - A T P a s e are also stimulated by acidic phospholipids (Caroni et al. , 1983; Niggli et al., 1981), and long chain fatty acids (Ronner et al., 1977). These enzymes are inhibited by vanadate (Caroni and Carafoli, 1981a; Niggli et al., 1981) and lanthanum (Caroni and Carafoli, 1981a; Schatzmann et al., 1986). The C a 2 + - A T P a s e of cardiac sarcolemmal and erythrocyte membranes can also be stimulated by protein kinase A and protein kinase C . Ca 2 + - transport i n 34 Introduction plasma membrane vesicles from both sarcolemma (Caroni and Carafoli, 1981b; Neyes et al., 1985) and erythrocytes (Neyes et al., 1985) was stimulated by the cAMP-dependent protein kinase. However, there is disagreement over whether this kinase affects the rate of Ca 2 + transport or the affinity for Ca 2 +. The purified enzyme can be phosphorylated to form a hydroxylamine-resistant phosphoprotein in the presence of the catalytic subunit of PKA (Neyes et al., 1985). In other studies, PKC in combination with TPA or diolein stimulated the Ca2+-ATPase, Ca2+-uptake and phosphorylation in erythrocyte plasma membranes (Smallwood et al., 1988). Similar findings were reported in smooth muscle cells, where the plasma membrane Ca2+-pump could be stimulated by TPA (Furukawa et al., 1989). Studies in isolated pancreatic acinar cells using protein kinase inhibitors and TPA, have also indicated that PKC is required for activation of the Ca 2 + pump (Muallem et al., 1988b). These reports suggest an involvement of PKC in the regulation of the Ca2+-transporting ATPase. As can be seen from the above discussion, our knowledge of the regulation of Ca 2 + homeostasis within the pancreatic acinar cell is still incomplete. 35 OBJECTIVE In erythrocytes and the cardiac sarcolemma, the role of the plasma membrane C a 2 + - A T P a s e i n C a 2 + extrusion from the cell is well known. However, the existence of such a relationship between C a 2 + - A T P a s e and C a 2 + -transport within the pancreatic acinar cell has not been established. Most of the studies to date have concentrated on ATPase activity, while little information is available concerning the C a 2 + transport process within these cells. In one study, a Mg 2 + -dependent and Ca 2 + - s t imulated ATPase activity i n the total particulate fraction of rat pancreatic acini was suggested to be the enzyme responsible for active C a 2 + translocation (Hurley et al., 1984). In another study, it was suggested that a C a 2 + ( o r M g 2 + ) - A T P a s e present i n rat pancreas plasma membrane-rich fractions was unlikely to be involved in active C a 2 + extrusion (Forget and Heisler, 1976). To our knowledge, only one laboratory has been able to study C a 2 + transport i n plasma membrane vesicles of pancreatic acinar cells (Bayerdorffer et al., 1985a; Kribben et al., 1983) and has proposed that this activity may be linked to a high affinity C a 2 + - A T P a s e . To date there has not been a successful systematic attempt to characterize and correlate the C a 2 + - A T P a s e and Ca 2 + - transport activities i n pancreatic acinar cell plasma membranes. The principal reason for this difficulty appears to be the presence of masking C a 2 + ( o r M g 2 + ) - A T P a s e or diphosphohydrolase-like activities within these cells. The extent of the problem may vary depending upon the animal species chosen for the study. Therefore, the aim of this study was to determine whether a high affinity C a 2 + - A T P a s e is responsible for Ca 2 + -extrusion and hence C a 2 + homeostasis in pancreatic acinar cells. To achieve this, the high affinity C a 2 + - A T P a s e and C a 2 + -transport activities were characterized i n the same plasma membrane preparations from guinea-pig pancreatic acini. These activities were compared 36 Objective with respect to their requirement for C a 2 + and M g 2 + , their substrate specificity for different nucleotide triphosphates, and their regulation by various intracellular mediators such as protein kinase A , protein kinase C , C a M and inositol phosphates. In addition, an attempt was made to purify the high affinity C a 2 + - A T P a s e . F r o m the results of our studies, conclusions were drawn regarding the possible correlation of the high affinity C a 2 + - A T P a s e with the C a 2 + transport activity which could be assayed within our membrane preparations. 37 MATERIALS AND METHODS MATERIALS a) . Radiochemicals: Y - 3 2 P - A T P (10-40 Ci/mmole) was purchased from Amersham Corporation (Oakville, ON) and 4 5 C a C l (10-40 mCi/mg calcium) was purchased from Amersham or New England Nuclear (Mississauga, ON) . b) . Reagents: The following chemicals were purchased from Sigma Chemical Company (St. Louis, MO): 2- mercaptoethanol, 3- (N-Morpholino)propanesulfonic acid (Mops), adenosine triphosphate (disodium), adenosine triphosphate (tris), ammonium bicarbonate, aprotinin, bromophenol blue, calmodulin, catalytic subunit of cyclic AMP-dependent protein kinase, C D T A , citric acid, compound 48/80, E D T A , E G T A , glucose, 38 Materials and Methods glycerol, glycine, H E P E S , hydroxylamine, Kodak G B X developer and replenishes Kodak G B X fixer and replenishes hthiurn dodecyl sulphate, magnesium chloride, P-nicotinamide adenine dinucleotide phosphate, reduced ( N A D P H ) , polyethylene glycol, silver nitrate, sodium azide, sodium bicarbonate, Soybean trypsin inhibitor, sucrose, 12-O-tetradecanoyl phorbol-13-acetate (TPA), tetraethyl-methylenediamine ( T E M E D ) , trichloroacetic acid (crystalline), trifluoperazine, Tris-base, Tris-hydrochloride, Triton X-100, Tween 20. The following chemicals were purchased from B D H Biochemicals (Toronto, ON): activated charcoal, calcium chloride, glacial acetic acid, methanol, potassium chloride, potassium phosphate (monobasic), sodium carbonate, sodium chloride, sodium dodecyl sulphate and sodium hydroxide. 39 Materials and Methods Bovine serum albumin, dithiothreitol, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4,5-trisphosphate and P M S F were purchased from Boehringer Mannhe im Canada L t d . (Laval, Quebec). The following electrophoresis chemicals were obtained from Bio-Rad Laboratories (Mississuaga, ON): acrylamide, N,N'-methylene-bis acrylamide, ammonium persulphate, Coomassie Bril l iant Blue R-250 and high and low molecular weight standards. The phospholipids, phosphatidylcholine, phosphatidylserine, and 1-stearoyl-2-arachidonoyl-SAi-glycerol (SA-DG) were purchased from Serdary Research Laboratories (London, ON). Formaldehyde, gluteraldehyde, and sodium phosphate (monobasic) were obtained from Fisher Scientific Co (Vancouver, B.C.) . Unisolve I® was purchased from Terochem Scientific (Edmonton, AB) . H A 1004 was from Seikagaku Kogyo Co. (Japan), while C G P 41 251 and C G S 9343B were generous gifts from Ciba-Geigy Corporation (Summit, NJ) . Fifty times concentrated amino acids containing L-glutamine were purchased from Gibco Laboratories (Burlington, ON) and collagenase (CLSPA) was obtained from Worthington Biochemical Corporation (Freehold, NJ) . 40 Materials and Methods METHODS Preparation of Pancreatic Acin i Pancreatic acini from guinea-pigs were prepared by the method of Williams et al. (1978) with slight modifications. Pancreata from four guinea-pigs (250-300 g each) were isolated and placed into ice-cold Kreb's Ringer Bicarbonate (KRB), containing 118 mM NaCl, 25 mM NaHC03, 4.7 mM KC1, 1.2 mM NaH2P04, 14 mM Glucose, 0.1 mg/ml soybean trypsin inhibitor, amino acids and 2.5 mM CaCl2 and equilibrated with 95% 02/5% C0 2 for 20 minutes. The pH was adjusted to 7.35 with NaOH as required. Ten milliliters of dissociation solution (KRB containing 70-75 U/ml collagenase, 0.1 mM CaCl2 and 0.1% bovine serum albumin) was then injected into the parenchyma of the pancreata. The organs were then transferred to a 50 ml conical flask and incubated for 15 minutes at 37°C in a water-bath oscillating at 120 cycles/min. After withdrawing the excess medium, 10 ml of fresh dissociation medium was added, followed by a further 45 minute incubation (as above). The acini were dissociated by drawing the tissue up and down through polypropylene pipettes having tip diameters of 1.5 mm and 0.9 mm. This and the following procedures were performed at room temperature. The dissociated tissue was filtered through a 150 micron Nytex® nylon mesh using an extra 20 ml of medium (without collagenase, but with 1% bovine serum albumin). The filtered acini were layered on KRB containing 4% bovine serum albumin and 0.5 mM CaCl2 and centrifuged at 50xg for 4 min. The pelleted acini were washed twice with the same medium followed by another wash in the isolation medium containing 1.25 mM CaCl2. Acini so prepared were used for isolating the plasma membranes as described below. 41 Materials and Methods Preparation of Plasma Membranes from Pancreatic Ac in i Plasma membranes were prepared by a modification of the method of Svoboda et al. (76). A flow-chart outlining the major steps i n the preparation is shown i n figure 4. A l l procedures were carried out at 4 ° C . The Acini were centrifuged at 500xg", and the pellet was suspended i n 5 volumes 10 m M Tri s -Cl , p H 7.4, containing 0.3 M Sucrose, 5 m M 2-mercaptoethanol and 1 m M ethylenediaminetetraacetic acid (EDTA) . This suspension was subjected to glass-teflon, followed by glass-glass (Dounce) homogenization. The homogenate was diluted to 10 volumes and centrifuged at 180xg for 10 minutes. In some preparations, the resulting pellet was re-homogenized i n a glass-glass homogenizer and centrifuged. The pooled supernatant was filtered through four layers of cheese cloth and centrifuged at l,000xg for 10 minutes. The second supernatant was again filtered through cheese cloth and centrifuged at 1 5 0 , 0 0 0 x £ for 30 minutes. The pellet was suspended i n the same buffer plus 2 m M E D T A , and layered on a discontinous sucrose density gradient consisting of 27%, 35% and 38% (w/w) sucrose layers. The tubes were centrifuged for 3 hours at 25,000 rpm i n an SW28 swinging-bucket rotor (Beckman). The 27/35% interface, containing the plasma membranes, was collected and washed in sucrose-free and EDTA-free buffer. The final pellet was suspended i n EDTA-free homogenization medium using a small glass-teflon homogenizer. The resulting plasma membrane vesicles were quick frozen and stored at - 8 0 ° C until use. Purification of C a 2 + - A T P a s e from Pancreatic Acinar Plasma Membranes C a 2 + - A T P a s e from pancreatic acinar plasma membranes was purified by the method of Bridges and Katz (1986). The entire procedure was performed at 4 ° C . The pelleted plasma membranes were solubilized (unless otherwise stated) 42 Materials and Methods Re-homogenize IX ^ -r PANCREATIC ACINI Homogenize with glass-teflon Homogenize with glass-glass Dilute to 1:10 P2 DILUTE HOMOGENATE 180xgfor 10 min SUPERNATANT SI l.OOOxgfor 10 min SUPERNATANT S2 150,000xgfor30 min PELLET P3 | Resuspend and layer on discontinuous sucrose gradient W 25,000 RPM for 3 lv in SW 28 rotor SUCROSE INTERFACES Collect separately and Wash WASHED SUCROSE INTERFACES 10/27%, 27/35%, 35/38% and PELLET P4 F i g u r e 4. Flow-chart for the preparation of guinea-pig pancreatic acinar plasma membranes. Details are given i n the text. 43 Materials and Methods in a medium containing 10 m M H E P E S , p H 7.4, 0.45% Triton X-100, 0.05% Tween 20, 300 m M KC1, 1 m M M g C l 2 , 100 u M C a C l 2 and 2 m M dithiothreitol. The solubilized membranes were centrifuged at 100,000xg for 30 minutes, and the supernatant was added to a CaM-agarose column pre-equilibrated with the solubilization buffer. Following binding, the column was washed with 25-100 bed volumes of Triton X-100-free solubilization buffer, and the bound protein was eluted with Ca 2 + - free , E D T A (2 mM)-containing washing buffer. The chelator i n the eluent was "neutralized" by 2 m M C a C l 2 . The enzyme was either assayed fresh or quick frozen for storage at - 8 0 ° C . Measurement of Calcium Uptake Activity ATP-dependent Ca 2 + - transport into guinea-pig pancreatic plasma membrane vesicles was measured at 3 7 ° C i n 40 m M K - H E P E S , p H 7.4, containing 110 m M KC1, 5 m M M g C l 2 , 5 m M N a N 3 and 5 m M A T P . The plasma membrane vesicles were preincubated for 6.5 minutes with the medium. The reaction was started by the addition of an appropriate C a 2 + solution containing 130 | i M E G T A and varying concentrations of CaCl2 (containing 4 5 C a C l 2 i 200,000 dpm/sample) to give the desired free C a 2 + concentrations. Free C a 2 + concentrations were determined using a Fortran program as described below. C a 2 + uptake was terminated after 5 minutes by filtering an aliquot of the reaction mixture through 0.45 (iM Millipore niters (HA 45, M i l l i p o r e ® Co.). The filters were washed with 20 ml of buffer containing 40 m M H E P E S , p H 7.4 and 0.25 M sucrose. The washed filters were dried, placed i n the liquid scintillation fluor (Unisolve 1®) and counted in a T R I - C A R B 4530 scintillation counter (Canberra-Packard Canada Ltd.). The rate of Ca^+ transport was determined using the following equation: 44 Materials and Methods (S.C. - B.G.) x D . F . x C a 4 5 C a 2 + - u p t a k e = (T.C. - B.G.) x P x T where: S .C. = 4 5 C a 2 + counts (dpm) i n the sample B . G . = counts (dpm) obtained from scintillant alone D . F . = correction for incubation volume sampled (=1.21) C a = nanomoles of total CaCl2 per tube T . C . = total radioactivity added to each sample (dpm) P = mg protein per sample T = reaction time i n minutes Assay of C a 2 + - A T P a s e Activity C a 2 + - A T P a s e activity was measured by a modification of the method of Blostein (1970). Unless otherwise stated, plasma membrane vesicles (50 Lig/ml) were preincubated for 3 minutes at 3 7 ° C i n a medium containing 40 m M H E P E S , p H 7.4, 130 u M E G T A , 5 m M N a N 3 , 0.1 m M ouabain, 500 u M M g C l 2 and different amounts of C a C l 2 to give the desired free C a 2 + concentrations as determined by a Fortran program described below. The reaction was started, in a final reaction volume of 0.2 ml, by the addition of 500 LiM A T P (containing y-3 2 P - A T P ; 2 x 10 5 dpm per sample) and terminated, after 30 minutes (except for time-course studies), with ice-cold " T C A stop solution" (consisting of 5% [w/v] trichloroacetic acid, 5 m M N a 2 A T P and 2 m M K H 2 P 0 4 ) . Unhydrolyzed A T P was removed from the reaction mixture by adsorption with a suspension of activated charcoal (0.15 g/ml i n 5% [w/v] T C A ) . Samples were shaken with the charcoal slurry for 5 minutes at room temperature in an Eppendorf Shaker (Brinkman Instruments), then centrifuged at 16,000xg for 5 minutes i n an Eppendorf microcentrifuge. A n aliquot of the supernatant was analyzed for liberated 3 2 P j , 45 Materials and Methods by liquid scintillation counting and used to calculate enzymatic A T P hydrolysis. Ca 2 + -dependent ATPase activity was calculated by subtracting the "basal" activity (in the absence of C a 2 + ) from the "total" activity (in the presence of In addition, a modification of the colourimetric assay of Raess & Vincenzi (1980) was used to study the substrate specificity of hydrolytic activity. The reaction was carried out under the same conditions as above, except 1.0 m M A T P or alternative substrates were used i n a final reaction volume of 0.4 ml. The reaction was stopped with 0.2 ml of 10% (w/v) sodium dodecyl sulphate (SDS). After the addition of 0.2 ml of 9% (w/v) ascorbic acid, followed by 0.2 ml of ammonium molybdate solution (1.25% [w/v] ammonium molybdate, 6.5% [v/v] H 2 S 0 4 ) , the absorbance of the samples was read at 660 nm. The phosphate concentration was extrapolated from standard curves performed for each experiment. ATPase activity, expressed in nanomoles per mg protein per minute, was calculated as follows: For radiometric assay: where: S .C. = 3 2 P j counts (cpm) in the sample B . G . = counts (cpm) obtained from scintillant alone A T P = nanomoles A T P present in each sample tube D . F . = dilution factor C a 2 + ) . (S.C. - B.G.) x A T P x D . F . ATPase Activity = T . C . x P x T reaction volume + stop solution + charcoal volume volume counted T . C . total radioactivity added to each sample (cpm) mg protein per sample reaction time i n minutes (=30) P T 46 Materials and Methods For colourimetric assay: (S.C. - B.C.) x V ATPase Activity = P x T where: S.C. = phosphate concentration in the sample as obtained from the standard curve (nmoles/ml) B.C. = phosphate concentration of the blanks V = reaction volume (0.4 ml) P = mg protein in the sample T = reaction time in minutes (=30) Assay of N a + / K + - A T P a s e Activity The Na+/K+-ATPase assay was similar to the Ca2+-ATPase assay. The assay medium consisted of 40 mM HEPES, pH 7.4, 100 uM EGTA, 500 uM MgCl 2, 20 mM KCL, 100 mM NaCl and 500 uM Tris-ATP (containing y-3 2P-ATP; 2 x 105 dpm per sample). The assay procedure was the same as Ca^+-ATPase. Na+/K+-ATPase was taken as the ATPase activity that could be inhibited by 1.5 mM ouabain. Determination of Endogenous Calmodulin The concentration of plasma membrane preparations was adjusted to approximately 1 mg/ml protein with 10 mM HEPES, pH 7.4 solution, containing 0.2 mM EDTA. The suspension was incubated for 5 minutes at 95°C and then centrifuged at 40,000xg for 30 minutes. The supernatant was collected, and the chelator "neutralized" with CaCl2 (0.2 mM final). The resulting extract was dialyzed against 10 mM NH 4 HC0 3 , pH 7.0 for 48 hours in the cold room. The 47 Materials and Methods extract was concentrated against crystalline polyethylene glycol. Concentrated samples were quick frozen and stored at - 8 0 ° C until use. The extracts were assayed for the presence of C a M using erythrocyte plasma membrane C a 2 + -ATPase , an enzyme known to be stimulated by C a M (Eibschutz et al., 1984). The CaM-depleted erythrocyte plasma membranes were a generous gift from Dr. Roufogalis's laboratory. These membranes were prepared using the method described by Wang et al. (1988). The assay procedure was similar to the ATPase assay described above. Phosphorylation of Guinea-pig Acinar Plasma Membranes Formation of the Ca 2 + -dependent phosphorylated intermediate of C a 2 + -ATPase was studied under conditions similar to those utilized in the ATPase assay described above. The phosphorylation medium consisted of 40 m M H E P E S , p H 7.4, 2 m M E D T A or 2 m M C a C l 2 and 2 u M A T P (containing y - 3 2 P -A T P ; 4 x 10 6 dpm per sample). The membrane preparation and the medium were preincubated separately at 1 0 ° C for 10 minutes. The reaction was started by the addition of plasma membranes to the medium. After 15 seconds at 1 0 ° C , the reaction was terminated with ice-cold 15% (w/v) T C A . Suspensions were mixed and centrifuged at l,500xg for 10 minutes at 4 ° C . Pellets were resuspended i n 0.5 ml of either 0.6 M hydroxylamine/0.8 M sodium acetate, p H 5.2 or 0.6 M NaCl/0.8 M sodium acetate, p H 5.2 (control). After a 10 minute incubation at room temperature, 1 ml of 15% (w/v) T C A was added to the samples, and the membrane protein was pelleted by centrifugation (10 minutes at l,500xg). The pellet was resuspended i n sample buffer and applied to polyacrylamide gels, as described below. 48 Materials and Methods Polyacrylamide Gel Electrophoresis and Autoradiography Sodium Dodecyl Sulphate Polyacrylamide gels Polyacrylamide slab gels (5-20%) were cast by the method of Laemmli and Favre (1973). The "separating" gel consisted of a 5-20% gradient of acrylamide-bisacrylamide mixture (30:0.8, w/w), containing 375 m M T r i s - C l buffer, p H 8.8, 0.1% SDS, 1.35-5.75% glycerol, 0.15 mg/ml ammonium persulphate (AP) and 0.03% tetraethyl-methylenediamine ( T E M E D ) . The "stacking" gel consisted of 5% acrylamide-bisacrylamide mixture, 315 m M Tri s -Cl , p H 6.8, 0.1% SDS, 0.4 mg/ml A P and 0.136% T E M E D . The samples were boiled for 4 minutes i n sample buffer (62.5 m M Tri s -Cl , p H 6.8 2% [w/v] SDS , 0.5 M 2-mercaptoethanol, 10% [v/v] glycerol and a small amount of Bromophenol Blue) and applied to individual wells. The gels were run at room temperature for approximately 1 hour under constant current of 25 mA, followed by 1.5-2 hours at 50 m A (Biorad Model 1000/500 Power Supply). The running buffer contained 25 m M Tri s -Cl , p H 8.3. The protein standards used for estimation of molecular weight (in kilodaltons) were: myosin (200), [3-galactosidase (116.25), phosphorylase b (92.5), bovine serum albumin (66.2), ovalbumin (45), bovine carbonic anhydrase (31), soybean trypsin inhibitor (21.5) and lysozyme (14.4). The gels were stained with silver stain to visualize the proteins (see below). Acid gels For electrophoresis of phosphorylated samples, the acid-gel system of Lichtner and Wolf (1979) was used. Polyacrylamide gels were prepared as above except T r i s - C l was substituted by 3-(N-Morpholino)propanesulfonic acid (Mops) 49 Materials and Methods and l i thium dodecyl sulphate (LiDS) replaced S D S in the gels. The p H of the "separating" gel was 7.0 and that of "stacking" gel was 6.5. Phosphorylated proteins were precipitated with 15% (w/v) T C A and centrifuged at l,500xg. Pellets were resuspended i n 25 ul of sample buffer (50 m M T r i s - P 0 4 , 2.5% L i D S , 0.12 M sucrose, 0.5 M 2-mercaptoethanol and 0.01% (w/v) Bromophenol Blue) and neutralized with 2 M Mops buffer. After incubation at 3 7 ° C for 4 minutes, the samples were applied to individual wells and gels were r u n as above, but at 4 ° C . The running buffer contained 20 m M Mops, p H 6.5 and 0.2% L i D S . Following electrophoresis, gels were stained with Coomassie Blue to visualize the proteins. Staining, Destaining and Drying Silver Staining When working with small amounts of proteins, the silver staining method of Morrissey (1981) was used due to its high sensitivity. Glass trays were used for staining and gels were handled only with gloves to avoid transferring fingerprints. A l l solutions were made fresh immediately prior to each step. The gels were fixed i n a mixture of 50% (v/v) methanol and 10% (v/v) acetic acid for 20 minutes. After a 20 minute incubation i n 5% (v/v) methanol and 7% (v/v) acetic acid mixture, the gels were transferred to 10% (v/v) glutaraldehyde for 30 minutes. The gels were washed i n deionized distilled water for at least 2 hours and then transferred to 5 Lig/ml dithiothreitol solution for a 20 minute incubation. After discarding the dithiothreitol, the gels were stained with 0.1% (w/v) silver nitrate for 20 minutes. Excess silver nitrate was removed by rinsing the gels once i n deionized distilled water. Superficial silver nitrate was removed by rinsing twice i n small volumes of developer (0.0185% [v/v] formaldehyde, 3% 50 Materials and Methods [w/v] N a C 0 3 ) . The gels were gently shaken i n fresh developer until all standards were visible. The developing process was stopped with the rapid addition of 2.3 M citric acid which was prepared before the addition of developer. After a 10 minute incubation, the gels were washed and stored i n deionized distilled water until drying. Coomassie Blue Staining The gels were stained i n a mixture containing methanol, acetic acid, water (5:1:5) and 0.25% (w/v) Coomassie Bril l iant Blue R-250 for 30 minutes at room temperature. The gels were then destained in a mixture of methanol, acetic acid and water (5:1:5), for one hour with 3 changes, and then in methanol:acetic acid:water (4:1:15) until the background became clear. Drying The gels were dried i n a sandwich of clear BioGelWrap sheets (BioDesign Inc., New York) i n a Plexiglass frame at room temperature. In some cases, to speed up the drying process, the frame was placed i n a fume hood or under a lamp. Autoradiography For autoradiography, the gels were dried immediately and exposed to X-ray film (Kodak X-Omat AR) using an intensifying screen (Cronex Lightning Plus, DuPont) for 2-7 days at - 8 0 ° C . The films were developed using Kodak G B X developer and replenisher and fixed i n Kodak G B X fixer and replenisher to visualize the phosphorylated proteins. 51 Materials and Methods Protein Assay The protein concentrations of membrane preparations were determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. A full standard curve (0-100 Lig protein) was produced for each assay, and the protein concentration was determined by extrapolating from the standard curve. Determination of Free Calcium Concentration Free calcium concentrations were determined using the Fortran program "CATIONS" written by Goldstein (1979). Association constants for cations and ligands were obtained from Martell and Smith (1979, 1982) and were corrected for ionic strength, pH and temperature according to the methods described by these authors. For the Ca 2 + transport assay, the corrected log association constants of chelating ligands (in order of first to fourth proton association) used were: 9.353, 8.733, 2.780 and 2.120 for EGTA; 12.246, 6.184, 3.650 and 2.540 for trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA); 6.664, 4.075, 0.0 and 0.0 for ATP. Association constants for unprotonated and monoprotonated cation-ligand complexes (in order) for the Ca 2 + transport assay were: 5.414 and 3.802 for Mg-EGTA; 11.225 and 0.0 for Mg-CDTA; 4.188 and 2.194 for Mg-ATP; 10.639 and 5.196 for Ca-EGTA; 13.049 and 0.923 for Ca-CDTA; 3.742 and 1.888 for Ca-ATP. The corrected log association constants of chelating ligands (in order of first to fourth proton association) used for the Ca2+-ATPase assay were: 9.323, 8.703, 2.750 and 2.090 for EGTA; 6.634, 4.045, 0.0 and 0.0 for ATP. Association constants for unprotonated and monoprotonated cation-ligand complexes for the Ca2+-ATPase assay were the same as above. 52 Materials and Methods Statistical Analysis Student's unpaired t-test was used to compare two means, as required. The null hypothesis was rejected only i f the two means were different at the 0.05 significance level. 53 RESULTS THE PLASMA MEMBRANE PREPARATION The viability of isolated acinar cells, as determined by trypan blue exclusion studies, was consistently 90% or higher. There was minimal bacterial contamination. Figure 5 shows the recovery data for protein, C a 2 + - A T P a s e and N a + / K + - A T P a s e activities in various fractions following homogenization and differential centrifugation. Almost 30% protein was lost in the first pellet, Pj^ (the 180xg pellet). This pellet also accounted for more than 50% of the N a + / K + -ATPase activity and 27% of the C a 2 + - A T P a s e activity. In later experiments, the pellet Pi was re-homogenized and centrifuged to recover some of this lost activity. The supernatants were pooled for the next step. Very little activity was lost i n the second pellet, P 2 (the l , 0 0 0 x £ pellet). The recovery of protein, C a 2 + -ATPase activity and N a + / K + - A T P a s e activity i n the P 3 fraction (the 150,000xg fraction) was 19%, 23% and 35%, respectively. After sucrose density gradient centrifugation, a large fraction of protein (7%) and the two ATPase activities (9% C a 2 + - A T P a s e activity and 5% Na+/K+-ATPase activity) was lost in the final pellet, P 4 . However, significant amounts of protein (1.3%), C a 2 + - A T P a s e activity (2.6%) and N a + / K + - A T P a s e activity (6.5%), were recovered i n the 27/35% sucrose interface. The other two sucrose interfaces contained very little protein or marker activities. Compared to the homogenate, P 3 (the fraction used for the source of plasma membranes) showed a 2-fold enrichment of ouabain-sensitive N a + / K + -ATPase activity, the basolateral plasma membrane marker, while ( C a 2 + + M g 2 + ) -ATPase activity was not enriched (Table II). However, material collected from the 27/35% interface following sucrose density gradient centrifugation was found to be enriched 17-fold i n Na+/K + -ATPase and 3.4-fold i n ( C a 2 + + M g 2 + ) -54 Results Figure 5. Recovery of protein, C a 2 + - A T P a s e activity and N a + / K + -ATPase activity in different centrifugation fractions of guinea-pig pancreatic acinar plasma membrane preparations. The results represent the mean + S . E . M . for three experiments, unless otherwise indicated by a number above the bar. D H - dilute homogenate, P : - 180xg pellet, P 2 - l , 0 0 0 x £ pellet, P 3 - 150,000xg pellet, L 1 ? L 2 , and L 3 - the 10/27%, 27/35% and 35/38% interfaces from the sucrose density gradient, P 4 - pelleted fraction from the sucrose density gradient. - Protein, - C a 2 + - A T P a s e , 7/777\ - N a + / K + - A T P a s e . 55 Results Table II. A comparison of the specific activities (SA) at different stages of the guinea-pig pancreatic acinar plasma membrane preparation. The results represent means of three experiments except where indicated within parentheses. Fraction ( C a 2 + + M g 2 + ) - A T P a s e S A Enrichment N a + / K + - A T P a s e S A Enrichment Dilute Homogenate 9.58 1.0 3.40 1.0 1 5 0 , 0 0 0 x £ Fraction 9.53 1.0 7.13 2.1 (P3> Plasma Membranes 32.33 3.4 58.68 17.2 (27/35% interface) The activities were measured as described i n the M E T H O D S and are expressed i n nanomoles A T P hydrolyzed/mg/min. The M g 2 + concentration was 0.5 m M and the free C a 2 + was 1.0 uM i n the assay for ( C a 2 + + M g 2 + ) - A T P a s e activity. N a + / K + - A T P a s e was taken as the ATPase activity that could be inhibited by 1.5 m M ouabain. 56 Results ATPase activity (Table II). This fraction was designated the plasma membrane-enriched fraction. Sodium azide (5 mM) was included i n all reaction media to inhibit any residual mitochondrial ATPase which may have been present i n this fraction. OPTIMIZATION OF ASSAY CONDITIONS To determine optimal conditions for assay of ATPase and Ca 2 + - transport activities, the effects of varying membrane protein concentration within reaction media were investigated. The 4 5 C a 2 + - u p t a k e studies showed a near linear increase with protein concentrations up to 180 ug/ml (Fig. 6). A linear increase was also observed i n the ATPase activity with protein concentrations up to 190 ug/ml (Fig. 7). Subsequent studies were, therefore, carried out at membrane protein concentrations of 15-50 ug/ml for the radiometric ATPase assay and 100-125 ug/ml for the colourimetric ATPase assay, while approximately 120 ug/ml protein was used i n the Ca 2 + - transport experiments. In other experiments, the time-course for the ATPase activity was studied. The Ca 2 + -dependent A T P hydrolysis was found to be approximately linear for at least 30 minutes (Fig. 8). A 30 minute reaction time was selected for all subsequent ATPase determinations. In our experiments, guinea-pig pancreatic acinar plasma membrane preparations typically showed maximal Ca 2 + -dependent ATPase activities ranging between 5 and 15 nmoles/mg/min. Freeze-thaw experiments were conducted i n an attempt to expose possible "latent" ATPase sites within the plasma membranes. Figure 9 shows the effect of freeze-thawing on C a 2 + -dependent, Mg 2 + -dependent and Total ATPase activities. Freeze-thawing the plasma membranes up to 5 times failed to produce a measurable increase in any of the three ATPase activities (Fig. 9). This suggested that the enzymatic sites 57 Results Figure 6. Effect of varying protein concentration on 4 5 Ca 2 + -uptake in guinea-pig pancreatic acinar plasma membranes. The experiments were done in the presence of 1.0 uM free C a 2 + and 5 mM added M g 2 + . Other reaction conditions are described in the M E T H O D S . The results represent the mean of two separate experiments. 58 Figure 7. Effect of varying protein concentration on C a 2 + -dependent ATPase activity in guinea-pig pancreatic acinar plasma membranes. The experiments were carried out i n the presence of 1.0 u M free C a 2 + and i n the absence of added M g 2 + . Other reaction conditions are described in the M E T H O D S . The results represent the mean of two separate experiments. 59 Results £ G • «-H . | — | > CD ' r l •+-> 0 2 < a £ so crj E DH \ TO. <; CD 1 rt + CM cd 10 20 30 40 50 React ion T i m e , m i n u t e d 60 Figure 8. Time-course of Ca2+-ATPase activity. The experiments were done in the presence of 1.0 |iM free C a 2 + and zero added Mg 2 + . The results represent the means of two to three separate experiments. 60 F i g u r e 9. Effect of freeze-thawing on Ca 2 + -dependent, M g 2 + -dependent and total ATPase activity. The results represent the mean ± S . E . M . for three experiments. - 1 freeze-thaw, 0 freeze-thaw, 2 freeze-thaw, V/y/y - 3 freeze-thaw, 4 freeze-thaw. 61 Results were already maximally exposed to the medium prior to freeze-thawing. Freeze-thawing the plasma membranes 2 or 4 times actually tended to cause inhibition of Mg 2 + -dependent and total ATPase activities. The Mg 2 + -dependent activity was also significantly inhibited after 3 freeze-thawings (Fig. 9). CHARACTERIZATION OF Ca2+ TRANSPORT Effects of C a 2 + and Calmodulin The ATP-dependent transport of 4 5 C a 2 + by guinea-pig pancreatic acinar plasma membrane vesicles was stimulated by C a 2 + i n a concentration-dependent manner (Fig. 10). Maximal uptake was achieved at approximately 1.0 u M free C a 2 + and remained at this level up to 6 u M C a 2 + . Double-reciprocal analysis of these data produced the following kinetic parameters: K Q & = 0.04 + 0.01 u M (n=5) and V m a x = 0.83 ± 0.09 nmoles/mg/min (n=5). Calmodulin (6 ug/ml) produced no significant effect on K C a (0.05 ± 0.01 u M , n=5) nor on V m a x (0.86 ± 0.14 nmoles/mg/min, n=5). Effect of M g 2 + Addition of M g 2 + to the reaction medium produced a concentration-dependent stimulation of ATP-dependent Ca 2 + - transport (Fig. 11). The rate of Ca 2 + - transport reached a maximum at approximately 2 m M total M g 2 + . The half-maximal rate of Ca 2 + -uptake was produced at 0.3 ± 0.04 m M total M g 2 + (4.3 ± 0.5 u M free M g 2 + ) . Approximately 25% of the maximal Ca 2 + -uptake activity was expressed i n the absence of added M g 2 + (Fig. 11). This level of C a 2 + transport activity may have resulted from the presence of endogenous M g 2 + . Therefore, experiments using C D T A , a chelator which is relatively more specific for M g 2 + than E G T A or E D T A , were carried out to examine this possibility. In 62 Results 0 . 0 0 . 5 1.0 1.5 2 . 0 [ C a 8 + ] f r e e , fJ-U 2.5 3 . 0 Figure 10. C a 2 + activation of 4 5 C a 2 + uptake by guinea-pig pancreatic acinar plasma membrane vesicles. ATP-dependent calcium transport i n the absence ( O ) and presence ( • ) of 6 Lig/ml calmodulin was measured as described i n the M E T H O D S . The experiments were carried out i n the presence of 5 m M added M g 2 + . The results represent the mean ± S . E . M . for five experiments. 63 Results Figure 11. Effect of M g 2 + on 4 5 C a 2 + - u p t a k e i n guinea-pig pancreatic acinar plasma membrane vesicles. Uptake of 4 5 C a 2 + was measured as described i n the M E T H O D S . The free C a 2 + concentration was maintained at 1.5 fiM. The results represent the mean ± S . E . M . for three experiments. 64 Results the absence of added M g 2 + , C D T A produced substantial inhibition of 4 5 C a 2 + transport (Fig. 12). The inhibition appeared to be biphasic: up to 30% of 4 5 C a 2 + -uptake was inhibited by as little as 0.1 m M C D T A , while roughly 60% could be inhibited by 0.8 m M C D T A . It was not feasible to use C D T A concentrations higher than 0.8 m M and still accurately determine free C a 2 + concentrations. Substrate Specificity Figure 13 shows the rate of 4 5 C a 2 + transport by guinea-pig pancreatic acinar plasma membrane vesicles i n the presence of different nucleotide substrates. The transport process appeared to be highly specific for A T P . Other substrates supported very little C a 2 + transport, the highest being 16% of maximum produced by cytosine 5'-triphosphate (CTP). As expected, p-nitrophenyl phosphate (pNPP) was unable to support any C a 2 + transport. CHARACTERIZATION OF Ca2+-ATPASE ACTIVITY To make a direct comparison, ATPase activity was studied i n the same membrane fraction as used to measure C a 2 + transport. As apparent from the results below, the ATPase activity showed somewhat different properties than the C a 2 + transport activity. Effect of M g 2 + In our studies, M g 2 + produced a concentration-dependent stimulation of "Basal" A T P hydrolysis (i.e. the activity measured in the absence of added C a 2 + ) (Fig. 14). The activity reached a maximum at approximately 500 L I M M g C l 2 . Figure 15 shows the effects of varying M g 2 + concentration on the C a 2 + activation of Ca 2 + -dependent ATPase activity. C a 2 + was able to stimulate the latter activity i n the complete absence of added M g 2 + . Increasing M g 2 + 65 Results 0.0 0.2 0.4 0.6 0.8 [ O D T A ] , m M Figure 12 . Effect of CDTA on 4 5 C a 2 + uptake in guinea-pig pancreatic acinar plasma membrane vesicles. The transport of C a 2 + was measured in the presence of 1.5 (J.M free C a 2 + and in the absence of added Mg 2 + . One hundred percent activity corresponded to 0.432 nmoles/mg/min. The results represent the mean ± S.E.M. for five experiments. 66 Results F i g u r e 13 4 5 C a 2 + - u p t a k e i n the presence of different nucleotide substrates in guinea-pig pancreatic acinar plasma membrane vesicles. Ca 2 + -uptake was measured at 1.5 u M free C a 2 + and i n the presence of the substrates indicated. The results represent the mean ± S . E . M . for three experiments. 67 Figure 14. Effect of M g 2 + on Basal ATPase activity. There was no added C a 2 + present. The results represent the mean ± S.E.M. for three experiments. 68 Results Figure 15. Effect of calcium on Ca 2 + -dependent ATPase activity i n the absence and presence of various magnesium concentrations, a) The ATPase activity was assayed in the absence ( O ), or presence of 1.0 L I M ( • ) , 10 |iM ( A ), 50 u M ( • ) or 100 |iM ( • ) M g C l 2 . b) The ATPase activity was assayed i n the presence of 500 L I M ( • ) or 1.0 m M ( • ) M g C l 2 . The C a 2 + -dependent activity was calculated by subtracting the basal activity (measured in the absence of C a 2 + and the presence of indicated M g 2 + ) from the total activity (measured i n the presence of both C a 2 + and M g 2 + ) . The results represent the mean + S . E . M . for three experiments. 69 Results concentrations appeared to inhibit Ca 2 + -dependent A T P hydrolysis i n a concentration-dependent manner (Fig. 15a). However, at high M g 2 + concentrations (0.5 and 1.0 mM), this inhibition was lost and Ca 2 + -dependent ATPase activity increased, peaking at 1 u M free C a 2 + . A t C a 2 + concentrations higher than 1 u M , the Ca 2 + -dependent activity declined (Fig. 15b). These data indicate the possible presence of two different Ca 2 + -dependent A T P hydrolytic activities - a low affinity C a 2 + ( o r Mg 2 + ) -st imulated ATPase and a high affinity Mg 2 + -dependent, Ca 2 + - s t imulated ATPase. Effect of C a 2 + , Calmodulin and K + Further investigation of the high affinity Mg 2 + -dependent, C a 2 + -stimulated ATPase (in the presence of 500 u M M g C l 2 ) showed that increasing C a 2 + concentrations stimulated A T P hydrolysis i n a concentration-dependent manner (Fig. 16). Kinetic analysis indicated a V m a x of 6.04 + 0.78 nmoles/mg/min and a K r j a of 0.076 ± 0.022 uM. Figure 16 also shows the effect of 6 ug/ml C a M on C a 2 + - A T P a s e activity. As apparent from the graphs, C a M did not significantly stimulate this activity. The calculated V m a x was 7.13 + 0.99 nmoles/mg/min, while the K C a was 0.072 + 0.018 u M . In other experiments, the effect of potassium on C a 2 + - A T P a s e was studied. Various potassium concentrations tested (1-300 mM) were unable to affect this Ca 2 + -dependent A T P hydrolysis (Fig. 17 shows partial data). Formation of the Phosphorylated Intermediate of the Calcium Pump Autoradiograms of phosphorylation experiments using plasma membranes from guinea-pig pancreatic acini showed the formation of a C a 2 + -dependent phosphoprotein with an apparent molecular weight of approximately 100,000 daltons (Fig. 18B, Lane 1). This phosphoprotein could be completely 70 Figure 16. Effect of C a 2 + on Ca 2 + -dependent ATPase activity in the absence ( O ) or presence ( • ) of 6 Lig/ml calmodulin. The experiments were carried out as described i n the M E T H O D S . The results represent the mean + S . E . M . for five experiments. 71 Figure 17. Effect of calcium on Ca 2 + -dependent ATPase activity i n the absence and presence of various potassium concentrations. The potassium concentrations used were: O - 0, • - 100 m M , A - 300 m M . The results represent the average of two experiments. 72 Results k D a 200-116-92.5-66.2-45-31-k D a Figure 18. Autoradiogram of phosphorylated proteins in guinea-pig pancreatic acinar plasma membranes. The phosphorylation was carried out in the presence of 2 mM Ca 2 + . A - Mg2+-dependent phosphorylation; Lane 1 - No M g 2 + added, Lane 2 - 0.5 mM MgCl 2, Lane 3 - 0.5 mM MgCl 2 + 0.6 M hydroxylamine. B - Ca 2 + -dependent phosphorylation in the presence of 0.5 mM MgCl 2; Lane 1 - No hydroxylamine, Lane 2 - 0.6 M hydroxylamine. Molecular weight markers (in kilodaltons) are shown on the left. 73 Results hydrolyzed by 0.6 M hydroxylamine (Fig. 18B, Lane 2). The presence of a second phosphoprotein ( M r approx. 50,000) was also detected. However, the phosphorylation of this latter protein appeared to be Mg 2 + -dependent and was not sensitive to hydroxylamine treatment (Fig. 18A,) suggesting the formation of an ester phosphate bond, instead of a typical ATPase acyl phosphate bond. No phosphorylation was observed in the absence of C a 2 + and M g 2 + . Substrate Specificity A colourimetric assay was used in a systematic study of the hydrolysis of different nucleotide phosphate substrates by Guinea-pig pancreatic acinar plasma membrane preparations. The results showed that i n addition to A T P , membranes were able to hydrolyze G T P , C T P , ITP and A D P (Fig. 19). Unlike Ca 2 + - transport , the nucleotide triphosphatase activity showed little specificity for A T P . G T P appeared to be the favoured substrate; the Ca 2 + -dependent hydrolysis of G T P , ITP and C T P (measured i n the presence of 5 m M M g 2 + ) was 172%, 74% and 48%, respectively, compared with A T P (Fig. 19a). The hydrolysis of A D P was 20% compared to A T P , and the enzyme showed 18% C a 2 + -dependent cleavage of p N P P . Although A T P appeared to be the most favoured substrate for Ca 2 + -dependent hydrolysis in the absence of M g 2 + , the enzyme did not show complete substrate specificity; hydrolysis of ITP, C T P and G T P was 73%, 66% and 56%, respectively, compared to A T P (data not shown). Mg 2 + -dependent hydrolysis of G T P also exceeded that of other substrates used (Fig. 19b). A T P appeared to be the least favourable triphosphate substrate for the Mg 2 + -dependent activity while A D P or p N P P were poorly hydrolysed. 74 Results C 6 cm a \ 01 CD o £ -i-> • i-H > • •—I H - > CJ < o • i-H H - 5 o T J 12 r-a) ATP OTP OTP ITPPNPPADP 60 4 0 2 0 b) 1 1 1 i~Ti i n lll l l l l l l ATP OTP OTP ITPPNPPADP Figure 19. Substrate specificity of Ca 2 + -dependent and M g 2 + -dependent hydrolytic activities i n guinea-pig pancreatic acinar plasma membranes. Hydrolytic activity was determined . spectrophotometrically as described in the M E T H O D S , a) C a 2 + -dependent hydrolysis was determined i n the presence of 5 m M M g 2 + and by subtracting the basal activity (i.e. activity measured i n the absence of M g 2 + ) from the total activity measured i n the presence of 1 | i M free C a 2 + . b) The Mg 2 + -dependent hydrolysis was determined as above, but i n the presence of 5 m M M g 2 + and zero C a 2 + . The results represent the mean + S . E . M . for four experiments. 75 Results REGULATION OF Ca2+-TRANSPORT AND Ca2+-ATPase BY PROTEIN KINASES AND INOSITOL PHOSPHATES Regulation by Protein Kinase A As shown i n Figure 20 and Table III, 400 units/ml catalytic subunit of protein kinase A (C-subunit) stimulated Ca 2 + - transport into plasma membrane vesicles more than two-fold ( V m a x = 1.26 ± 0.29 nmoles/mg/min for controls; 2.50 ± 0.33 nmoles/mg/min for C-subunit stimulated, P < 0.05). The affinity for C a 2 + was unaffected (0.18 ± 0.07 | i M Vs . 0.17 ± 0.1 LIM ) . In other experiments, 300 units/ml C-subunit produced no statistically significant stimulation of C a 2 + -dependent ATPase activity except at 0.08 | i M C a 2 + (Fig. 21). The V m a x (control = 4.35 ± 0.94 and C-subunit = 5.18 ± 0.82) and KCa (control = 0.20 + 0.15 L I M and C-subunit = 0.05 + 0.01 | iM) were also unaffected. To determine whether stimulation by an endogenous protein kinase A contributed to the Ca 2 + - transport or C a 2 + - A T P a s e activity observed, the effects of a specific protein kinase A inhibitor, H A 1004, were studied. Under our experimental conditions H A 1004 failed to inhibit either Ca 2 + - transport (Table III) or C a 2 + - A T P a s e (Fig. 21) activities. The inhibitor also failed to affect the stimulatory effect of exogenously added C-subunit on C a 2 + - u p t a k e (Table III). Regulation by C a M As reported above, exogenous C a M had no effect on either Ca 2 + - transport (Fig. 10) or Ca 2 + -dependent ATPase activity (Fig. 16) in guinea-pig pancreatic acinar plasma membranes. To further understand the regulation of these activities, effects of three putative C a M inhibitors, trifluoperazine (TFP), compound 48/80 and C G S 9343B were investigated. 76 Results 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 [ d a ] , , / xM L J f r e e ^ Figure 20. Effect of the catalytic subunit of protein kinase A on C a 2 + activation of 4 5 C a 2 + uptake. The results represent the mean + S . E . M . for three experiments. O - Control, • - 400 units/ml C -subunit. 77 . Results Table III. Effect of protein kinase inhibitors on 4 5 C a 2 + uptake in plasma membrane vesicles. The transport of C a 2 + was measured i n the presence of 0.5 u M free C a 2 + and 5 m M M g 2 + . The results represent mean ± S . E . M . for three experiments except where indicated within parentheses. 4 5 C a 2 + - U p t a k e (nmoles/mg/min) Basal ATP-dependent Control 0.08 ± 0 . 0 1 0.35 ± 0 . 0 2 CGS9343B (10 LIM) 0.08 ± 0 . 0 1 0.32 ± 0 . 0 2 C G P 41 (0.5 uM) 0.08 ± 0 . 0 1 0.34 ± 0.04 HA1004 (10 |±M) 0.08 ± 0 . 0 1 0.34 ± 0.03 C-subunit (300U/ml) 0.08 ± 0.01(4) 0.88 ± 0 . 1 2 ( 4 ) * C-subunit + HA1004 0.08 ± 0 . 0 1 ( 4 ) 0.87 ± 0 . 1 0 ( 4 ) * significantly different from Control (P<0.01) 78 Results Figure 21. Effect of catalytic subunit of protein kinase A and the inhibitor H A 1004 on Ca 2 + -dependent ATPase activity. The M g 2 + concentration was 0.5 m M . The results represent the mean + S . E . M . for three experiments. O - Control, • - 300 units/ml C -subunit, • - 10 u M H A 1004. 79 Results Both T F P and compound 48/80 were found to inhibit 4 5 C a 2 + - u p t a k e activity i n a concentration-dependent manner (Figs. 22 and 23). The inhibitory effects were observed with as little as 10 | i M T F P (Fig. 22) and 10 Lig/ml compound 48/80 (Fig. 23). T F P completely inhibited C a 2 + transport activity at 100 L I M , while compound 48/80 produced almost complete inhibition at 1 mg/ml. The effects of these agents on C a 2 + - A T P a s e activity were somewhat more complex; T F P did not significantly inhibit the Ca 2 + -dependent ATPase activity at two different C a 2 + concentrations (Fig. 24). Inhibition of Ca 2 + -dependent ATPase activity by compound 48/80 was less consistent when tested at 3 different C a 2 + concentrations (Fig. 25). This activity was significantly inhibited by 1 | ig/ml of compound 48/80 at all C a 2 + concentrations. Ten Lig/ml did not affect the Ca 2 + -dependent ATPase, while 100 Lig/ml only inhibited it at 1.0 LIM free C a 2 + . It was very interesting to observe that compound 48/80 reproducibly stimulated "Basal" (Mg 2 + -ATPase ) and "Total" ATPase activity (the activity i n the presence of both C a 2 + and M g 2 + ) i n a concentration-dependent manner (Fig. 26). A supposedly more potent inhibitor of C a M , C G S 9343B (Norman et al., 1987), produced no inhibition of C a 2 + 1 u p t a k e (Table III) or Ca 2 + -dependent ATPase (Table IV) activities in our preparations. Determination of Endogenous CaM To determine whether guinea-pig pancreatic acinar plasma membrane preparations contained endogenous C a M , membranes were boiled i n a medium containing 0.2 m M E D T A and the extracts were then assayed for the presence of C a M . The indicator system used to detect C a M within guinea-pig pancreatic acinar plasma membrane extracts was erythrocyte plasma membrane C a 2 + -ATPase , an enzyme stimulated by C a M . Figure 27 shows the results from a 80 Results [Trifluoperazine], /J,M Figure 22. Effect of trifluoperazine on 4 5 C a 2 + uptake i n guinea-pig pancreatic acinar plasma membrane vesicles. The transport of C a 2 + was measured as described i n the M E T H O D S i n the presence of 1.5 [iM free C a 2 + and 5 m M M g 2 + . The results represent the mean ± S . E . M . of three experiments. • - Basal, • -ATP-dependent uptake. 81 Results [Compound 4 8 / 8 0 ] , M g / m l Figure 23. Effect of compound 48/80 on 4 5 C a 2 + uptake in guinea-pig pancreatic acinar plasma membrane vesicles. The transport of C a 2 + was measured as described in the METHODS in the presence of 1.5 |iM free C a 2 + and 5 mM Mg 2 + . The results represent the mean ± S.E.M. of three experiments. • - Basal, • -ATP-dependent uptake. 82 Figure 24. Effect of trifluoperazine (TFP) on Ca 2 + -dependent ATPase activity at different C a 2 + free concentrations. The M g 2 + concentration was 0.5 m M . The results represent the mean ± S . E . M . for four experiments. Y///X - Control, - 1.0 (iM T F P , - 10 u M T F P , 100 u M T F P , T F P . - 1 m M 83 Figure 25. Effect of compound 48/80 on Ca 2 + -dependent ATPase activity at different C a 2 + free concentrations. The M g 2 + concentration was 0.5 m M . The results represent the mean ± S . E . M . for four experiments. X^/Z/yX Control, ^ 1.0 Hg/ml pi - ioo compound 48/80, [^^| - 10 ug/ml compound 48/80, (ig/ml compound 48/80. * - significantly different from Control at P<0.005, ** - significantly different from Control at P<0.05. 84 Figure 26. Effect of compound 48/80 on total ATPase activity at different C a 2 + free concentrations and in the presence of 0.5 m M M g 2 + . The results represent the mean of four experiments. • - 0 C a 2 + (Basal activity), V - 0.03 u M C a 2 + , T - 0.1 p M C a 2 + , • - 1.0 u M C a 2 + . 85 Results Table IV. Effect of inhibitors C G S 9343B and C G P 41 251 on C a 2 + -dependent ATPase activity at different C a 2 + concentrations. The results represent mean ± S . E . M . of three experimens. C a 2 + - A T P a s e Activity (nmoles/mg/min) | i M C a 2 + Control C G S C G P 0.08 3.40 ± 1 . 0 6 4.11 + 1.25 4.11+ 1.11 0.30 3.56 ± 0 . 7 4 4.25 ± 1.13 4.01 ± 0 . 5 0 1.50 4.09 ± 1 . 1 6 4.90 ± 0 . 8 9 3.88 ± 0 . 7 1 86 Figure 27. Stimulation of erythrocyte C a 2 + - A T P a s e activity by exogenous C a M . The experiments were carried out i n the presence of 1.0 LIM free C a 2 + and 1.0 m M M g 2 + and in the absence ( • ) or presence ( O ) of 60 LIM trifluoperazine (TFP). Superimposed on these curves are resultant activities measured when erythrocyte membranes were treated with boiled guinea-pig pancreatic acinar plasma membrane extracts in the absence ( A ) and presence ( • ) of T F P . These data are results of a representative experiment. 87 Results representative experiment. Exogenous C a M produced a concentration-dependent stimulation of erythrocyte C a 2 + - A T P a s e activity. This red cell marker activity was also stimulated by guinea-pig pancreatic acinar plasma membrane extracts. Stimulation by both the exogenous C a M and plasma membrane extracts was blocked by 60 (iM T F P . Removal of Endogenous CaM Attempts were made to deplete the endogenous C a M so that possible stimulation by exogenous C a M could be determined and C a M affinity chromatography could be utilized for the purification of C a 2 + - A T P a s e . Hypertonic-hypotonic treatment i n the presence of E G T A , as described by Caroni and Carafoli (1981a), proved unsuccessful in removing endogenous C a M from plasma membranes (not shown). A promising strategy utilizing Sephadex G-75 column chromatography of detergent-solubilized plasma membrane proteins was also attempted for depletion of endogenous C a M . As shown in figure 28, proteins were eluted from the column i n 2 peaks. As expected, when column eluates were analyzed by S D S - P A G E , the first peak contained high molecular weight proteins, while the second peak represented smaller proteins close in size to C a M (not shown). Enzymatic assay of these fractions was precluded by the presence of high concentrations of Triton X-100 arising from the solubilization and equilibration media. Regulation by Protein Kinase C It has been suggested that P K C is involved i n the stimulation of C a 2 + efflux from pancreatic acinar cells (Muallem et al., 1988b). In our studies, 70 fig/ml purified P K C from rat brain and 1.0 u M of the phorbol ester T P A failed to stimulate Ca 2 + -uptake , in the absence or presence of 80 ug/ml 88 Results 0.05 r a d 0.04 o CO CV2 at 0.03 d) o C cd 0.02 o m JO 0.01 < 0.00 Fraction Number Figure 28. Protein profile (absorbance at 280 mm) of fractions eluted from a Sephadex G-75 chromatography column after applying solubilized guinea-pig pancreatic acinar plasma membranes. The elution medium was identical to the solubilization buffer described in METHODS. 89 Results phosphatidylserine (PS) (Fig. 29). PS alone also produced no significant effect on this activity (Fig. 29) or on the Ca2 +-ATPase activity (Table V). Both PKC and TPA were unable to affect the PS-stimulated Ca 2 +-ATPase activity. However, a combination of PS and TPA produced a significant stimulation of control C a 2 + -ATPase activity at both C a 2 + concentrations tested, whereas the PS and PKC combination produced stimulation only at 1.0 \iM. free C a 2 + . Another activator of PKC, l-stearoyl-2-arachidonoyl-sn-glycerol (SA-DG), failed to affect either activity in the presence of PS at low C a 2 + concentration (Fig. 30 and Table V), but it significantly stimulated Ca 2 +-ATPase at 1.0 | iM C a 2 + (Table V). CGP 41 251, a potent and selective inhibitor of PKC (Meyer et al., 1989), did not inhibit Ca2+-transport (Table III) or Ca2 +-ATPase (Table IV) activities. Regulation by Inositol Phosphates The effects of 1 | iM inositol 1,4,5-trisphosphate (IP3) and 5 \xM inositol 1,3,4,5-trisphosphate (IP4) were studied on Ca2 +-transport only. Figure 31 shows that neither the individual nor a combination of inositol phosphates produced a statistically significant effect on Ca2+-uptake. SOLUBILIZATION AND PURIFICATION OF Ca2+-ATPase The presence of endogenous CaM in our preparations and the previous demonstration of a 133 kDa CaM-binding protein in rat pancreatic acinar plasma membranes (Ansah et al., 1984) suggested that this protein may have a direct role in stimulating the Ca2 +-ATPase or C a 2 + transport activities of guinea-pig pancreatic acinar plasma membrane, in a manner similar to that seen in erythrocytes. The possibility of a direct interaction between CaM and the ATPase indicated that CaM-affinity chromatography might be utilized in an attempt to purify the enzyme. 90 Results Uptake Uptake Figure 29. Effect of Protein kinase C or 12-O-tetradecanoyl phorbol-13-acetate (TPA) on 4 5 C a 2 + uptake by guinea-pig pancreatic acinar plasma membrane vesicles. Experiments were carried out at 0.5 uM free Ca 2 + . The concentration of PS used was 80 uM/ml. The results represent the mean ± S.E.M. for three experiments..^^ - Control, - 70 ug/ml PK-C, -1.0 |iM TPA. Cntrl Blk - control basal activity, PS Blk - basal activity in the presence of phosphatidylserine, Cntrl Uptake - ATP-dependent uptake in the absence of phosphatidylserine, PS Uptake - ATP-dependent uptake in the presence of phosphatidylserine. 91 Results Table V. Effect of Protein kinase C, SA-DG and TPA on Ca 2 +-dependent ATPase activity of guinea-pig pancreatic acinar plasma membranes. The effects of PKC, SA-DG and TPA were studied in the presence of PS. The results represent mean + S.E.M. of three experiments. Ca2+-ATPase Activity (nmoles/mg/min) 0.1 |iM Ca 2 + 1.0 |iM Ca 2 + Control 2.73 ± 1.02 3.36 ±0.85 PS (80 |ig/ml) 4.98 ± 1.88 7.11 ±1.63 PK-C (70 Lig/ml) 6.59 ±2.68 8.21 ±2.02* SA-DG (8 Lig/ml) 4.70 ± 1.32 8.27 ±0.85** TPA (1.0 LIM) 9.34 ± 1.31* 13.38 ±3.51* *Significantly different from Control (P<0.05) **Significantly different from Control (P<0.01) 92 Results fi Bagal Uptake Figure 30. Effect of l-stearoyl-2-arachidonoyl-sn-glycerol (SA-DG) on 4 5 C a 2 + uptake. The results represent the mean ± S.E.M. for three experiments. Experiments were carried out in the presence of 0.5 uM free Ca 2 + and 80 ug/ml phosphatidylcholine. -Control, - 8 ug/ml SA-DG. 93 Results Figure 31. E f fec t of 5 u M I P 4 a n d 1 u M I P 3 on 4 5 C a 2 + - u p t a k e i n gu inea -p i g panc rea t i c ac i na r p l a s m a m e m b r a n e s . T h e resu l t s rep resen t the m e a n + S . E . M . for th ree exper imen ts . 94 Results In early studies, we were able to separate two major proteins from the solubilized guinea-pig pancreatic acinar plasma membranes using the C a M -afrinity column. However, it was discovered that most of the ATPase activity, comprising the greater part of the applied protein, did not bind to the column, but came out i n the void volume (Fig. 32). Further attempts to increase the protein binding to the column and optimize purification proved unsuccessful. However, careful analysis of the eluted fractions by S D S - P A G E and ATPase assay was carried out. The following is a brief summary of some of the strategies used and the results obtained. 1. Attempts at removing solubilized endogenous C a M by using Sephadex G-75 prior to applying the solubilizate to a CaM-agarose column failed to increase the binding of any proteins to the latter column. 2. A number of protease inhibitors, including Leupeptin, Pepstatin A , T P C K and P M S F were added to the buffers in an attempt to retard possible proteolysis of the C a M binding site of the enzyme by endogenous proteases. This strategy too was unproductive in increasing the protein binding to CaM-agarose or the ATPase activity i n the eluted fractions. 3. Excluding Asolectin (an acidic phospholipid-rich mixture) from buffers or substituting it with phosphatidylcholine did not appear to increase the binding of proteins or ATPase activity in fractions eluted from the C a M -affinity column. In another attempt to increase the yield of eluted proteins, cruder starting material ( P 3 instead of 27/35% sucrose interface) was used. This procedure resulted i n broader bands observed i n the polyacrylamide gels. However, it failed to produce any new bands or increase the ATPase activity in eluted fractions. 4. To overcome the problem of low binding to CaM-agarose, we tested Cibacron Blue 3GA-agarose and Reactive Red 120-agarose affinity columns. 95 Results V o l u m e After Load ing , m l Figure 32. ATPase activity and absorbance at 280 nm of fractions collected from CaM-agarose column following loading and washing. The proteins were solubilized using a method similar to that described by Caroni and Carafoli (1981a). The graph represents a typical experiment. 96 Results As seen with CaM-agarose, most of the protein and ATPase activity showed negligible retention by the column. However, we were able to specifically elute three major proteins from both columns using A T P in the elution buffer. Unfortunately, these proteins did not appear to represent the ATPase. 5. A n attempt to use gel filtration (using Ultrogel) to separate the proteins by molecular size was unsuccessful. A l l solubilized plasma membrane proteins appeared to migrate together in the micelles and were eluted in the void volume or shortly thereafter. 6. The use of higher KC1 concentration (0.5 M) and 0.6 M sucrose in the solubilization buffers, according to the method of Caroni and Carafoli (1981a), failed to increase binding. O n the other hand, lowering the KC1 concentration from 0.5 M to 0.1 M and compensating the resulting loss of ionic strength to some extent by increasing the buffer concentration to 100 m M from 20 m M H E P E S , p H 7.4 produced somewhat improved binding. However, the ATPase activity and the protein yield were too small to allow further characterization (Fig. 33). EXPERIMENTS WITH PLASMA MEMBRANES FROM HUMAN PANCREAS The human pancreatic tissue was obtained from the Pacific Organ Retrieval for Transplantation program. The experiments depended on the availability of tissue. Hence, only a few experiments were possible. It was found that the tissue was very difficult to digest with collagenase to obtain the acini. Preliminary experiments showed that plasma membranes prepared from human pancreatic acini were somewhat more buoyant than those from guinea-pigs. This was illustrated by an enrichment of the plasma membrane marker N a + / K + -97 Results Volume After Load ing , m l Figure 33. ATPase activity and absorbance at 280 nm of fractions eluted from the CaM-agarose affinity column. The solubilization medium was modified by lowering KC1 concentration to 100 mM and raising HEPES concentration to 100 mM. The activity was measured in the presence of 1 mM MgCl2 and 1 (iM free Ca 2 +. The graph represents a typical experiment. 98 Figure 34. Enrichment of Ca 2 + -dependent ATPase and N a + / K + -ATPase activities i n human pancreatic acinar plasma membranes. Experiments were carried out at 1.0 m M free C a 2 + . *Y//fy - C a 2 + -ATPase activity in the absence of M g 2 + , j ^ ^ j - C a 2 + - A T P a s e activity i n the presence of 0.5 m M M g C l 2 , jggg^ - N a + / K + - A T P a s e activity. The results represent a typical experiment. 99 Results ATPase i n the 10/27% sucrose density interface (Fig. 34). A Ca 2 + -dependent ATPase activity was also observed i n this fraction. In other experiments, C a M was found to stimulate the C a 2 + - A T P a s e activity more than 5-fold at low C a 2 + (Fig. 35). Trifluoperazine alone had no effect on C a 2 + - A T P a s e activity. However, it completely inhibited the stimulation by C a M (not shown). A t higher free C a 2 + concentration, neither agent produced an effect on the Ca 2 + -dependent ATPase activity. 100 Results Figure 35. Effect of CaM and TFP on Ca2+-dependent ATPase in human pancreatic acinar plasma membranes. J - Control, - 6 ng/ml CaM, - 60 LIM TFP. The results represent a typical experiment. 101 DISCUSSION Previous efforts to characterize pancreatic acinar plasma membrane C a 2 + - A T P a s e have centered on the use of rat tissue. However, this species shows high levels of diphosphohydrolase (Ansah et al., 1984; LeBel et al., 1980), making characterization of the high affinity C a 2 + - A T P a s e difficult. In an attempt to find an alternative source of pancreatic plasma membranes with little or no diphosphohydrolase activity, both bovine and guinea-pig pancreata were investigated. Plasma membranes from bovine pancreas showed up to a 12-fold enrichment of N a + / K + - A T P a s e and C a 2 + - A T P a s e activities (data not shown). However, this tissue source was abandoned due to problems with bacterial contamination, difficulties with tissue digestion and low cellular viability. Although the guinea-pig pancreatic acinar plasma membranes displayed a low affinity non-specific ATPase activity similar to the previously reported diphosphohydrolase, we were still able to reproducibly demonstrate the presence of a high affinity C a 2 + - A T P a s e activity. In addition, the presence of a high affinity Ca 2 + - transport activity i n pancreatic acinar plasma membranes was demonstrated i n this species. This species was therefore chosen for our studies. The plasma membrane preparation showed a good enrichment of the plasma membrane marker, N a + / K + - A T P a s e . Since the plasma membrane of the pancreatic acinar cell has been reported to account for only 4.8% of the total membrane surface area (Bolender, 1974), a protein recovery of 1.3% i n our preparations (Fig. 5), compared to the homogenate was considered good. This recovery also compared well with the plasma membrane yield (approximately 2% of the homogenate) from the preparative method of another laboratory (Bayerdorffer et al., 1985a) 102 Discussion BASIC CHARACTERIZATION The ATP-dependent Ca 2 + - transport activity present i n our plasma membrane fraction required very low C a 2 + for activation ( K ^ a = 0.04 uM) and was saturated at 1 u M free C a 2 + (Fig. 10). To our knowledge, a similar high affinity for C a 2 + has only been reported for the plasma membrane C a 2 + - A T P a s e of rat hepatocytes (Lin and F a i n , 1984; Lotersztajn et al., 1981; Pavoine et al., 1987; Prpic et al., 1984), human hepatocytes (Lotersztajn et al., 1982) and human small intestine basolateral membranes (Kikuchi et al., 1988). The guinea-pig pancreatic acinar plasma membrane C a 2 + - p u m p , though, showed a 20-fold higher affinity for C a 2 + compared to that in rat tissue (Bayerdorffer et al., 1985a). Such a high affinity may suggest that the pump is active both in resting acinar cells, when the reported cytosolic free C a 2 + concentration ranges between 0.09 and 0.18 uM (Muallem et al., 1988b; Ochs et al., 1985; Powers et al., 1985) and during stimulation by secretagogues, when the free C a 2 + concentration approaches 0.8-1.3 u M (Muallem et al., 1988b; Pandol et al., 1985a; Powers et al., 1985). A role for the plasma membrane C a 2 + - p u m p in controlling intracellular C a 2 + at rest and during stimulation has been previously proposed for pancreatic acinar cells (Dormer et al., 1987; Mual lem et al., 1988a, b, c, d). Most of these studies have been carried out using whole cells. The increase i n intracellular free C a 2 + i n response to stimulation by secretagogues was found to be transient (Muallem et al., 1988c; Pandol et al., 1985a; Streb and Schulz, 1983), and this might suggest that either the E R or plasma membrane Ca 2 + - transporter or both are activated during stimulation. The rate of 4 5 C a 2 + efflux has been shown to increase i n the presence of secretagogues (Muallem et al., 1988b). However, a rise i n intracellular free C a 2 + concentration was not required for activation of the C a 2 + - p u m p (Muallem et al., 1988b). 103 Discussion The affinity of the guinea-pig pancreatic acinar plasma membrane C a 2 + -pump for C a 2 + was higher than that reported for the C a 2 + - p u m p of pancreatic acinar endoplasmic reticulum from rat (Bayerdorffer et al., 1985a; Brown et al., 1987) or guinea-pig (Galvan and Lucas, 1987). Rat pancreatic acinar plasma membrane C a 2 + transporter (Bayerdorffer et al., 1985a) and Ca 2 + -act ivated, Mg 2 + -dependent ATPase (Al-Mutairy and Dormer, 1985; Dormer and A l -Mutairy, 1987) had been reported to have a lower affinity for C a 2 + ( K ^ a = 0.9 | iM) than rat pancreatic acinar E R C a 2 + - A T P a s e ( K ^ = 0.16-0.17 LIM) (Brown et al., 1987; Richardson and Dormer, 1984). Therefore, it was suggested that the plasma membrane C a 2 + - p u m p may not play a significant role i n the maintenance of cytosolic free C a 2 + at rest (Al-Mutairy and Dormer, 1985; Dormer and Al-Mutairy, 1987). The high affinity of the plasma membrane C a 2 + -pump for C a 2 + i n our preparations leads to a different conclusion. This property suggests that the enzyme may be involved in the fine regulation of intracellular C a 2 + i n pancreatic acinar cells at rest, while the function of E R C a 2 + - p u m p may be the rapid sequestration of raised cytosolic C a 2 + to terminate the secretagogue signal. The maximal rate of Ca 2 + - transport in guinea-pig pancreatic acinar plasma membrane vesicles (0.83 nmoles/mg/min) compares well with that of rat panreatic acinar plasma membrane vesicles (Bayerdorffer et al., 1985a) and rat liver plasma membranes vesicles (Prpic et al., 1984), viz. 0.66 and 0.65 nmoles/mg/min, respectively. Similar to the human erythrocyte C a 2 + - A T P a s e pump prototype, the guinea-pig pancreatic acinar plasma membrane C a 2 + - p u m p showed an apparent requirement for M g 2 + (Fig. 11). This M g 2 + requirement can be satisfied to a certain extent by endogenous M g 2 + , as has been shown previously for the C a 2 + -ATPase activities of plasma membranes prepared from rat pancreatic acini 104 Discussion (Ansah et al., 1984), rat liver (Lin and Fa in , 1984; Lotersztajn et al., 1981), rat adipocytes (Pershadsingh and McDonald, 1980) and rat corpus luteum (Verma and Penniston, 1981). In our experiments, 25% of the maximal 4 5 C a 2 + transport into plasma membrane vesicles could be observed i n the absence of added M g 2 + . C D T A , a cation chelator with higher affinity for M g 2 + than either E G T A or E D T A (Verma and Penniston, 1981), inhibited a major portion of this residual 4 5 C a 2 + uptake (Fig. 12). These results support the contention that sufficient endogenous M g 2 + is present i n the plasma membrane preparations to support partial activation of the C a 2 + - p u m p i n g ATPase in the absence of added M g 2 + . However, for optimum stimulation of the pump, additional M g 2 + was required. Therefore, measurement of Ca 2 + -dependent ATPase by others i n the absence of added M g 2 + (see below) does not represent the optimal conditions for studying the C a 2 + - p u m p i n g activity. The A T P hydrolytic activity displayed by guinea-pig pancreatic acinar plasma membrane preparations was complex. These preparations contained a low affinity ATPase activity which could be stimulated by either C a 2 + or M g 2 + (Fig. 14 and Fig . 15a, open circles). This activity appeared to be competitively inhibited by the other cation, as shown by the apparent inhibition of C a 2 + -dependent activity by increasing M g 2 + (Fig. 15a). Similar ATPase activities have previously been reported i n plasma membranes from many rat tissues including pancreas (Forget and Heisler, 1976; Lambert and Christophe, 1978; Mart in and Senior, 1980), liver (Lin and Fain , 1984; Lotersztajn et al., 1981), corpus luteum (Minami and Penniston, 1987), cardiac muscle (Anand-Srivastava et al., 1982; Zhao and Dhalla, 1988), osteosarcoma (Murray et al., 1983) and kidney cortex (Parkinson and Redde, 1971). In many cases, this C a 2 + ( o r M g 2 + ) - A T P a s e has been suggested to be an ecto-enzyme (Hamlyn and Senior, 1983; L i n , 1989; L i n and Russell, 1988; Tuana and Dhalla, 1988). Unlike 105 Discussion other ATPases , the liver ecto-ATPase has been shown to have a high-affinity for C a 2 + and M g 2 + (Lin and Russell, 1988). The Ca 2 + -dependent and M g 2 + -dependent activities are not distinct, but appear to reside within the same protein (Lin, 1985). In addition to the C a 2 + ( o r M g 2 + ) - A T P a s e , guinea-pig pancreatic acinar plasma membranes demonstrate a high affinity Ca 2 + -dependent ATPase ( K ^ a = 0.076 ± 0.022 uM) which was dependent on external M g 2 + (Fig 15b). In fact, this latter activity was only observed i n the presence of high M g 2 + (0.5 m M or higher). A high affinity C a 2 + - A T P a s e has previously been reported i n plasma membranes from a number of tissues including rat pancreas (Ansah et al., 1984; Dormer and Al-Mutairy , 1987; Ochs et al., 1988), liver (Iwasa et al., 1982) and corpus luteum (Minami and Penniston, 1987; Verma and Penniston, 1981). However, these other workers had been unable to demonstrate this activity i n the presence of high, physiological M g 2 + concentrations. To overcome the difficulty i n studying the high affinity C a 2 + - A T P a s e caused by the presence of an overwhelming low affinity C a 2 + ( o r M g 2 + ) - A T P a s e activity in their membrane preparations, some investigators have utilized low or zero added M g 2 + (Ansah et al., 1984; M i n a m i and Penniston, 1987; Verma and Penniston, 1981). However, in our studies i n guinea-pig pancreatic acinar plasma membranes, we failed to observe the high affinity C a 2 + - A T P a s e under these conditions. In the presence of a high M g 2 + concentration, the demonstration of high affinity C a 2 + - A T P a s e activity may depend on the assay conditions used. For example, in our experiments, where lower amounts of protein (16.7 ug/ml vs. 50 ug/ml) were used in a larger reaction volume (0.6 ml vs. 0.2 ml) and i n which reactions were started with diluted membranes instead of A T P , the Ca 2 + -dependent ATPase activity observed was not saturable and showed a 10-fold lower affinity for C a 2 + (data not shown). 106 Discussion The formation of a Ca 2 + -dependent, hydroxylamine-sensitive phosphoprotein i n guinea-pig pancreatic acinar plasma membranes (Fig. 18) also suggests the presence of a C a 2 + - p u m p i n g ATPase similar to that found within the erythrocyte plasma membrane. Phosphorylation of a 100,000 daltons protein has also been reported i n other tissues and organelles such as corpus luteum plasma membranes (Minami and Penniston, 1987) and rat pancreatic acinar E R (Imamura and Schulz, 1985). The E R protein of Imamura and Schulz was also Ca 2 + -dependent and hydroxylamine-sensitive. Although extensive E R marker studies were not performed, we found that the plasma membrane fraction of our preparations was not enriched in N A D P H cytochrome c reductase (not shown), confirming the findings of Svoboda et al. (1976), whose modified method was followed. Furthermore, our preparation showed no stimulation of Ca 2 + -dependent ATPase activity by potassium (Fig. 17), whereas the E R enzyme has been reported to be stimulated by this cation (Galvan and Lucas, 1987). Therefore, it seems unlikely that the protein phosphorylated i n our preparations was from an E R contaminant. In addition to the 100,000 daltons phosphoprotein, a smaller protein ( M r 50,000) was also phosphorylated. However, this phosphoprotein was stable in hydroxylamine and appeared to be Mg 2 + -dependent. Phosphorylation of a similar (albeit lower M r ) Mg 2 + -dependent protein has been described i n liver E R (Fleschner et al., 1985). The transport of 4 5 C a 2 + showed strong substrate specificity for A T P (Fig. 13). However, the hydrolytic activity was somewhat non-specific for the different nucleotide triphosphate substrates tested (Fig. 19), displaying a difference between the high-affinity C a 2 + - A T P a s e and the Ca 2 + - transport activities. The substrate specificity of C a 2 + transport i n our preparations was similar to that reported for the C a 2 + - p u m p of rat pancreatic plasma membranes (Bayerdorffer et al., 1985a) However, it differed from the substrate specificty of the C a 2 + 107 Discussion transporter of guinea-pig pancreatic endoplasmic reticulum (Galvan and Lucas, 1987). The substrate specificity of the Ca 2 + -dependent hydrolytic activity in our preparations generally appeared to be different from the specificities of similar activities found in some other systems studied, such as the liver plasma membrane C a 2 + - A T P a s e (Lotersztajn et al., 1981), or even guinea-pig pancreatic acinar E R (Galvan and Lucas, 1987). Only the liver plasma membrane C a 2 + -ATPase enzyme characterized by L i n and coworkers showed a broad substrate specificity similar to that of our preparation (Lin, 1985; L i n and Russell, 1988). This lack of specificity for hydrolytic activity is better illustrated by the ' M g 2 + -ATPase' activity (Fig 19b), which compares well with that observed i n rat liver plasma membranes (Lin and Russell, 1988). The low hydrolytic activity of the guinea-pig enzyme towards A D P indicates that it is distinct from the plasma membrane ectopic diphosphohydrolase activity previously described i n the rat (Ansah et al., 1984) and pig pancreas (LeBel et al., 1980). The function of ecto-ATPases is not known. The pancreatic diphosphohydrolase has been shown to be associated with zymogen granules and may be involved i n storage and exocytosis of zymogens (LeBel and Beattie, 1985). REGULATION OF Ca2+-ATPASE AND Ca2+-TRANSPORT The effects of potential regulators on the Ca 2 + -uptake and C a 2 + - A T P a s e activities i n guinea-pig pancreatic acinar plasma membrane preparations are summarized in Table VI . Although C-subunit has been shown to stimulate the erythrocyte and cardiac sarcolemmal C a 2 + - p u m p (Caroni and Carafoli, 1981b; Neyes et al., 1985), there is disagreement as to whether it affects the rate of C a 2 + transport or the affinity for C a 2 + . Whereas one study reported an increased affinity for C a 2 + without an effect on the maximal velocity (Neyes et al., 1985), the other 108 Discussion Table VI. Summary of the effects of different potential regulators on the Ca 2 +-uptake and Ca 2 +-ATPase activities of guinea-pig pancreatic acinar plasma membrane preparations. S - stimulation, I - inhibition, NC - no change, nd - not determined. Regulator Ca 2 +-Uptake Ca 2 +-ATPase PROTEIN KINASE A C-Subunit S NC H A 1004 NC NC C-Subunit + HA 1004 S a nd CALMODULIN CaM NC NC Compound 48/80 I I b TFP I NC CGS 9343B NC NC PROTEIN KINASE C PS NC NC SADG NC S TPA NC S PK-C NC S CGP 41251 NC NC Stimulation compared to control, i.e. no change in C-subunit-stimulated activity bInhibition at some concentrations; see text 109 Discussion study showed increased maximal velocity with no effect on affinity (Caroni and Carafoli, 1981b). In our experiments, C-subunit stimulated the maximal velocity of C a 2 + transport into plasma membrane vesicles more than two-fold without affecting the affinity of the pump for C a 2 + (Fig. 20). While these results are in agreement with those of Caroni and Carafoli (1981b), they conflict somewhat with the findings of Neyes et al. (1985). However, they do suggest that the C a 2 + efflux from pancreatic acini mediated by the C a 2 + - p u m p may be regulated by protein kinase A . The exact mechanism of this regulation is as yet unknown and requires further investigation. One possibility is a direct phosphorylation of the ATPase as suggested for the sarcolemmal C a 2 + - p u m p (Neyes et al., 1985). The lack of stimulation of C a 2 + - A T P a s e activity by C-subunit demonstrates another difference between this activity and Ca 2 + - transport activity within our preparations. H A 1004, a specific inhibitor of protein kinase A , was found to be ineffective i n blocking the control and C-subunit-stimulated C a 2 + transport and C a 2 + - A T P a s e activities in pancreatic acinar plasma membranes (Table III and Fig . 21). In whole cell experiments, Mual lem et al. had noted no effect of H A 1004 on the ability of rat pancreatic acinar cells to 'resist' the increased permeability induced by ionomycin (Muallem et al., 1988b). From these results, they concluded that P K A has no role in the regulation of the pancreatic acinar plasma membrane C a 2 + - p u m p . However, our results with C-subunit disagree with that interpretation. We suggest that H A 1004 may not be a useful inhibitor of the actions of P K A , at least in pancreatic acinar plasma membrane preparations. Both the C a 2 + - p u m p and Ca 2 + -dependent A T P hydrolytic activity of guinea-pig pancreatic acinar plasma membranes were insensitive to exogenous C a M (Figs. 10 and 16). This contrasts to the C a M stimulation observed for a 110 Discussion number of other plasma membrane Ca 2 + - transport ing ATPases, such as the enzymes of erythrocytes (Carafoli et al., 1986), bovine sarcolemma (Caroni et al., 1983), toad and pig stomach smooth muscle (Lucchesi et al., 1988) and rat pancreatic acinar cell (Ansah et al., 1984). In a more recent study of rat pancreatic acinar plasma membrane C a 2 + - A T P a s e (Ochs et al., 1988), C a M was shown to have no effect. Furthermore, the plasma membrane C a 2 + - A T P a s e s from rat liver (Lin and F a i n , 1984; Lotersztajn et al., 1981) and corpus luteum (Verma and Penniston, 1981) have also been reported to be insensitive to C a M . Therefore, it would appear that the universitality of CaM-stimulation of C a 2 + -ATPase has not been unequivocally established. Nevertheless, the lack of stimulation of Ca 2 + -dependent A T P hydrolysis and C a 2 + transport activity of our guinea-pig preparations by exogenous C a M may simply reflect the presence of saturating levels of endogenous C a M which may be fully activating the pump in situ. Further experiments were undertaken to investigate this possibility. A concentration-dependent inhibition of 4 5 C a 2 + - u p t a k e by T F P and compound 48/80 (Figs. 22 and 23) suggests the presence of endogenous C a M . However, the complete inhibition of C a 2 + transport activity seen in the presence of 1 m M T F P was probably due to non-specific changes in the microenvironment of the enzyme (Seeman, 1977). O n the other hand, the two inhibitors failed to produce an inhibition of Ca 2 + -dependent ATPase activity at different C a 2 + concentrations tested (with the exception at 1 (J.g/ml compound 48/80). The reason for the inhibition with 1 Lig/ml compound 48/80 (Fig. 25) is not clear. Compound 48/80 is reported to be much more selective than T F P at inhibiting C a 2 + - A T P a s e activity (Gietzen et al., 1983). However, this would not explain the lack of inhibition at higher concentrations of compound 48/80. Furthermore, we currently have no explanation for the reproducible concentration-dependent stimulation of "Basal" (Mg 2 + -ATPase ) and "Total" (in the presence of both C a 2 + 111 Discussion and M g 2 + ) ATPase activities by compound 48/80 (Fig. 26). It is assumed, however, that this effect is not due to inhibition of endogenous C a M . Whether this effect is somehow related to the histamine releasing properties of compound 48/80 or its recently reported ability to stimulate GTPase activity (Mousli et al., 1990), is not immediately apparent. The dissimilar effects of T F P and compound 48/80 on Ca 2 + - transport and Ca 2 + -dependent ATPase activities once again suggest that the two activities are distinct. The presence of endogenous C a M i n our preparations was confirmed in experiments showing stimulation of CaM-sensitive erythrocyte membrane C a 2 + -ATPase by the boiled extracts of guinea-pig pancreatic acinar plasma membranes (Fig. 27). The unusually high affinity of C a 2 + - u p t a k e for C a 2 + i n our preparations may also indicate the presence of endogenous C a M ; a 50-fold difference i n the apparent affinity for C a 2 + has been reported for purified sarcolemmal C a 2 + - A T P a s e assayed in the presence and the absence of C a M (Caroni et al., 1983). The pancreas contains one of the highest known concentrations of C a M in mammalian cells (Klee et al., 1980). For example, dog pancreas has been reported to contain 190 mg C a M / k g of tissue (Bartelt et al., 1986). Furthermore, the presence of a multifunctional C a M - P K , presumably responsible for many of the actions of C a M , has been reported i n pancreas from rat (Burnham and Williams, 1984; Gorelick et al., 1983) and dog (Bartelt et al., 1986). This protein kinase has been purified from rat pancreatic acinar cells (Cohn et al., 1987). Another kinase, C a M - P K III, having a somewhat different and unique substrate specificity, has also been reported to be present i n rat pancreas (Nairn et al., 1985). However, it still remains to be determined whether C a M acts v ia a C a M - P K or via a direct interaction with the C a 2 + -transporting ATPase molecule, as has been observed i n the erythrocyte (Carafoli et al., 1986). The suggestion that a 133,000 daltons CaM-binding protein in rat 112 Discussion pancreatic acinar plasma membranes may be the C a 2 + - A T P a s e (Ansah et al., 1984) implies a direct interaction between the two proteins. However, no evidence was provided by these workers to support the proposal that this C a M -binding protein was indeed the C a 2 + - A T P a s e . Attempts to deplete possible endogenous C a M by use of the E G T A -washing method of Caroni & Carafoli (1981a)) were unsuccessful, indicating that this protein may be tightly bound, as seen i n liver plasma membranes (Gazzotti et al., 1985; Gloor and Gazzotti, 1986) and heart sarcolemma (Caroni and Carafoli, 1981a). Further studies using Sephadex G-75 column chromatography appeared promising (Fig. 28), but were abandoned due to low protein recoveries using this procedure. It has been suggested that P K C may regulate C a 2 + extrusion by the plasma membrane Ca 2 + - transport ing ATPase i n erythrocytes (Smallwood et al., 1988), cultured vascular smooth muscle cells (Furukawa et al., 1989) and rat pancreatic acinar cells (Muallem et al., 1988b). Studies by Mual lem and colleagues used whole cells to determine the effects of H-7 (a protein kinase C inhibitor) and T P A on ionomycin-induced increases i n intracellular free C a 2 + levels. The changes i n free C a 2 + were interpreted by those authors as effects of these agents upon P K C to alter the ability of the cell to extrude C a 2 + via the C a 2 + - p u m p . To investigate the possible role of P K C i n the stimulation of C a 2 + efflux from pancreatic acinar cells, we studied the effects of purified rat brain P K C , as well as its activators T P A and S A - D G and an inhibitor C G P 41 251, directly on the 4 5 C a 2 + transport into plasma membrane vesicles. Purified P K C , the phorbol ester T P A and the diacylglycerol analog S A - D G failed to stimulate C a 2 + - u p t a k e (Figs. 30 and 31). These data suggest that P K C may not be involved i n the regulation of the plasma membrane C a 2 + - p u m p i n guinea-pig pancreatic acinar cells. B y contrast, purified P K C stimulated C a 2 + - A T P a s e 113 Discussion activity i n the presence of PS at 1 u M free C a 2 + (Table V) . The potentiation of C a 2 + - A T P a s e activity by T P A and S A - D G in the presence of P S may represent a stimulation of an endogenous P K C . The stimulation by S A - D G and purified P K C noted only at high C a 2 + concentrations (Table V) agrees with previous reports that demonstrated an increased V m a x of Ca 2 + - transport with P K C without concomittent alteration i n KQ& (Furukawa et al., 1989; Smallwood et al., 1988). C G P 41 251, a potent and selective inhibitor of P K C ( I C 5 0 for P K C = 50 n M , for P K A = 2.4 uM) (Meyer et al., 1989), had no effect on either the C a 2 + -transport or the C a 2 + - A T P a s e activity (Tables III and IV). Therefore, our studies provide evidence contrary to the conclusions drawn by Mual lem et al. (1988b) who implicate P K C in the regulation of the plasma membrane C a 2 + - p u m p . These results also point to another difference between Ca 2 + - transport and C a 2 + -ATPase activities i n guinea-pig pancreatic acinar plasma membranes. As discussed in the I N T R O D U C T I O N , I P 3 and I P 4 have both been suggested to stimulate C a 2 + influx into various cell types by a possible action on C a 2 + channels in the plasma membrane. To investigate whether these pathways might operate i n pancreatic acinar cells, the effects of these two inositol phosphates were studied on Ca 2 + - transport . If the inositol phosphates were acting on C a 2 + channels within pancreatic acinar plasma membranes, 4 5 C a 2 + -uptake into membrane vesicles should be impaired due to the opposing leakage through these channels. We found that neither agent produced a significant effect on C a 2 + - u p t a k e (Fig 32), suggesting that IP 3 - and/or I P 4 - mediated C a 2 + releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. 114 Discussion IS THE HIGH AFFINITY Ca2+-ATPASE THE Ca2+-PUMP? The similar high affinity of the Ca 2 + -uptake and the Mg 2 + -dependent, Ca 2 + - s t imulated ATPase may indicate a possible involvement of this latter activity i n C a 2 + transport. Furthermore, both activities were insensitive to C a M stimulation and showed an apparent dependency on external M g 2 + . However, despite these similarities, the high affinity C a 2 + - A T P a s e and the C a 2 + transport activities appear to differ i n the guinea-pig pancreatic acinar plasma membrane vesicles: The maximal velocity for ATPase activity was seven-fold higher than that for Ca 2 + - transport ( V m a x = 6.04 vs. 0.83 nmoles/mg/min, respectively). Whereas Ca 2 + - transport was highly specific for A T P as substrate, the hydrolytic activity was somewhat non-specific. The two activities also differed with respect to their regulation: While Ca 2 + -uptake is stimulated by C-subunit, C a 2 + - A T P a s e is not. Although C a M failed to stimulate both activities, C a M antagonists only inhibited Ca 2 + - transport . Finally, the C a 2 + - A T P a s e activity showed apparent stimulation by both endogenous and exogenous P K C , while Ca 2 + - transport was unaffected. The lack of correlation between the Ca 2 + - transport and the ATPase activities observed i n guinea-pig pancreatic acinar plasma membrane preparations suggests that the two activities are probably not related. Similar conclusions were recently drawn by Ochs et al. (1988). Rat liver and corpus luteum have also been suggested to contain two different activities (Birch-Machin and Dawson, 1988; L i n and Russell, 1988; M i n a m i and Penniston, 1987). The suggestion that a high affinity C a 2 + - A T P a s e i n liver plasma membranes is a C a 2 + transporter (Lotersztajn et al., 1981; Pavoine et al., 1987) is still under dispute (Lin, 1985; L i n and Russell, 1988). This latter controversy may have arisen as a result of the use of different methods of membrane isolation which affect the relative yield of canalicular Vs . sinusoidal plasma 115 Discussion membranes (Birch-Machin and Dawson, 1988). It is suggested that the high affinity C a 2 + - A T P a s e observed in the present studies is not the biochemical expression of the C a 2 + pump. C a 2 + - u p t a k e i n guinea-pig pancreatic acinar plasma membrane vesicles may be due to an enzyme of low activity whose ATPase activity is not detected in the presence of the higher specific activity Ca 2 + -dependent ATPase . A similar situation has been observed previously i n corpus luteum (Minami and Penniston, 1987) and liver plasma membranes (Birch-Machin and Dawson, 1988). Assuming that the major portion of the high affinity C a 2 + - A T P a s e activity is due to a non-specific ATPase, which is masking the C a 2 + - p u m p i n g enzyme activity, Kel ly and Smith (1987) devised a simple strategy to dissect out the latter activity. Using G T P to block the non-specific ATPase , these workers were able to show the presence of a high affinity Mg 2 + -dependent C a 2 + - A T P a s e activity with V m a x and K ^ a similar to the C a 2 + transporter. PURIFICATION OF THE Ca2+-PUMP Because of the heterogeneity of ATPase activity within pancreatic acinar plasma membranes, a complete characterization of the high affinity C a 2 + -ATPase which drives transmembrane C a 2 + transport must await its isolation to homogeneity and its subsequent reconstitution into defined lipid systems. Accordingly, purification of the Ca 2 + - transport ing ATPase was attempted using CaM-affinity chromatography (Caroni and Carafoli, 1981a; Niggli et al., 1979a) and dye-ligand affinity chromatography (Coll and Murphy, 1984; Scopes, 1987). CaM-affinity chromatography was chosen for two reasons: First, the demonstration of indigenous plasma membrane-bound C a M indicated the possibility of its direct interaction with C a 2 + - A T P a s e ; second, the demonstration of a 133,000 daltons CaM-binding protein i n 1 2 5 I - C a M gel-overlay experiments 116 Discussion i n rat pancreatic acinar plasma membranes which was suggested to be a C a 2 + -ATPase candidate (Ansah et al., 1984). Dye-affinity chromatography was chosen because dye-ligands, including Cibacron Blue 3 G A and Reactive Red 120, are known to bind proteins with a dinucleotide-binding fold such as ATPases (Coll and Murphy, 1984; Scopes, 1987). After a number of attempts with both types of affinity chromatography systems, we were unable to purify the C a 2 + - A T P a s e . STUDIES WITH THE HUMAN PANCREATIC ACINAR PLASMA MEMBRANES Preliminary studies with human pancreatic acinar plasma membranes demonstrated the presence of a Ca 2 + -dependent ATPase activity (Fig. 34). Exogenous C a M produced a pronounced stimulation of this activity (Fig. 35), indicating that either these membranes are devoid of endogenous C a M or the enzyme is not fully stimulated by it. The observed lack of inhibition of C a 2 + -dependent ATPase by T F P tends to favour the former possibility, i.e. the absence of endogenous C a M from our human pancreatic acinar plasma membrane preparations. This CaM-activated, Ca 2 + - s t imulated ATPase activity may represent the C a 2 + extrusion system of human pancreatic acinar cells. ORIGINAL CONTRIBUTIONS TO THE LITERATURE 1. Demonstrated a highly specific high affinity Ca 2 + - transporter and a high affinity broad specificity C a 2 + - A T P a s e i n the same preparation of guinea-pig pancreatic acinar plasma membrane vesicles. 2. Demonstrated that these two activities are not related. 3. Demonstrated that Ca 2 + - transport in guinea-pig pancreatic acinar plasma membrane vesicles is stimulated by protein kinase A and endogenous C a M , but not by P K C . 117 SUMMARY AND CONCLUSIONS 1. These studies demonstrate the presence of an A T P requiring high-affinity C a 2 + - p u m p in guinea-pig pancreatic acinar plasma membranes. This pump may be fully stimulated by endogenous C a M . 2. The unusually high affinity of the C a 2 + - p u m p for C a 2 + would indicate that it may play a role in C a 2 + extrusion from acinar cells, both at rest and during stimulation. 3. Also present i n these preparations was a high-affinity Ca 2 + -act ivated, M g 2 + -dependent ATPase activity which could only be observed i n the presence of high M g 2 + . 4. Ca 2 + - transport was highly specific for A T P as substrate, but the high affinity C a 2 + - A T P a s e was somewhat less specific. 5. Ca 2 + - transport was stimulated 2-fold by catalytic subunit of protein kinase A , while C a 2 + - A T P a s e was unaffected. C a M antagonists, compound 48/80 and T F P inhibited Ca 2 + - transport . Purified P K C and its activators, T P A and S A - D G failed to stimulate Ca 2 + - transport . The results suggest a possible role for C a M and protein kinase A , but not protein kinase C , i n the regulation of C a 2 + efflux from pancreatic acinar cells. 6. The exact relationship of the high affinity C a 2 + - A T P a s e activity to C a 2 + -transport is unclear at present. 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Calcium transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes
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