THE HYDROLYSIS OF INOSITOL PHOSPHOLIPID IN IVIOUSE EXOCRINE PANCREAS ,,r'ir:::'r]. *. DEGREE OF DOCTOR OF PHILOSOPHY of The Department of Physiology Uni vers i ty of Adel ai de , South Austra'l i a by KARIN ANNE TENNES B.Sc.(Hons.) 1984 fi.6sx'-eL,'( I ? - S" 8:'" TABLE OF CONTENTS Page SUMMARY . (v.ii) DECLARATI0N (x) AC KNOI,ILEDGEMENTS (xi ) LIST OF TABLES (xii) LIST 0F FIGURES (xiv) CHAPTER 1 : INTRODUCTION 2 CHAPTER 2 LITERATURE REVIEI^, I 2.t PANCRIATIC STRUCTURE AND FUNCTION 9 2.2 INTERACTION BETI^IEEN ENDOCRINE AND EXOCRINE PANCREAS 23 2.3 PANCREATIC SECRETAGOGUES 27 2.3 (a) Secretagogues which Increase the Intra- cel I ul ar Concentrati on of Cal ci um 27 2.3 (b) Secretagogues wh'ich Cause an Increase in the Intracel I ul ar Concentrati on of cAl'lP 28 2.3 (c) Pancreatic Secretagogues which do not Appear to Act via Increas'ing the Intra- cellular Concentration of Calcium or cAMP 42 2.4 THE ROLE OF CALCIUM IN PANCREATIC ENZYIrIE SECRETION 47 2.a G) Evidence for the Involvement of Calcium in Pancreatic Enzyme Secretion 47 2.4 (b) Evidence for the Involvement of Changes i n l,lembrane Potenti al i n Protei n Secreti on 49 2.a (c) Cytop'lasmic Calcium Concentrations in Pancreatic Acinar Cells 56 ( j ) (i i ) Page 2.5 CALCIUM FLUXES IN PANCREATIC ACINAR CELLS : THE EVENTS LEADING TO INCREASID INTRACELLULAR CALCIUM CON CENTRATI ON 60 2.6 THE RELATIONSHIP BETI^IEEN INHIBITION OF PROTEIN SECRETION, CYTOSOLIC CALCIUM CONCENTRATION AND RECEPTOR OCCUPATION IN THE EXOCRINE PANCREAS 70 2.7 CALCIUM-ACTIVATED STIMULUS-SECRETION COUPLING IN EXOCRINE PANCREAS 76 2.7 (a) Protein Kinase C and Pancreatic Exocrine Secreti on 77 2.7 (6) Calcium-activated, Calmoduì'in-dependent Protein Kinase and Pancreatic Exocrine Secreti on B5 2.8 : PHOSPHATIDYLINOSITOL : THI INITIAL DISCOVERY 88 2.9 : "PTDINS EFFECT" 1953-1980 90 2.9 (a) Phosphatidylinositol Metabolism 91 2.9 (b) The Subcellular Distribution of Phosphol i pase C 95 2.9 (c) Activation and l4odulation of Ptdlns- speci fi c Phosphol i pase C 97 2.9 (d) The Calcium-Dependency of Phospholipase C Acti vi ty 99 2.I0 z "PTDINS EFFECT" 1981-1984 101 2.10(a) Po'lyphosphoinosi tide : Metabolism t02 2.10(b) Po'lyphosphoinositide Phosphodiesterase : Activation and Modulation 106 Z.tt : SITE OF INOSITOL PHOSPHOLIPID METABOLISM 110 2.12 : MEASUREMENT OF THE AGONIST.STIMULATED HYDROLYSIS OF INOSITOL PHOSPHOLIPID 119 (iii) lgge 2.T3 : THE CALCIUM DEPENDENCY OF INOSITOL PHOSPHOLIPID METABOLISM IN EXOCRINE PANCREAS L32 2.I4 z THE ROLE 0F IN0SITOL PH0SPHOLIPID HYDR0LYSIS IN MEDIATING STIMULATION OF THE CELL RESPONSE 140 2.1a(a) The Role of Inosi tol Phosphol ipid Hydrolysi s i n Mobi I i zi ng Cal ci um 141 2.I4(b) The Role of Inositol Phospho'lipid Hydroìysìs in Activation of Protein Kinase C 153 2.T5 : THE RELATIONSHIP BETWEEN INOSITOL PHOSPHOLIPID HYDROLYSIS AND RECEPTOR OCCUPATION 154 2.16 : INOSITOL PHOSPHOLIPID METABOLISM AND CYCLIC AMP 158 2.t7 : LITERATURE REVI EI^i : CONCLUSION 161 CHAPTER 3 MATERIALS AND METHODS t62 3.1 EXPERIMENTAL ANI¡IALS 163 3.2 I4EASUREMENT OF THE HYDROLYSIS OF INOSITOL PHOSPHOL IP ID 163 3.2 (a) Assay Procedure 163 3.2 (b) Radioactive Counting 165 3.3 EXTRACTION AND CHROI4ATOGRAPHIC SEPARATION OF PHOSPH 0L I P I DS LAB E LLED in uiuo hIITH nty o - Q-e g'1 INOS I TOL 165 3.4 EXTRACTION AND CHROMATOGRAPHIC SEPARATION OF 3H. LABELLTD COMPOUNDS RELEASED FR}M mso-(2-3H) INOSITOL- LABELLED INOSITOL PHOSPHOLIPID 166 3.5 : INDUCTION OF DIABETES AND MAINTENANCE OF DIABETIC MICI r67 (iv) !gg-e- 3.5 (a) STZ-induced Diabetes r67 3.5 (b) Blood Glucose Determination 168 3.5 (c) Insulin Maintenance of Diabetic Mice 169 3.5 (d) Inositol Phospholipid Hydrolysis in Diabetic Mice T7L 3.6 : DISSOCIATI0N 0F PANCREATIC ACINAR CELLS 173 3.7 : AMYLASE SECRETI0N 174 3.7 (a) Amy'lase Secretion in Pancreatic Sl'ices 174 3.7 (b) Amyìase Secretion in Pancreatic Acini 176 3.8 DETERMINATION OF ADENOSINE TRIPHOSPHATE (ATP) CONTENT 177 3.9 : SOLUTIONS AND CHEI{ICALS L7B 3.9 (a) Solutions 178 3.9 (b) Chemicals 181 (i ) Radiochemicals 181 (i i ) Chemicals I82 3.10 : STATISTICS 184 CHAPTER 4 VALIDATION OF THE METHOD DEVELOPED TO MEASURE THE HYDROLYSIS OF INOSITOL PHOSPHOLIPID IN MOUSE EXOCRINE PANCREAS 186 4.1 INTRODUCTION t87 4.2 MATERIALS AND I{ETHODS 191 4.3 RESULTS t92 4.4 D ISCUSS ION 2I0 CHAPTER 5 INVESTIGATION OF INOSITOL PHOSPHOLIPID HYDROLYSIS AS AN EARLY EVENT IN ACTIVATION OF THE EXOCRINE PANCREAS 217 (v) Page 5.1 INTRODUCTI ON 218 5.2 MATERIALS AND METHODS 22I 5.3 RESULTS 225 5.4 DISCUSS I ON 242 CHAPTER 6 : THE ROLE OF CALCIUM IN THE HYDROLYSIS OF INOSITOL PHOSPHOLIPID IN EXOCRINE PANCREAS 25r 6.1 INTRODUCTION 252 6.2 MATERIALS AND METHODS 256 6.2 (a) Inositol Phospho'lipid Hydro'lys'is and Intracel I ul ar Ca2+ 257 6.2 (b) Inositol Phospholipid Hydrolysis and Extracellular Ca2+ 26L 6.3 : RESULTS 262 6.3 (a) Inositol Phospholipid Hydroìysis and Intracellular Ca2+ 263 6.3 (b) Inositol Phospholipid Hydrolysis and Potentiation by Ca2+ 280 6.4 DISCUSS ION 296 6.4 (a) The Effect of Lanthanum in Agonist- stimulated Tissue 313 6.4 (b) The Effect of Lanthanum in Unstimulated Ti ssue 316 6.a (c) The Effect of l4anganese in Agonist- stimulated Tissue 317 6.4 (d) The Effect of Manganese in Unstimulated Ti ssue 318 (vi ) Page 6.4 (e) Concluding Summary 319 (i) The site associated with potentiation of basal breakdown 319 (ii) The site associated with potentiation of agonist-stimulated breakdown 319 CHAPTER 7 THE RELATIONSHIP BETI^JEEN THE HYDROLYSIS OF INOSITOL PHOSPHOLIPID AND THE SECRETION OF AMYLASE IN MOUSE EXOCRINE PANCREAS 324 7.L INTRODUCTION 325 7.2 : MATERIALS AND METHODS 326 7.3 RESULTS 328 7.4 DISCUSSION 337 CHAPTER 8 CONCLUSION 345 8.1 CONCLUDING DISCUSSION 345 BIBLIOGRAPHY 358 SUMMARY 1 A nethod was developed which allows the rapid, reproducible measurement of the hydrolysis of inositol phospho'lipid in mouse exocrine pancreas. The technique involves the in uiuolabelling of pancreati c ti ssue wi th rnao -(2-3 H) i nos'i to] and does not requi re the time-consum'ing extraction and chromatographic separation of 'l'ip'ids which has been necessary in the majority of assays of phos phoi nos i ti de turnover. The measurement of the release of myo-(2-3H) inositol-labelled products from inositol phospholìpid provides a direct assay of phosphoinosi t'ide breakdown and not of a combinati on of hydro'lysis and resynthesis, since the (3H)-inositol released is not re- incorporated into inositol phospholipid. In addition, the increase in (3H)-'inos'itol released is not due to agonist-act'ivat'ion of the phosphatidyìinositol : rn7o-inositol exchange enzyme which would cause a complication in measuring lipid breakdown due to an increase in exchange of inositol between lip'id-bound and free states. Since the method measures the ('tt)-inositol-label'led products of phosphoinositide breakdown the vaìidity of the method does not depend on whether the I i pi d hydro'lysed i s phosphati dy'l i nosi to'l , phosphatidyl inositol-4 phosphate or phosphatidylinositol-4,5 bi sphosphate. This assay facilitates the study of inositol phospholipid hydrolysis. In particular, an improved examination of the dose-dependent (vi i ) (vìii) relationship between'lipid hydro'lys'is and pancreatìc function is poss i b'le . 2 The agonist-stimulated breakdown of inositol phospholipid was found to be closeìy linked to receptor activation. This lipid response is not dependent on the agon'ist-activated influx of Na+ and does not requ'ire an intact cytoske'leta'l network or protein synthes'is. A dependency of the agonist-stimulated lipid breakdown on ATp was identified, which supported the proposal that phosphatidy'linositol- 4,5 bisphosphate and not phosphat'idyìinositol may be the initial lìpid hydro'lysed folìowing receptor activation. 3 The agonìst-stimulated breakdown of inositol phospholipid ìs not Ca2+-dependent since 'it occurs in the absence of extracellular Ca2+ and does not require the release of Ca2+ from intracellular stores. Inositol lipid breakdown is not activated by ca2+ since an increase in intracellular ca2+ with ionophore A23r87 does not stimulate the tipid response. 4 The presence of Ca2+ in the extracelìular medium potentiates inositol phospho'lipid hydroìysis. This potentiation ìs not due to the movement of Ca2+ through the plasma membrane since neomycin, an agent which blocks Ca2+ influx, does not alter the potentiation effect. 5 Two sites of potentiation by Ca2+ of inositol ìipid hydrolysis have been identified on the external surface of the pancreat'ic cell membrane. One site is associated with potentiation of unstimulated (ix) inositol lipid hydrolysis. This site is lanthanum insensitive but manganese can replace Ca2+ at this site. The second site is associated with potentiation of agonist-stimulated inositol ìipid hydroìys'is. Lanthanum and manganese compete with Ca2+ at this site to remove the potentiation effect. 6 Results show that at h'igh concentrations of agonists the degree of inositol phospholipid hydrolys'is correlates with the degree of inhibition of amylase secretion. Since this effect on secretion is due to Ca2+ the result suggests that inositol lipid breakdown and the increase in intracellular Ca2+ are correlated. 7 At low concentrations of agonists, when amyìase secretion is stimulated, the degree of inositol 'lipid hydrolysis is very small but may be sufficient to cause release of Ca2+ from intracelìular s tores .