MSU LlBRARlES .- RETURNING MATERIALS: P1ace in book dro p to remove this checkout from your record. FINES wi11 be charged if boo k is returned after the date stamped beIow. REGULATION AND MECHANISM OF PHOSPHOINOSITIDE AND INOSITOL POLYPHOSPHATB METABOLISM IN RAT HEPATOCYTES by Mark Allan Seyfred A DISSERTATION submitted to Michigan State University in partial fullfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1984 ABSTRACT REGULATION AND MECHANISM OF PHOSPHOINOSITIDE AND INOSITOL PHOSPHATE METABOLISM IN RAT HEPATOCYTES BY Mark Allan Seyfred Vasopressin stimulated breakdown of phosphoinositides in rat hepatocytes has been demonstrated by a number of workers. however. the subcellular site of this induced breakdown has not been determined. To examine the subcellular site of vaSOpressin induced breakdown of phOSphoinositides. rat hepatocytes were isolated and incubated with 32?i for 90 min. The r1—32P1ATP specific radioactivity has reached isotopic equilibrium. as well as the rates of 32? incorporation into phosphatidic acid. phosphatidylinositol 4-phosphate (PI-P). and phosphatidylinositol 4.5-bisphosphate (PI-P2) have approached steady state within this labelling time. VasOpressin. at 200 nM. was incubated with the labeled hepatocytes for 0-10 min. At various times. the cells were fractionated into partially purified plasma membranes. mitochondria. lysosomes. and microsomes using Percoll density gradient centrifugation. The 32 P-phOSpholipid levels were examined in each fraction by deacylating the labeled phospholipids with mild alkali. and then analyzing the 32P-glycerOphosphate esters by anion exchange high pressure liquid chromatography. It was found that breakdown of 91-? and PI-Pz. in response to vasopressin. occurs at the Mark Allan Seyfred plasma membrane. Further investigation led to the finding that a calcium-dependent polyphOSphoinositide-Specific phosphodiesterase was responsible for the breakdown of PI-P and PI-Pz. This phosphodiesterase is inactive under normal cellular conditions of ionic strength and calcium concentrations. but can be activated by the addition of vasOpressin. The metabolism of inositol trisphOSphate (1-P3)' the product of phosphodiesterase action on 91-?2 and the proposed second messenger of hormone-induced calcium mobilization. was also examined. It was found that 1-?3 is degraded to form inositol bisphosphate (1-92) by a phosphatase. located almost exclusively in the plasma membranes of rat liver. Further phOSphatase catalyzed breakdown of 1-?2 occurs primarily in the cytosolic and microsomal fractions. It was determined that I-P3 phosphatase (1-? ase) is a specific enzyme and not a general 3 acid or alkaline phOSphatase by pH optimum determination and its insensitivity to phoSphatase inhibitors such as LiCl and flat. The enzyme was inhibited by physiological levels of Spermine. but not putrescine or spermidine. This suggest that the activity of I-PBase. and thus cytosolic calcium levels. may be associated with the regulation of intracellular spermine concentrations. To my wife. Linda H ACKNOLEDGEMENTS I would like to thank Dr. William Wells for his support and guidance during the course of my graduate studies. and the other members of the laboratory. particularly Charles Smith and Jeffrey Nickerson. for their helpful discussion. and also Lynn Farrell for her technical skills. I would also like to express my appreciation to Dr. Rodney Boyer of Hope College for Sparking my interest in biochemical research. and to my parents. who provided encouragement and financial SUpport during my educational career. Most of all. I would like to thank my wife. Linda. for her suPport and patience. TABLE OF CONTENTS Page List of Tables.... ...... . ..... ...... .......... . ..... . vii List of Figures .......... ....... ......... .. ....... ...viii Abbreviations ..... . ..... . ............ .. ...... ... ..... x Introduction .................... . ................. ... 1 Literature Review The effect of calcium or calcium—calmodulin complexes upon cellular processes............... 3 Molecular regulation of intracellular calcium concentrationSOOOOOOOOOO0.0000000000000000. ..... 5 Agonist—induced increases in intracellular free calCiumOOOOO0.0............OOOOOOOOOOOOOOOI 7 PhOSphoinositide biosynthesis and degradation... 8 Stimuli-induced changes in cellular phosPhoinositide levels.................. ....... 16 PhoSphoinositide effects on cellular function... 16 ReferenceSOOOOOI......OOOOOOOOOOOOOOOCOO ........ .0... 23 Chapter I Subcellular Incorporation of 32? into PhoSphoinositides and Other PhoSpholipids in Isolated Hepatocytes Abstract.... ...................... ... ...... ..... 35 FootnoteSOOOOOOOOOOO... ........ O. ........ O ...... 36 Introduction......... ........... . ........ . ...... 37 Experimental Procedures "ateriGISOOIOOOOO ..... O OOOOOOOOOOOOOOO 0...... 39 Hepatocyte isolation......................... 39 Hepatocyte incubation..... .......... ......... 40 Cell homogenization and subcellular fractionationOOOOOOIOIOOO00.0.0000... ........ 40 PhOSpholipid isolation and deacylation....... 43 iv Analysis of deacylated phospholipids by high pressure anion exchange liquid chromatography...............32............ .Determination of cellular TV— PlATP specific radioactivity..................... Results Purity of subcellular fractions............ Anion exchange HPLC analysis of deacylated phosPholipids.............................. Estimation of possible post—homogenization artifacts.................32............... Rates of incorporation of P into phOSpholipidS of various subcellular fractions.................................. Discussion... ................................. ReferenCGSOOOOOOO......OOOOIOOOOOOOOO ..... 0... Chapter II Subcellular Site and Mechanism of Vaso— pressin Stimulated Hydrolysis of Phosphoinositides in Rat Hepatocytes Abstract 0 O ..... O O O O O O O ........ O O O O O O O O O O ....... O Footnot‘s O O O O O O O O O O O O O O O O O O O O O O I O ......... O O O O O O Introauction. O O O O O O O O O ........... 0 O O O O O O O ....... EXperimental Procedures Hepatocyte incubations..................... Subcellular fractionation of hepatocytes and phospholipid analysis..............32.. Phospholipid degradation studies using P- labeled plasma membranes................... Other methods.............................. Materials......................... ....... .. Results Effect of VSEOptOSSin treatment of rat hepa- tocytes on P-labeled phospholipids isolated from various subcellular ggganelles. p- Time course of the degradation of labeled plasma membrane phospholipids: Effect of calcium............................ Dose response of calcium on plasma membrane polyphosphoinositide phOSphodiesterase activity: Effect of ionic strength.......... 44 44 46 48 49 52 64 7O 76 78 79 82 82 83 84 84 94 100 Effect of vasopressin on 32P—labeled phosphoinositide levels in isolated plasma m.mbranes.00.0.0.0....00.0....000000000000. Discussion.. ........................... .... ..... R.fer.nc°s0000.0..0.0000.000.00.00.00000000... Chapter III Characterization of myg-Inositol Tris- phosphate Phosphatase in Rat Liver Plasma Membranes Abstract 0 0 0 0 0 ...... 0 . 0 . 0 0 0 0 0 0 0 0 0 ..... 0 0 0 0 . ...... Footnot.s0 0 0 . . 0 0 . 0 0 0 OOOOOOOOOOOOO . 0 0 . 0 0 0 0 0 . 0 0 0 0 0 Introauction. 0 . 0 0 0 0 0 . 0 ....... . 0 0 0 0 . 0 0 0 . 0 ........ Experimental Procedures Hepatocyte incubations and 32P—labeled plasma membrane isolation.. ..... ........... Isolation of subsellular organelles........ Preparation of P-labeled I-Pzand I-P3.... Assay for the degradation of I PII-P2 and 1.? ...000..0.......0...000.00.00.00... 0th.: m.th°d8eeeeeeeeeeeeeeeeeeeeeeeeeeeeee Mat.rials.00..000......00...00 ..... 0 ........ . Results Breakdown of endogenously produced [32PlI-P by partially purified rat hepatocyte plasma membranes ....... ..... ........ .. ..... ......... Subcellular distribution of I-Pzase and I-P ase activities......................... Metal requirement for plasma membrane I—P Tim; course of the breakdown of [ PII-P3 by plasma membranes........................ Effect of pH on plasma membrane I-P3ase activity.................. ................. Effect of various agents on plasma membrane I-P3ase activity........................... Discussion........................ ..... ....... R‘f.r.nc.s0000000.0 ...... 00.000.000.0000000000 summary ..... ...0...000000.0.00.0 ..... 0.00.0...00000 vi as. actiVitYeeeeeeeeeeeeeeeeeazeeeeeeeee 105 106 111 116 117 118 120 120 120 122 122 123 124 127 130 130 130 135 140 144 148 LIST OF TABLES Literature Review Table Page Receptors Whose Stimulation Provokes a I ‘PI' Response (PI breakdown or enhanced PI labelling) in ApprOpriate Target Tissue..................................... 17 Chapter I I Marker Enzymes and Estimate of Fraction Purity000000000000.000000000000000000000... 47 Distribution of 32P Incorporation into II DPI and TPI of Various Subcellular Fractions...0.0.0.00000000.000..........0.0 63 Chapter III I Marker Enzyme Analysis..................... 128 II Subcellular Distribution of I-P2 and I-P3 Phosphatase Activity.................. 129 III Effect of Various Agents on I-P3 Phos- phatase Activity........................... 138 vii LIST OF FIGURES Literature Review Page Structure of Inositol Lipids............... 10 Pathways for the Biosynthesis and Degradation of Phosphoinositides.. ..... .... 13 Chapter I Flow Diagram for the Subcellular Fractionation of Rat Hepatocytes........... 41 Anion Exchange HPLC Elution Profile of Polar Deacylated PhOSpholipid Products..... 49 Time Course of 32? Incorporation into Various PhoSpholipids Isolated from Sub- cellular Fractions of Rat Hepatocytes...... 53 Chapter II Effect of Vasopssssin Treatment of Rat Hepatocytes on P-Labeled Phospho- lipids Isolated from Various Subcellular Fractions.................................. 86 Time Course of the Degradation of 32P- Labeled Plasma Membrane Phospholipids: Effect of Calcium.......................... 95 Dose Response of Calcium on Plasma Membrane Polyphosphoinositide Phospho- diesterase Activity: Effect of Ionic Strength................................... 101 viii Figure Chapter III Page + Endogenous M92 -Dependent I-P ase Activity in Partially Purifie Plasma Membranes Isolated from Rat Hepatocytes.... 12$ Dose ReSponse of Magnesium on Plasma Membrane I-P3ase Activity.................. 131 Time Course of I-P3 Breakdown.............. 133 Effect of pH on the Plasma Membrane I-PBOSG ActiVitYeeeeeeeeeeeeeeeeeeeeeeeeeee 136 ix PC PE PI PG PA PS PI-P. DPI PI-P TPI 2. GPC GPE GPI GPG GP GPS GPIP GPIP ABBREVIATIONS phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylglycerol phosphatidic acid phOSphatidylserine phosphatidylinositol 4-phosphate phosphatidylinositol 4.5—bisphosphate glycerophosphorylcholine glycerOphosphorylethanolamine glycerophosphorylinositol glycerophosphorylglycerol glycerol phosphate glycerOphosphorylserine glycerOphosphorylinositol 4-phosphate glycerophosphorylinositol 4.5—bisphosphate inorganic phosphate axe-inositol phosphate gig-inositol bisphosphate gig-inositol trisphosphate gig-inositol bisphosphate phosphatase myo-inositol triSphosphate phosphatase HEPES TRICINE MES BICINE EDTA EGTA PMSF TCA HB HPLC N-2-hydroxyethylpiperazine-N°-2-ethane- sulfonic acid N-trislhydroxymethyllmethyl glycine 2-[N-morpholino]ethanesulfonic acid N.N—bis[2-hydroxyethyllglycine ethylenediaminetetraacetic acid ethyleneglycol-bis-(B-aminoethyl ether)- N.N.N'.N'-tetraacetic acid phenylmethylsulfonyl flouride trichloroacetic acid homogenizing buffer high pressure liquid chromatography xi Introduction In many different tissues and cell types. the stimulation of various receptors leads to an increase in the concentration of cytosolic free calcium. and also induces changes in phoSphoinositide metabolism (1.2). In rat hepatocytes. the addition of vasopressin increases cytosolic free calcium (3-7) and decreases the level of phoSphatidylinositol (PI). phOSphatidylinositol 4—phOSphate (PI-P). and phOSphatidylinositol 4.5-bi5phosphate (PI-P2) (8.9.10). However. neither the subcellular site of the agonist-induced breakdown nor the mechanism of breakdown is known. Therefore. the subcellular site and mechanism of vasopressin-induced breakdown of phOSphoinositides in rat hepatocytes was investigated and is described in Chapters 1 and 2. Chapter 1 describes the methodolgy for the subcellular fractionation of rat hepatocytes. and the analysis of the polar products of mild alkali deacylation of phospholipids by anion exchange high pressure liquid chromatography. The rates of 32P incorporation into the phospholipids of various subcellular fractions isolated from rat hepatocytes are also described. The subcellular site and mechanism of vaSOpressin-induced phOSphoinositide breakdown are described in Chapter 2. Both of these chapters are preesented in manuscript form as outlined by the Journal of Biological Chemistry and are to be published as separate. but adjoining papers in one of the June or July issues of the Journal. They are reprinted here by permisssion of the publisher. The role of myg-inositol triSphosphate (I-P3). the product of plasma membrane phOSphodiesterase action on PI-P2 (11). as a second messenger for the hormonal mobilization of cellular calcium. has been described in saponin- permeabilized pancreatic acinar cells (12) and rat hepatocytes (13). The cytosolic level of I-P is determined 3 by not only its rate of release from PI-Pz. but also the rate of its degradation. Therefore. in Chapter 3. the subcellular distribution and properties off a specific myg—inositol triSphoSphate phOSphatase from rat liver are described. This chapter is also in manuscript form and has been submitted to the Journal of Biological Chemistry for publication. Literature Review In this literature review. I would like to review two main tOpics. The first tOpic involves cell calcium: its role in modulation of cellular processes. and the mechanisms by which intracellular calcium concentrations are regulated. In the second topic. the biosynthesis and degradation phoSphoinositides. and the effect of hormones on these processes. will be described. Also. the effect of phosphoinositides and its metabolites Upon various enzymes and intracellular calcium concentrations will be reviewed. The Effect of Calcium or Calcium-Calmodulin Complexes Upon Cellular Processes - A vast number of enzymes and cellular processes are affected by calcium—calmodulin complexes or by calcium alone. CDP-diacylglycerol-inositol transferase from rat liver (14) and rabbit lung (15) as well as the liver mitochondrial enzymes. pyruvate carboxylase and carbamoyl—phOSphate synthetase. are inhibited by calcium (16-18). Microtubule dissassembly is also inhibited by calcium-calmodulin (19). However. a majority of the enzymes and cellular processes are stimulated by calcium or calcium-calmodulin. These include cytosolic phOSpholipase C from liver (20.21): phOSpholipase A2 from liver (22) and platelets (23); mitochondrial glycerol phOSphate dehydrogenase from insect flight muscle (24): and NAB-linked isocitrate dehydrogenase. obketoglutarate dehydrogenase (25). and pyruvate dehydrogenase phoSphatase (26) from mitochondria. Cyclic AMP phOSphodiesterase in brain (27). liver (28). and heart (29) is calcium-calmodulin dependent. Brain adenylate cyclase is stimulated by low concentrations of calcium. but inhibited at higher calcium concentrations (30). Pancreatic islets also contain a calcium-calmodulin sensitive adenylate cyclase (31). Calcium is also involved in enzyme. catecholamine. and hormone secretion in a variety of cell types (32-39). A number of protein kinases are be dependent Upon or activated by calcium or calcium-calmodulin. These include myosin light chain kinase (40) and tryptOphan hydroxylase kinase (41). Garrison et al. (42) demonstrated that angiotensin II and vaSOpressin. hormones which increase intracellular calcium levels without increasing cAMP levels (3). stimulate the phOSphorylation of 10—12 cytosolic proteins in rat hepatocytes. Schulman and Greengard (43) have described a calcium-calmodulin protein kinase system in a variety of other tissues. Protein kinase C. the phospholipid—dependent protein kinase which is stimulated by phorbol esters. requires calcium for maximum activity (44.45). Mg2+-dependent ATPase's are also stimulated by calcium-calmodulin. The best characterized systems are the sarcoplasmic reticulum (46-49). which may be regulated through the phOSphorylation of a 22k dalton membrane protein (phospholamban). and the erythrocyte membrane (Ca2+-MgZ+)ATPase (50-53). Other (Ca2+-MgZ+)ATPase activities are found in adipocyte plasma membranes (54) and endOplasmic reticulum (SS). pancreatic islet plasma membranes (56). heart plasma membranes (S7). and microsomes (58-60) and plasma membranes (61.62) from rat liver. PhoSphorylase kinase and glycogen synthase control the degradation and synthesis of glycogen (63). In skeletal muscle. calcium-calmodulin stimulate phOSphorylase kinase to activate glycogen phOSphorylase and inhibit glycogen synthase (64). In liver. phosphorylase kinase appears to be regulated by phOSphorylation of the enzyme by a cAMP-dependent protein kinase. as well as by a calcium-mediated activation (65-67). PhoSphorylation of liver glycogen synthase by phOSphorylase kinase has also been shown to inhibit glycogen synthase (68). Molecular Regulation of Intracellular Calcium Concentrations Cells. in general. contain between 1 and 2 umol calcium per gm wet weight. Most of the total cellular calcium is bound to fixed anionic binding sites of glycoproteins. proteins and phOSpholipids. with a smaller fraction complexed with mobile anions such as inorganic phoSphate (3). In intact tissues. there is a rapidly exchangeable cellular calcium pool. representing 10-20% of the total cell calcium. which is bound to glycoproteins. glycolipids and muc0polysaccharides on the external cell surface (69). Rapid cell fractionation has shown that 70-80% of the total intracellular calcium is located in the mitochondria of hepatocytes (70.71) and synaptosomes (72). Free calcium concentrations in the mitochondrial matrix is about 16 uM (73). The endoplasmic reticulum appears to account for most of the remainder of the organelle-bound calcium. Cytosolic free calcium in isolated hepatocytes is about 0.2 uM (70). while cytosolic bound calcium is 700-800 times greater (73). The question now arises as to what maintains the intracellular calcium levels of the cytosol. mitochondria and endoplasmic reticulum.. The concentration of free calcium in the blood is 1.25 mM. while the free calcium concentration in the cytosol of most cells is 0.1-0.2 uM (70.74). Consequently. there is a gradient across the plasma membrane of about 104. Two mechanisms have arisen which maintain this calcium gradient. The first is the 2+ 2+ Na+-Ca exchange or Na+/Ca antiporter of excitatory and secretory cells (75.76). However. studies in liver show no 2+ appreciable Na+-Ca exchange (77.78). The second mechanism for calcium efflux is by a unidirectional MgZ+-dependent Ca2+-ATPase. as demonstrated in erythrocytes (SO-S3). adipocytes (S4). pancreatic islets (56). heart (57). and liver (61.62). The endOplasmic reticulum also contains calcium pumps similar to those observed in plasma membranes. Calcium tranSport by the sarcoplasmic reticulum (Ca2+—Mg2+)ATPase has been extensively studied in skeletal and cardiac muscle (46-49). A calcium pump in endOplasmic reticulum of liver has also been demonstrated (58-60). but is poorly understood. Uptake and efflux of calcium in mitochondria occurs by different mechanisms. Uptake of calcium takes place by an electrOphoretic uniport mechanism. which is energetically driven by the electrogenic proton pump of the reSpiratory chain (75.79.80). The efflux carrier is electroneutral and catalyzes the exchange of one Ca2+ for two H+ in liver mitochondria (81). and one Ca2+ for two Na... in heart and brain mitochondria (79). The vmax for calcium Uptake is 100 times greater than the vmax efflux rate (82.83). It has been suggested that the mitochondria may be reSponsible for buffering the cytosolic free calcium in intact cells (84). Agonist-Induced Increases in Intracellular Free Calcium - A wide variety of agonists can act on various tissues and cell types to increase the level of intracellular calcium. The cholinergic agonist. acetylcholine. stimulates an increase in cytosolic free calcium in adrenal medulla (35). pancreas (85). parotid gland (86) and iris smooth muscle (87). Glucose stimulates calcium accumulation in the Islet of Langerhans (39). IgE. concanavalin A or Specific antigens act on mast cells to release histamine. with a correSponding increase in intracellular calcium (88). Cholecystokinin-pancreozymin acting on pancreas (33). thrombin. collagen. and ADP on platelets (37). and 5-hydroxytryptamine (89) acting on blowfly salivary glands. all induce an increase in the level of intracellular calcium. Whether the increase in free cytosolic calcium is due to an increase in the influx or a decrease in the efflux of calcium from the external media. or an increase in calcium efflux from intracellular sites. is still a subject of controversy. In these tissues. most of the evidence points to a depolarization of the plasma membrane leading to an opening of calcium channels and. hence. increased influx of extracellular calcium. The addition of vaSOpressin. angiotensin II or cX-adrenergic agonists (phenylephrine. epinephrine and norepinephrine) to rat liver increases intracellular calcium levels (3-7). VaSOpressin and phenylephrine induce the release of calcium from intracellular stores. probably mitochondria and endoplasmic reticulum (6). and also inhibit calcium tranSport by plasma membrane vesicles. presumably by inhibiting the (Ca2+-MgZ+)ATPase (7). Glucagon. when added at supraphysiological concentrations to rat liver. increases cytosolic free calcium levels (5). The fl-adrenergic agonist iSOproternol. also increases cytosolic calcium levels when added to mouse kidney cortex slices (90). PhoSQhoinositide Biosynthesis and Degradation - In almost all eucaryotic cells. the predominant inositol lipid is phosphatidylinositol (PI) (Fig. 1). Typical concentrations of this lipid range from 0.5-2.5 umol per gm of tissue. which is equivilant to about 2-12% of the total phOSpholipids of the cell (91-93). The phOSphorylated derivatives of PI. phoSphatidylinositol 4-phOSphate (PI—P) and phOSphatidylinositol 4.5-bi5ph05phate (PI—P2) (Fig. l). are also found in a wide range of animal cells (94.95). yeast (96) and higher plants (97). although only in trace quantities (0.1% of the total phOSpholipids). It is difficult to assess the concentration of the phosphorylated derivatives of PI since they break down rapidly post—mortem. and are difficult to extract quantitatively (94.95). The brain appears to have the highest level of PI-P and PI—P2 at 0.153 umol and 0.083 umol per gm of tissue. respectively. in unmyelinated brain. and 0.219 umol PI-P and 0.280 umol PI—P2 per gm of tissue in myelinated brain (98). Platelets (99) and kidney (98) also appear to be rich sources of polyphoSphoinositides (PI-P and PI-Pz). Inositol lipids in animal cells possess uncommonly high prOportions of stearoyl residue at position 1 and arachidonyl at position 2 (100). The phosphoinositide biosynthetic and degradative pathways are outlined in Figure 2. The biosynthesis of phoSphatidylinositol occurs by two mechanisms. both of which take place in the endOplasmic reticulum (1.101). The first involves the formation of CDP-diglyceride from phosphatidic acid and CTP. catalyzed by CDP-diglyceride synthetase. PI 10 Figure 1. Structure of inositol lipids. 11 OH OH 0 9 Ptd Ins o-f-o-cu2 on OH 0' CHOCOR cnzocon' 2- oeo3 0“ a ‘3. Ptd Ins 4-P O-P-O-CHZ OH OH 0" CHOCOR CHZOCOR' z— oeo3 0H Q 9 PM Ins 4, 5-P2 o-g-o-cu2 OH on 0' cHocoa cnzocoa' Figure 1 12 Figure 2. Pathways for the biosynthesis and degradation of phosphoinositides. 13 Gttcerol __> Glycerol-3- 4-— Dthydroxyacetone-<_ Glucose phosphate ——-> phosphate Fatty ac:d Phosphatidic acid Acyt-CoA t cw 0 ADP 1 C 1 - l I 1. ATP CDP Dtacy g ycero PP PhOSphatIdyICholine InOSitOI 1- P 2 r|nosltol + Inositol 1:2- 1.2-Dnacyl- cyclic-P \ 7 Phosphattdyt- ethanotamine Q'YCGVO' ‘ pl + CMP Inositol- ATP dtphosphate 3 TnaCylglyceroI ADP InOSIiOI- p|_p trtphosphate 5 ATP 4 ADP Pl-bts P Figure 2 14 is then formed by the transfer of inositol to CDP-diglyceride to form PI and CTP in a reaction catalyzed by CDP-diglyceride-inositol transferase. PI can also be formed by a base exchange reaction (PI + I*v——A'PI* + I). PI-kinase. which catalyzes the phoSphorylation of PI by ATP to form PI-P. is found in a variety of subcellular fractions and tissues. Among these are lysosomal membranes (102.103). golgi (104). microsomes (102.105-107). nuclear enveIOpes (102.105) and plasma membranes (102.105.107) of rat liver: and plasma membranes of rat brain. kidney. heart. skeletal muscle. testis. and human erythrocytes (107). PI-P kinase is associated with nuclear envelOpes. plasma membranes and microsomes of rat liver (102.105): erythrocyte membranes (108); and is a soluble enzyme in brain (109). In rat liver. the highest Specific activity of PI kinase is found in microsomes. while plasma membranes contain the highest Specific activity of PI-P kinase. The mitochondria of rat liver possess little or no phoSphoinositide or polyphoSphoinositide synthetic activity (101.102). The breakdown of phOSphatidylinositol procedes primarily by cleavage of the glycerol—phoSphate bond by a phoSpholipase C activity. PI-Specific phOSpholipase C activity is localized in the cytosolic fraction and requires calcium for maximal activity. This activity exist in a number of tissues including platelets (110.111). rat liver (112.113). sheep seminal vesicle glands (114). iris smooth muscle (115) and rat brain (116). Various reports suggests 15 that a Specific phoSpholipase A in platelets (117). 2 neutrophils (118). and pancreatic acini (119) acts on PI causing the release of arachidonic acid. The polyphoSphoinositides are degraded by two mechanisms: 1) directly by a phOSpholipase C activity to form inositol polyphOSphates and diacylglycerol. or 2) by action of phoSphomonoeSterases to form PI which is then degraded. PhOSphomonoesteraseS. which act on PI-P are 2. found in brain (120.121). kidney (122). iris smooth muscle (123). and human erythrocytes (124.125). PI-P2 phOSphatase from brain (121) and kidney (122) hydrolyzes PI-P as well as PI-Pz. A metal independent. PI-P Specific phOSphatase has been described in human erythrocyte membranes (126) and rat liver nuclear enve10pes (127). PolyphOSphoinositide phoSphodiesterase. like PI phoSphodiesterase. requires calcium for Optimal activity. This activity exist in brain (128-130). erythrocyte membranes (131.132). platelets (133.134). rat hepatocytes (11) and iris smooth muscle (123). Further descriptions and references concerning the phOSphoinositide biosynthetic and degradative enzymes can be found in a recent review by Irvine (135). Stimuli—Induced Changes in Cellular PhOSphoinositide Levels Hormonal regulation of phOSphatidylinositol metabolism was first observed by Hokin and Hokin (136) in pancreas. Since that report. a variety of tissues and cell types have been examined for agonist induced changes in intracellular PI 16 levels. A list of these tissues and cell types. and the stimuli which induce the "PI reSponse" has been compiled by Michell (1.2) and is Shown in Table 1. Among these stimuli are vaSOpressin. angiotensin II and dradrenergic agonists Operating in liver: muscarinic and cholinergic agonist in secretory tissue and smooth muscle; S-hydroxytryptamine in blowfly salivary glands; thrombin in platelets: and platelet activating factor (l-O—alkyl-Z-acetyl-gg-g1yceryl-3- phOSphorylcholine) in platelets (137) and rat hepatocytes (138). Further investigation shows that the metabolism of the polyphOSphoinositides is also influenced by the same agents (8—lO.l39-l46). The evidence suggest that these agents stimulate a phOSphodiesterase to produce diacylglycerol and the correSponding inositol phOSphate (11.134.146-148). PhOSphoinositide Effects on Cellular Function - The possible role of phoSphoinositides in cell function can be classified into three catagories: 1) effects on Specific enzymes. 2) effects on membrane structure. and 3) phOSphoinositide metabolites effect on cellular calcium homeostasis and protein kinase C activity. PhoSpholipid-protein interactions are known to affect the activities of many membrane-associated enzymes (149). Acidic phoSpholipids are associated with the (Na+-K+)ATPase protein in isolated microsomal membranes from rabbit kidney (150) and membranes isolated from Torpedo marmorata electric 17 Table I Receptor Whose Stimulation Provokes a ‘PI response' (PI breakdown or enhanced PI labelling) Type of Receptgg Cholinergic (muscarinic) Adrenergic (an) Histamine (H1) S-Hydroxytryptamine (S-HTI) VasOpressin (V1) Pancreozymin Substance P Bombesin Angiotensin II Bradykinin ThyrotrOpin f-Met-Leu-Phe. phagocyticable particles Secretagogues (antigens. etc.) Thrombin. collagen. ADP Membrane depolarization Glucose in ApprOpriate Target Tissue TlSSU. Brain. synaptosomes. iris smooth muscle. parotid gland Hepatocytes. adipocytes. brain. iris smooth muscle. parotid and submaxillary gland Smooth muscle. brain Smooth muscle. brain. blowfly salivary gland Hepatocytes. smooth muscle Pancreas. smooth muscle Parotid. hypothalmus Pancreas Mepatocytes Endothelia. kidney Therid NeutrOphils. macrOphages Mast cells Platelets Smooth muscle. ganglia. synaptosomes Islets of Langerhans 18 organ (151). PhoSphoinositides may be involved in the regulation of canine renal (Na+-K+)ATPase (152). The (Ca2+-MgZ+)ATPase in erythrocyte membranes (50.153.154) and sarcoplasmic reticulum (155) is stimulated by increased levels of polyphOSphoinositides. Studies done by Smith and Wells (156) demonstrated that the nuclear membrane ATPase can be synergistically activated by RNA and polyphOSphoinoSitides. PolyphoSphoinositides also activate cholesterol side chain cleavage with purified cytochrome P-450 (157). and inactivate low Km cAMP phOSphodiesterase in rat adipocyte microsomes (158). PhOSphatidylinositol has been proposed to act as an unCOUpler of‘fl—adrenergic receptors from the remainder of the adenylate cyclase complex (159). Acidic phOSpholipids. along with divalent cations are important factors in membrane fusion (160). Sundler and PapahadjOUpoulos (161) have prOposed that head-group hydration is critical in the fusion process. Thus. the polyphOSphoinoSitideS. which are highly hydrated. would tend to block fusion. Indeed. the fusion of chick embryo myoblast. initiated by raising the medium calcium levels. also results in polyphoSphoinositide breakdown (162). Pickard and Hawthorne (163) suggested that the breakdown of phoSphatidylinositol to form diacylglycerol. promotes the fusion of vesicles and plasma membranes during exocytosis. The lateral mobility of glycoproteins in erythrocyte membranes is increased by the addition of PI-P2 (164). 19 In 1975. Michell (1) reviewed phOSphatidylinositol metabolism and suggested that phoSphatidylinositol breakdown may somehow influence intracellular calcium concentrations. The stimulation of many different types of recceptors in different tissues. brought about PI breakdown and also increased calcium concentrations in the cytosol. PI breakdown does not require the presence of extracellular calcium. is unaffected by the addition of the calcium ionOphore A23187 in the presence of calcium. and stimulation of glucagon or fiLadrenergic receptors does not provoke PI breakdown. The same agonist. which were involved in stimulating PI breakdown and intracellular calcium elevation. also stimulate polyphoSphoinositide breakdown. but at a much faster rate (165). Although the breakdown of polyphoSphoinositides is thought not to be regulated by calcium. it does appear to be calcium dependent (8.9.11). Exactly how polyphOSphoinositides are involved in cellular calcium homeostasis is just now becoming apparent. Agonists. which stimulate the breakdown of polyphoSphoinositides. also inhibit calcium tranSport in plasma membrane vesicles isolated from rat liver (7). Furthermore. PI-P and PI-P2 stimulate the (Ca2+—Mg2+)ATPase (50.153.154). Eye-Inositol triSphoSphate (I-P3)' the product of phoSphodiesterase action Upon PI-P mobilizes 2! intracellular non—mitochondrial calcium stores in saponin-permeabilized pancreatic acinar cells (12) and rat hepatocytes (13). Therefore. phOSphodiesterase catalyzed 20 breakdown of PI-P2 would inhibit the plasma membrane calcium pump. decreasing the calcium efflux rate out of the cell. and the product. I-P3. would act to mobilize intracellular calcium Stores. The combined result would be to increase cytosolic free calcium concentrations. PolyphOSphoinositide breakdown may also be involved in protein phOSphorylation. Jolles et al. (166) demonstrated that corticotrOpin stimulates both protein phOSphorylation and polyphoSphoinositide turnover in brain. Thrombin can stimulate polyphOSphoinositide turnover in platelets and also phOSphorylation of an endogenous 40k dalton protein (167). which results in serotonin secretion. This phOSphorylation is catalyzed by a calcium-activated. phoSpholipid-dependent protein kinase (protein kinase C). Protein kinase C can be activated igzgitgg by diacylglcerol (45) suggesting that polyphOSphoinositide breakdown. which produces diacylglycerol and elevates cytosolic calcium. activate protein kinase C (168). leading to some cellular reSponse. A similar kinase activity has been described in rat hepatocytes where a 16k dalton protein present in purified plasma membranes and cytosol can be phoSphorylated by an endogenous protein kinase C activity (169). This activity is Stimulated in intact hepatocytes by the addition of vaSOpressin. The turnover of phOSphoinositideS may be involved in controlling cell growth. It was recently reported that the purified Rous sarcoma virus (RSV) transforming gene product. 21 v—src pp60 . which has tyrosine kinase activity. can also phoSphorylate PI. PI-P. and 1.2—diacylglycerol (170). The transforming protein of avian sarcoma virus UR2. psev-ros’ also possesses tyrosine kinase and PI kinase activity (171). Transformation of fibroblast by a temperature sensitive mutant of RSV led to a large increase in the level of 32 P-labelled PI-P. PI-P2 and phOSphatidic acid only at the permissive temperature (170). Cells transformed by UR2 showed both an increase in 32P-labelling of PI-P and PI-P2 relative to other phoSphlipids and an accumulation of I-P2 and I-P3 (171). The link between phoSphoinositide turnover and cell transformation may occur through protein kinase C. The tumor promoting phorbol esters can Stimulate protein kinase C activity in the absence of phOSphatidylinositol turnover or production of diacylglycerol (172). Thus. the phorbol esters may be imitating the action of diacylglycerol by intercalating into the cell membrane and activating the kinase. The kinase may remain activated for prolonged periods of time Since the phorbol esters are very slowly metabolized (173). In contrast. production of diacylglycerol occurs transiently during phOSphoinositide turnover in normal cells and disappears rapidly. 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(1980) CRC Crit. Rev. Toxicol. 9. 153-234. Michell. R.H. (1982) Cell Calcium 9. 429-440. CHAPTER I Subcellular Incorporation of 32P into Phosphoinositides and Other Phospholipids in Isolated Hepatocytes 34 35 ABSTRACT Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophos- phate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32p incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid. This data suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32F into ph05phatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-biSphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32F into DPI occurs in other subcellular sites, especially lysosomes. 36 FOOTNOTES 1The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, ph05phatidylinositol; PG, phosphatidyl- glycerol; PA, phosphatidic acid; PS, ph05phatidylserine; DPI (diphos- phoinositide), phosphatidylinositol 4—phosphate; TPI (triphospho- inositide), phosphatidylinositol 4,5-bisphosphate; GPC, glycerophos- phorylcholine; GPE, glycerophosphorylethanolamine; GPI, glycerophos- phorylinositol; GPG, glycerophosphorylglycerol; GP, glycerol phosphate; GPS, glycerophosphorylserine; GPIP, glycerophosphorylinositol 4-phos- phate; GPIPZ, glycerophosphorylinositol 4,5-bisphosphate; HEPES, N-Z-hydroxyethylpiperazine—N'-2-ethanesulfonic acid; EDTA, ethylene- diaminetetraacetic acid; TRICINE, N-trisEhydroxymethylJmethyl glycine; PMSF, phenylmethylsulfonyl fluoride; HB, homogenizing buffer; HPLC, high pressure liquid chromatography. 37 INTRODUCTION Hormonal regulation of phosphatidylinositol (PI)1 metabolism was first observed in the pancreas by Hokin and Hokin (1). Since that report, the influence of hormones upon the phosphatidate-inositide cycle has been studied in a number of tissues and cell types. Agonist induced changes in phosphoinositides and phosphatidic acid have been reported in kidney (2,3), iris smooth muscle (4,5), pancreas (1,6,7), parotid gland (8,9), adrenals (10), adipose tissue (11,12), platelets (13,14), brain (9,15,16), insect salivary glands (9,17) and liver (18-23). However, nearly all these studies measured whole cell phos- pholipid metabolism and not in specific organelles where the site of hormone action may occur. Evidence suggests that the plasma membrane is the subcellular site of increased PI hydrolysis in response to vasopressin (20,24). However, vasopressin stimulated hydrolysis of ph05phatidylinositol 4,5-bisphosphate (TPI) could not be attributed to any specific subcellular fraction presumably due to the extreme lability of this ph05pholipid (23). Studies on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) metabolism have suggested regulation by insulin (25), glucagon (25,26), cAMP analogues (27), vasopressin and angiotension (28) in rat hepatocytes. However, the intracellular site of hormone action was presumed to be the endoplasmic reticulum although this was not fully documented. 38 In this report, a rapid subcellular fractionation was used to prepare microsomes, nuclei, mitochondria, lysosomes and plasma membranes from isolated rat hepatocytes which induced minimal post-homogenization artifacts. Phospholipid metabolism in these fractions was examined by incubating the hepatocytes with 32Pi, isolating the organelles and extracting their component phospholipids. These phospholipids were deacylated and the glycerophosphate esters analyzed by a new method utilizing anion exchange high pressure liquid chromatography. 39 EXPERIMENTAL PROCEDURES Materials - (32P)0rthophosphate, carrier free, was purchased from ICN. Phospholipid standards were obtained from either Sigma or Serdary Research Laboratories. Glass distilled organic solvents were obtained from MCB. Collagenase (E.C. 3.4.99.5) CLSII was purchased from North- ington. Penicillin-streptomycin solution (10,000 units/ml penicillin, 10 mg/ml streptomycin), MEM non-essential amino acids solution (100X), MEM amino acids solution with 100 mM L-glutamine (50X) and MEM vitamin solution (100X) were obtained from Grand Island Biological Co. CD-Sprague Dawley male rats were acquired from Charles River Laboratories. Roller bottles (490 cm2) were a product of Corning. Percoll was purchased from Pharmacia. All other chemical were of reagent grade. Hepatocyte Isolation - Hepatocytes were isolated from 200-250 gm fed male rats by the method of Seglen (29) as modified by Kurtz and Wells (30). The procedure was further modified by providing the perfusion buffer with 10 mM glucose and the normal concentration of MEM non-essential and essential amino acids. Typically, 4.0-6.0 x 108 cells of 85—95% viability, as judged by Trypan blue exclusion, were obtained from a single animal. The cells were suspended at a concentration of 1.0-2.0 x 106 cells/ml in media modeled after Dulbecco's modified Eagle's medium. This medium contained (per liter) 40 6.4 9 NaCl, 3.7 g NaHC03, 0.4 9 KCl, 0.2 g anhydrous CaClz, 98 mg anhydrous M9504, 0.11 g sodium pyruvate, 1.0 9 glucose, 4.8 g HEPES, 15 mg phenol red, 40 ug FeCl3, 14.0 mg NaH2P04-H20, 20.0 9 bovine serum albumin (Fraction V), 10 ml of penicillin-streptomycin solution, 10 ml of 100x MEM non-essential amino acids solution, 20 ml of 50 HEN essential amino acids solution and 10 ml (100X) MEM vitamin solution at a pH of 7.3. Cell suspensions of 35 ml were placed in 490 cm2 tissue culture roller bottles which were flushed every 30 min with a 10 sec burst of 95% 02/5% C02, sealed and rotated at 2 r.p.m. at 37°. Under these conditions, the cells remained well suspended and exhibited no loss in viability over a 3 hr period. Hepatocyte Incubations - Carrier-free 32Pi was added to the cell suspensions at the level of 5-10 uCi/IO6 cells. At various times of incubation up to 120 min, the cells were diluted with 7 volumes of ice-cold homogenizing buffer (HB) which consisted of 250 mM sucrose, 1 mM EDTA, 10 mM NaN3, 20 mM NaF, 75 mg/l PMSF and 10 mM TRICINE pH 8.0. The cells were pelleted at 75 x g for 5 min at 4°C. The cell pellet was resuspended in 5.0 ml of ice-cold HB. Cell Homogenization and Subcellular Fractionation - The cell suspension was homogenized and fractionated at 0-4°C as described in Figure 1. After the second homogenization step, cell breakage exceeded 95% as judged by phase contrast microscopy. The stock 45% Percoll solution was prepared in homogenizing buffer (HB). Resuspension of the 1500 x g pellet and subsequent "nuclear" pellets was accomplished by 3 strokes of a tight fitting glass-glass Potter-Elvehjem homogenizer. All other 41 Figure 1. Flow diagram for the subcellular fractionation of rat hepatocytes. Hepatocytes were isolated and labeled with 32P1 for various times as described under "Experimental Procedures”. The cells were transferred into 7 volumes of ice cold HB and pelleted at 4°C. The cell pellet was gently reSUSpended in 5.0 ml of ice cold HB and subjected to the fractionation scheme at 4°C depicted to the right. 42 F 0000.. .0000 0e .0_.00 CE 3 0 3.. IE ....::.. E .. r. .... .. ...; 2.52:2...» 3.00092! pit P . 0.... 0 0 x 000. 0020.000 .... 0.. 0 0.0.0. 0. . ...... .0. 0.0. o. 0.2.0 2 20.00.0000 Luv”... . x 000.0. 0 0.0.000 ... . 000 . 0.. ....s . .... 0 0 . u. ..0 0.0. 0. 0.2.0 . fl 00.00.0000 ..0 0.. .06.. .....molflam F300 3000000025 . H a 0 .000 00 .0 . 000.00. 00... ..000 .0 . 0.4. 00 :0... ...... 0.0 2000.0... 0.05. . .00.. 0 x 000 00. 0.... 0 .0 x 0000 000.5000 .0 2.00. 600.000 an.“ .0. 0.0 03 .0 0030.000 0.... 00 0.. ...... 0.0.... 00.0.... 0.0.0. . .00.. SE0. .0 x 08.0.“ eo:.t.ceo 8 .E 0.0. 0. 05:0 3 6090.5 268.4. .E O.“ c. 0:000:00: 3 :03 ...: :0. . 13:00.00 20.00.0000 .0000 5.0.. 89 .E O.‘ 8‘ .N F . ar-SCEa-dm C 0: V =0 0.. 0. 00000000.. .. .000 0... 20.00.0000 .0000 .0 x 000.0. 0020.000 .0 w b :03 ...! fio. . ...m. .....0 0. .0 x 80. 000.5000 8 .3200 .... 0.0 004 .0 0.010 050..» 8:5. .000 0. . . 0.. .....00... 0.0.0. 68.000 3.000... .0 x 000.0. 000.5000 .0. 0 0 0. 00003000 .. ..0 0.0. 0. 000000000 2 28050....” 6.3% 3:00 .000 00 .0 . 000.0. 0030.000 .0 ....E r. .0 x oven 000.5000 :0! 0.2 .mo. . mm. .303... 000.0 .... 0.. . not 8 8:82:63 02.3600 a... .E of. ... sceneseec .. r «2.0% 0.0. 0 .0 .. 000. 000.5000 .0 . : 0. 00 0: ..0 0.0 0. .0000 0000006000.. .0 0.0. 0 .0 x 000. 000.5000 a 8.0.. on ..000 on 30.62.00; how .253» 050: 00300060: A. .0... .0000 0030002000 .0. 0.0 0. 0:00 00. x . mmtoohklmx h<¢ HO ZOFdZOFOSun. Isadamomam 43 pellets were resuspended using 3 strokes of a Teflon-glass Potter-Elvehjem homogenizer. The final pellets were resuspended in 1.0 ml of HB. Complete fractionation of five groups of cells was accomplished in 3.5-4.0 hrs. Using the marker enzymes hexosaminidase (31) for lysosomes, fumarase (32) for mitochondria, alkaline phosphodiesterase I (33) for plasma membrane and glucose 6-phosphatase (34) in the presence of 40 mM L-tartrate (35) for microsomes, the organelle content of the various fractions was determined using the method reported by Leighton gt al. (36). Protein was determined by fluorescamine (37) using bovine serum albumin as the standard. DNA was measured using the method of Karsten and Wollenberger (38). Phospholipid Isolation and Deacylation - The total phospholipid fraction was isolated by a modification of the method of Schacht (39). Briefly, 3.0 ml of methanolzchloroform (2:1 v/v) were added to the resuspended pellets followed by 1.0 ml of 2.4M HCl and 1.0 ml of chloroform, and the contents were vortexed for 15 sec. The resulting two phases were separated by centrifugation. The lower phase was set aside and the upper phase washed first with 2.0 ml chloroform and then with l.0 ml chloroform. The lower phases were combined and washed with 5.0 ml of methanol:1N HCL (1:1 v/v). The lower phase was removed and evaporated to dryness using a N2 stream at 37°C. The residue was dissolved in 0.5 ml of methanolzchlorofonm (3:2 v/v). The phospholipids were deacylated using the mild alkaline hydrolysis procedure described by Kates (40). A minor modification was made which allowed the pH of the polar fraction to remain slightly 44 alkaline (pH 93, 8.0) by using only 0.1 ml of Dowex 50 [HT]. This modification resulted in increasing the recovery of glycerolphosphoryl- choline. The polar fraction was evaporated using a N2 stream at 37° and dissolved in 100 pl of 20 mM ammonium borate, 100 mM ammonium formate, pH 9.5. Analysis of Deacylated Phospholipids by High Pressure Anion Exchange Liquid Chromatography - The deacylated products were analyzed by high pressure liquid chromatography (HPLC) on a 250 x 4 mm BioRad Aminex A-27 anion exchange column using a modification of the ion exchange procedure described by Dittmer and Wells (41). A polyphasic gradient beginning with 100 mM ammonium formate, 20 mM ammonium borate, pH 9.5 and ending with 750 mM ammonium formate, 20 mM ammonium borate, pH 9.5 was established by a Beckman Model 342 Gradient Liquid Chromatograph and used to elute the deacylated phospholipids (Fig. 2A). Fractions of 0.78 ml were collected at a rate of 0.6 ml/min in Pyrex tubes. The fractions were either counted by Cerenkov radiation or evaporated overnight at 150°C in a vented hood. The residue which remained in the tubes after evaporation was digested with 0.3 ml of 14% H2804:12% HClO4 (2:1 v/v) for 90 min at 180°C after placing glass marbles on top. The phosphate content of the fractions was determined by the method of Ames (42) with detection of the reduced phospho-molybdate complex at 820 nm. Determination of Cellular EV-3EPJ-ATP Specific Radioactivity - A 3.5 ml aliquot of cell suspension was removed after various times of 3291 labeling and placed in 500 pl of ice cold 5% HCl04. The HCl04-treated samples were homogenized using a glass-glass 45 Potter-Elvehjem homogenizer. placed on ice for 15 min and then centrifuged. The supernatant was removed and neutralized with 2M KHC03 at 4°C. The KClO4 was pelleted and the supernatant removed. The EV-32PJ-ATP specific radioactivity in the supernatant was determined using the method of Hawkins gt al. (43). 46 RESULTS Purity of Subcellular Fractions - Subcellular fractions were obtained by using the fractionation scheme outlined in Figure 1 and analyzed for various marker enzymes as summarized in Table I. The plasma membrane fraction was enriched in alkaline phosphodiesterase I activity greater than 4-fold over the whole homogenate. Fumarase activity in the mitochondrial fraction was enriched 2.2-fold over the whole homogenate. In the lysosomal fraction, hexosaminidase was enriched approximately 13—fold over the whole homogenate while glucose 6-phosphatase, in the presence of L-tartrate, was enriched nearly 2.8-fold in the microsomal fraction compared with the whole homogenate. DNA/protein was enriched 3-fold in the nuclear fraction compared with the whole homogenate. After measuring the contamination of other marker enzymes within each fraction and using the formula described by Leighton §t_gl, (36), the purity of the various fractions was-estimated (Table I). The fractionation process resulted in a plasma membrane fraction which consisted of 28% plasma membrane but also contained 57% microsomes. The mitochondrial fraction was composed of 60% mitochondria, 36% microsomes but only 2% plasma membrane. The lysosome fraction contained 19% lysosomes, approximately equal amounts of mitochondria and microsomes but only 2% plasma membranes. The microsome fraction was the purest fraction obtained and was composed of nearly 86% microsomes and 9% plasma membranes. 47 .0.0. .0.v. .n.0. .0mnw0 .0MUO. .wwwo. .0mnw0 0.00 «.00 000.0 + N0..0 0000.0 + 00.0.0 0.0.0 + 0000.0 «000.0 + 0000.0 .0500000.z .n.0n. .v.0v. .0... .wa.. .nmm00 .me.. .0mn0. 0.0 0.0. «0.0.0 + 0000.0 0000.0 + 0.0.0 000.0 + 000.0 mn00.0 + 00.0.0 .05000000 .0.nn. .0.n. .0.~. .0rww. ..mww. .mmww. .0mnw. 0.0 0.00 0000.0 + nn00.0 0000.0 + 0000.0 000.0 + 000.0 0000.0 + 00.0.0 .0.00500000.z .v.0n. .n.N. .0.N.. .0rw.. .00H0. .mmwo. .0nmwv ..0 0.00 0000.0 + 0000.0 .000.0 + .0n0.0 000.0 + ~0..0 0000.0 + 00..0 0500050: 0500.0 .nth. .NOHb. ..on.. .mmwo. .0mnb. 0.0. 0N0. H «0.0 0000.0H 900.0 0000.0 H. 0.00.0 00..0H 00n.0 0000.0 H 00.0.0 000.002 000. + n~.0 0.00.0 + 0000.0 0000.0 + n.n0.0 .n0.0 + 000.0 0000.0 + 0nn0.0 000500050: u u 00.000 5.00000\0.>.00< 0.0.0000 050 0050050 00x00: ..l .050.00.>00 00005000 «050.00000000 0000000000 0500000.0 0000 00 05005 000 000.0> ..00. ..0 00 50000.00 00 000005 000 00 0000.00.00 003 00..05000o 05.005.500500 00 0000500000 000 .uoo. 5000 0000000000 00..05000o 05.005.500000 00 0000500000 000 0. 00.000 50.00000 000 0..0x 50.005.500500 00 0000500000 000 0. 0.000050000 5. 000505 050000 000 ..00.>.000 0.0.0000 0>.00.00. 0005000500 000 00 >0.>.000 0.0.0000 000 00 0>.00.00 50.00000 05000505 50>.0 0 5. >0.>.000 0.0.0000 000 00 050500.050 000 00>.0 0.000050000 5. 000505 000.0 000 ..rxrv 5.00000\<20 000 05.5.500000 00 00005.000 00: 50.00000 000.005 000 5. .0.005 00 050500.050 .00000000000t0 00000.0 000 5.00000 00 05\5.5\000000000I0 00000.0 5000 00000.00 000000000 0.500005. 00 00.05000.5 050 «00000500 000 5.00000 00 05\5.5\000.05 5000 00000000 00000500 00 00.05000.5 .. 00000000.00000000 05..0x.0 000 0000000000505.n|05.0.5>00I.05000000.5I0 5000 050 0000.5.5000x00 000 00.5.500000.0|0| I.>0000|2I.>5000000.5u0 5000 5.00000 00 05\5.5\00000.00 .05000000.5n0 0o 00.05000.5 000 >0.>.000 0.0.0000 000 00.50 .00.000 05.00.00.00 5003 0.0.0000 00 00 0050000 003 000005 00.00.0000 0000 00.00 000 50.00000 000 00.0500. 005>~50 00x005 000 00 0000 0>000 050.0050.000 50.00000 000 .00000.0>0 050.00000 000 00 00000 550.00 0500:000. 000 5. 050.00000 00.000 50.00000 00 0005.000 050 0050050 00000: . 0.000 48 Anion Exchange HPLC Analysis of Deacylated Phospholipids - PhoSpholipid standards were deacylated and subjected to anion exchange HPLC as described under "Experimental Procedures". Eight glycerophosphoryl species were separated including glycerophosphorylinositol 4-phosphate (GPIP) and glycerophosphorylinositol 4,5-bisphosphate (GPIPZ) (Fig. 2A). Greater than 95% recovery of deacylated phospholipids was obtained. 32P-labeled phospholipids isolated from rat hepatocyte cell homogenate were similarly deacylated and chromatographed. As shown in Figure 28, radioactivity corresponded very well with lipid phosphorous with the exception that very little radioactivity was incorporated into PS (glycerophosphorylserine, GPS). Also, there was no detectable lipid phosphorous associated with DPI (GPIP) or TPI (GPIPZ), however, there was substantial 32F incorporation into these minor phospholipids. Estimation of Possible Post-Homogenization Artifacts - Although the subcellular fractionation process was relatively rapid, enzyme inhibitors were present in the buffer, and the fractionation was conducted at 0-4°C, the possibility of large changes in the level of 32P-labeled phospholipids after homogenization did exist. To investigate this possibility, an aliquot of cell homogenate was placed into methanolzchloroform (2:1, v/v) immediately after homogenization. Another aliquot was kept on ice and methanol:chloroform (2:1, v/v) was added following the completion of the subcellular fractionation. The phospholipids were isolated, deacylated and analyzed by anion exchange HPLC as described under "Experimental Procedures". In three different cell preparations, no change in the amount of 32p in PC, PG and TPI 49 Figure 2. Anion exchange aPLC elution profile of polar deacylated phospholipid products. Phospholipids were 32P isolated from hepatocytes, labeled for 60 min with i o deacylated with mild alkali and the polar products were analyzed on an Aminex A—27 anion exchange column as described under "EXperimental Procedures”. A. Deacylated phospholipid standards. O-—-O. absorbance at 820 nm of reduced phOSphomolybdate complexes; ---. ammonium formate concentration. 8. Deacylated phOSpholipids from hepatocyte cell homogenate. O-—-O. radioactivity as determined by Cerenkov radiation; O-—-O, nanomoles lipid phoSphorous. The position of elution of polar deacylated standards is indicated. 50 GPC GPE GPIGPG GP GPS GPIP GPIP2__ | I l | I l | /| AO.40~ A ’ “0.70M E C o N g - 3 0150 0 g 30.20- /’ 3 3 ’ 9L q . 0.30 a? b 2 , I x, I ’/ OILfiitJII I (lIO 0 IO 20 60 Fraction No.(0.78m|) GPC GPEGPIfiPG GP GPS GFilF’ GFIIPZ I I l I I I I B o —4 '9 e—A A *3 x h, J..” o- ”If ' =r2'o 'o I o .. ..0... X 3 ' ° _ E _ I o. o o E I- °fi I “I O C ' _ o I I \I ° ' m4 0 l IO Fro ciion No. (0.78 ml) Figure 2 51 occurred during fractionation. Only a 1-2% decrease in 32P-labeled PE, PI and P5 was observed. PA appeared to increase an average of 7% during fractionation while DPI decreased an average of 13%. The isolation of purified nuclei require the use of divalent cations, usually M92+ or Ca2+, to prevent aggregation of nuclei with other subcellular organelles (44). Therefore, a similar experi- ment was performed to determine if any changes in 32P-labeled phospholipids occurred in the crude nuclear fraction in the presence of MgZ+. After removal of plasma membranes, the crude nuclear fraction was resuspended in 250 mM sucrose, 10 mM MgCl2 and 10 mM Tricine pH 8.0 (Fig. 1). An aliquot was removed and placed in methanolzchloroform (2:1, v/v). Another aliquot was placed on ice and then placed in methanolzchloroform (2:1, v/v) following isolation of the nuclear pellet. Analysis of the deacylated phospholipids showed that large changes in the amount of 32p in PC, PE, PI and PA occurred during the isolation of nuclei in the presence of buffer containing MgClz even at 4°C. Only small changes (ca. 5-10%) were seen in the amount of 32F in PG, PS, DPI and TPI (data not shown). Therefore, the incorporation rates of 32P into phospholipids isolated from partially purified nuclei that were measured are only valid for PG, PS, DPI and TPI. The possibility of exchange of labeled phospholipids between organelles after homogenization was also investigated by mixing 32P-labeled microsomes obtained using the scheme depicted in Figure 1 with unlabeled rat hepatocyte cell homogenate. The homogenate was fractionated by the standard procedure and an analysis of the partially purified fractions revealed 81.5% of the radioactivity was recovered in 52 the microsomal fraction, 9% in the plasma membrane, 8% in the mitochon- drial fraction and 1.4% of the radioactivity was recovered in the lysosome fraction. Rate of Incorporation of 333 into Phospholipids of Various Subcellular Fractions - Isolated hepatocytes were labeled with 32Pi for various times and fractionated. The phospholipids were isolated, deacylated and analyzed by anion exchange HPLC as described under "Experimental Procedures". The results are shown in Figures 3A-H. The data shown are the levels of 32P in the various glycerophosphate esters which are a measurement of the amount of 32p incorporated into various phospholipids. The rates of 32F incorporation per mg protein into PC, PI, PG, and PA were similar between the lysosomal, microsomal and plasma membrane fractions but slightly slower in the mitochondrial fraction. Similar rates of 32P incorporation into PE were seen in the lysosomal, plasma membrane and mitochondrial fractions but 32P incorporation rates into PE were twice as fast in the microsomal fraction compared with the other subcellular fractions. The nuclear fraction has the fastest rate of 32F incorporation into P5 of all the fractions analyzed. However, the most striking result was the rapid and extensive amount of 32P incorporated into DPI and TPI in the plasma membrane fraction compared with the other subcellular fractions. After 30 min of 32Pi labeling, the amount of 32P incorporated per mg protein into TPI was approximately 5 and 2.5 times greater than the amount of 32F incorporated into PI and DPI, respectively, in the plasma membrane fraction. 32F incorporation into DPI was nearly 2 times greater than PI after 30 min of labeling 53 Figure 3. Time course of 32? incorporation into various phospholipids isolated from subcellular fractions of rat hepatocytes. Hepatocytes were isolated and labeled for various times with 32?i as described under ”Experimental Procedures." The 32P-labeled hepatocytes were homogenized and fractionated as described in Figure l. The phospholipids were isolated from the various subcellular fractions. deacylated with mild alkali and analyzed by anion exchange HPLC as described under ”EXperimental Procedures.” A. Glycerophosphorylcholine; B. GlycerOphosphorylethanolamine3 C. Glycerophosphorylinositol3 D. Glycerophosphorylglycerol2 E. Glycerol phosphate; F. GlycerOphOSphorylserine; G. GlycerOphosphorylinositol d-phosphate; F. GlycerOphosphorylinositol 4.5-bisphosphate. O—O. Cell homogenate: D-..-D, Nuclear fraction: H: Plasma membrane fraction: O-——O, Hicrosomal fraction: I—I. Lysosomal fraction: ‘—A: Mitochondrial fraction. and x---x. [8-32P1ATP. The results shown are from one of three similar hepatocyte preparations. 54 Glycerophosphorylcholine 54o - - A - microsomal \ J: l0 0 I _ plasma membrane \lysosomai— mitochondrial cpm X1 0'2/ mg protein .. o: 8 8 l T cell homogenate 60— . - l L 1 O 60 90 120 Time (min) Figure 3A 55 Glycerophosphorylethanolamine membrane\ ‘ A 270 r c .— B .- c: '5 21 o— , — fl 2 microsomal \ Q _ - O! E ‘2' 1 5O - - 2 plasma X E a 0 / l0 0 r / / lysosomal - I /\—mltochondrial _ / 4‘) 30~ K / . cell " / homogenate é.’ ' l l l 0 3O 60 90 1 20 Time (min) Figure 3B 56 360 _ Glycerophosphoryllnositol _ -(3 _ .5 0 280 — " g A 2 \ g P microsomal \ £13m; E N\ 200 ’ \Iysosomal " 23 Inna. F T O X F. o 1 20 " '- _ o . cell homogenate 40 ~ — / 1 l l l 0 3O 60 90 1 20 Time (min) Figure 3C 57 1 20 _ Glycerophosphorylglycerol C _ u .8 D microsomal\ H 2 90 0- _ I O! lysosome \/_I A E - /' K nuclear - N plasma '0 , membrane F 60 I— :; \_,rnno]. Q _. o O 30 T \cell homogenate“ / 21./J l l J O 30 60 90 1 20 Time (min) Figure 3D 58 GI ceroi hos hate 320— y p p -+ 2.0 ‘ ~< _ 0) E Io ‘ .g 240- ,,/”‘\\ -1 5 5 x ‘v 8 7 329,“? / lysosomal / 'U h- __ / 1 ca- : \' {microsomal ‘ 3 SE 160i —'IJ) ;; N xplasma 'o membrane 3 P 2 X ° T' E . ,1 8 80 mitochondrial '* 0'5 : >< cell homogenate . q l 1 l " 1 O 30 60 90 120 Time (min) Figure 3E 59 Giycerophosphorylserine _ 30‘ F I C - nuclear 4.0 O h- a' 20" I " D E \ ‘2‘ CD ” " lyso\‘ ‘ ;2 \\nflu1 a . A O 10 _ micro. '- O / plasma membrane . ' \cell homogenate 4,55” 1 1 I O 30 60 90 120 Time (min) Figure 3F 60 Glycerophosphorylinositol 4-phosphate 120*- \ plasma membrane cpm X1 0'2/ mg protein a o l l 7 I l O 30 60 90 120 Time (min) Figure 3G 270 cpm X1 6ng protein Figure 3H 61 L Glycerophosphorylinositol 4,5- bisphosphate cell homogenate \ ' 60 \ plasma membrane nuclear\ . Kmicro. \lysosomal . A \mitochondLlal 90 1 20 Time (min) 62 with 32Pi and only slightly less than the amount of 32P incorporated into PA in the plasma membrane fraction. Analysis of cell homogenate 32P-labeled phospholipids illus- trates that after 120 min of 32Pi labeling, the rates of 32F incorporation into PC, PE, PI, PG, and PS were still increasing. However, the incorporation of 32P into PA, DPI and TPI approached a steady state, reflecting the specific radioactivity of B1—32PJ-ATP which reached steady state after 60-90 min of 32Pi labeling. Table II illustrates the high level of 32F per mg protein in DPI (GPIP) and TPI (GPIPZ) in the plasma membrane compared with the other organelles after 90 min. The lysosomal fraction had 20% while the other fractions had approximately 10% of the amount of 32P in DPI in the plasma membrane fraction. The amount of 32P-labeled TPI in the nuclear, lysosomal, mitochondrial and microsomal fraction was only 1-4% of the amount of 32P-labeled TPI isolated from the plasma membrane fraction. Attempts to chemically measure the mass of GPIP and GPIPZ by phosphate analysis were unsuccessful because of the low quantity of DPI and TPI in the various subcellular fractions. 63 o.H H m.m um.o u m.~ Hm.o w e.H m.~ a H.¢ ooH usage ~.H n m.o~ H.m H H.o~ ¢~.o a m.o m.~ « H.0H ooH swam a a a a a Pmeomocuwz pmsomomau _w_cc:o;oou_z campuzz mcmcnsmz mammpa .mcowumw>mp ucuucmum u meowuccwamga wpxuouoqm; ucwcmeepu mugs» we new: use are mm:_m> .covuumce «corneas osmmpg as» gas: pecaneou mcopuumce Pmeomocups use pesomoma. .pmwcucozooups .cmm—usc as» ea newborn me\=o_uucoacou=F a m we mmcucmucma mg» ecu vmuconmc camp mg» .uoczmmme mo: copuuace cops—pouaam some cm campers co as can “masses Hae use Aasawv *ao ages a co cossmtoatoucs .m at=m_u cw umnwcumwu me pummouocg use cps om Low gum gar: mmppoamp are: mouxuoueam: .mcopuomew cmpappmunsm maopgw> mo Hap pew He: can? cowaoeoacoucp sun so cowuanpcumwo HH epoch 64 DISCUSSION In this study, we used a rapid subcellular fractionation method to prepare plasma membrane, mitochondrial, lysosomal, microsomal and nuclear fractions that are enriched by at least two-fold based on the fraction's marker enzyme (mitochondrial-fumarase) or as much as 13-fold (lysosomal-hexosaminidase). The yields of organelles, although as low as 4.5% for the lysosomes, were adequate to accurately measure 32P incorporation into various phospholipids. Using Percoll instead of sucrose as the density gradient centrifugation medium, greatly decreased the time required to obtain the mitochondrial, plasma membrane, nuclear and lysosomal fractions. Thus, the procedure allows one to process a number of samples in a relatively short period of time. The enzyme inhibitors present in the homogenizing buffer in combination with decreased temperature and rapid fractionation prevented major changes in 32P-phospholipid levels during fractionation. Changes in 32P-labeled PC, PE, PI and PA occurred in the crude nuclear fraction probably due to the presence of Mg2+ in the buffer which is a known cofactor for many of the enzymes involved in the metabolism of these phospholipids (45). Only small changes were observed in 32P—labeled PG, PS, DPI and TPI. Determination of the rate of 32P incorporated into PC, PE, PI and PA must await the development of an alternative procedure to isolate nuclei, for example, by using divalent cations other than MgZ+. 65 Very little phospholipid exchange occurred between the microsomes and other organelles during the fractionation. The small percentage of radioactivity recovered in the plasma membrane, mitochondrial and lysosomal fractions was probably due to contamination of these fractions with added labeled microsomes. There are a number of techniques available for the analysis of phosphoinositides. These include chromatography on formaldehyde treated paper (46) or oxalate impregnated silica gels (47), silica gel chromatography using multi-component solvent systems (2,15,41,48,49) and affinity chromatography on immobilized neomycin columns (39). Although polyphophoinositides can be analyzed quite well using these systems, the ability to separate other phospholipids that may be present in the mixture is limited. Quite often it is necessary to use a second or third solvent system to completely analyze all the phospho- lipids in the mixture (10). Normal pressure anion exchange chromatog- raphy was also used to separate deacylated phospholipids, but it was either very time consuming (41) or analysis of only the deacylated phosphoinositides was reported (21,23). The anion exchange HPLC procedure used in this study separated all the major deacylated phospholipids including those of polyphosphoinositides within 70 minutes. Recovery of the deacylated phospholipids from the anion exchange column exceeded 95% and the compounds remained in solution during the separation making it easier to determine radioactivity or phosphate levels as compared to working with silica gel or cellulose scrapings. However, one obvious drawback of the analysis procedure is that it gives no information on the level of lyso-phospholipids present in the mixture but rather analyzes them together with diacyl-phOSpho- 66 lipids. In addition, sphingomyelin and alkenoxy and alkoxy ether phospholipids are omitted from analysis. The rates of synthesis and degradation of phospholipids would influence the amount of 32F incorporation into phospholipids. Synthesis of major phospholipids has been attributed to the microsomal fraction of rat hepatocytes (45,50). Of the major biosynthetic phos- pholipid enzymes, only glycerol-3-phosphate phosphatidyltransferase in the mitochondria (45) and diglyceride kinase in the cytoplasm (51) are found primarily outside of the endoplasmic reticulum. Phosphatidyl- inositol kinase is now known to be present in a number of subcellular sites including the plasma membrane (52,53), Golgi (54), lysosomal membranes (55), microsomes (53,56) and nuclear envelopes (53). DPI kinase is associated with nuclear envelopes, plasma membranes and microsomes of rat liver (53) and is a soluble enzyme in brain (57). Various phospholipases are present in virtually all subcellular organelles (58) including a phosphatidylinositol-specific phospholipase C (59) which is predominantly located in the cytosol. Therefore, specific subcellular locations for breakdown of specific phospholipids has not been established. This present study demonstrates that the microsomal fraction has the highest rates of 32P incorporation per mg protein into phospholipids with the exception of PS, DPI and TPI. This finding agrees well with published reports that the endoplasmic reticulum contain the highest level of phospholipid biosynthetic enzymes compared with other subcellular fractions (46). Nuclear envelopes are reported to have a high level of PS biosynthetic enzymes, second only to the endoplasmic reticulum (45), which is in agreement with this study which shows that the nuclear fraction has the highest 67 rate of 32P incorporation per mg protein into PS compared with to the other subcellular fractions. The similarity of 32P incorporation rates into the major phospholipids between the microsomal, plasma membrane and lysosomal fractions (Fig. 3) may be due to similar turnover rates or possibly due to contamination of the lysosomal and plasma membrane fractions with microsomes. However, the two-fold higher 32P incorporation rate into PE seen in the microsomal fraction compared with the plasma membrane or lysosomal fractions would argue against contamination causing the similar incorporation rates between the fractions. Therefore, the presence of 32P-labeled phospholipids in the plasma membrane and lysosomal fractions in the absence of their synthetic enzymes is probably due to breakdown of existing phospholipids and rapid translocation of newly synthesized phospholipids from the microsomes to the other organelles via transport proteins (60) or some other process (61,62) such as membrane flow in the case of plasma membranes (63). The lower rates of 32F incorporation into the major phospholipids in the mitochondrial fraction compared with the microsomal fraction is probably due to slower turnover of the mitochon- drial phospholipids. Bygrave (64) reported that microsomal phospha- tidylcholine turns over at twice the rate of mitochondrial phospha- tidylcholine. Despite the multiple sites within the cell where PI kinase and DPI kinase have been reported (52-56), this study indicates that the plasma membrane has the fastest rate of 32P incorporation into polyphos- phoinositides per mg protein of all the subcellular fractions analyzed. The rate of 32p incorporation per mg protein was 5-10 and 25-50 68 times faster into DPI and TPI, respectively, in the plasma membrane fraction compared with the other subcellular fractions. This would suggest that an active phOSpholipase C or ph05phomonoesterase which acts on polyphosphoinositides is present in the plasma membrane of rat hepatocytes. Such enzymes are present in erythrocyte membranes (65,66). The very low amounts of 32F incorporated into TPI in other subcellular fractions compared with the plasma membrane fractions may reflect the plasma membrane contamination in these fractons which ranged from 2-9% (Table I). It is interesting to note that although plasma membranes composed 9% of the microsomal fraction as determined by alkaline phosphodiesterase 1 activity (Table I), 32P incorporation into TPI in the microsomal fraction is only 3% of that seen in the plasma membrane fraction (Table II). This suggests that turnover of TPI may occur in a specific region of the plasma membrane which preferentially pellets under the influence of lower g-forces. Contamination by plasma membranes cannot account for the 32P incorporation into DPI in the other subcellular fractions suggesting that DPI turnover may occur in parts of the cell other than the plasma membrane. Of particular interest is the lysosome where the rate of 32P incorporation is only 20% of the plasma membrane rate but nearly 2-fold greater than the rate seen in other subcellular fractions. Although the amount of DPI and TPI present in various subcellular fractions is still unknown, this study provides evidence that the plasma membrane may contain the most metabolically active pool of DPI and TPI. Published reports which describe hormonal effects on 32P-TPI metabolism in the whole hepatocyte (18-23), therefore, 69 closely reflect changes in plasma membrane 32P-TPI levels. However, analysis of 32P-DPI metabolism in the whole hepatocyte nay not accurately reflect changes in 32P-DPI levels in the plasma membrane because of previously unaccounted contribution of 32P-DPI from other subcellular organelles especially lysosomes. Acknowledgements - We wish to acknowledge the technical assistance of Lynn Farrell, and Carol Fenn and Theresa Fillwock for preparation of the manuscript. 10. 11. 12. 70 REFERENCES Hokin, M.R., and Hokin, L.E. (1953) J. Biol. Chem. 292, 549-558. Benabe, J.E., Spry, L.A., and Morrison, A.K. (1982) J. Biol. Chem. .252, 7430-7434. 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Acta. 222, 95-117. Yatke, M.P., and Kennedy, E.P. (1983) Biochemistry 22, 1497-1507. Sleight, R.G., and Pagano, R.E. (1983) J. Biol. Chem. 222, 9050-9058. Morre', D.J. (1977) Cell Surface Rev. 2, 1-83. Bygrave, F.L. (1969) J. Biol. Chem. 222, 4768-4772. Downes, C.P., and Michell, R.H. (1981) Biochem. J. 222, 133-140. Koutouzov, S., and Marche, P. (1982) FEBS Lett. 222, 16-20. CHAPTER I I Subcellular Site and Mechanism of VasOpressin Stimulated Hydrolysis of PhOSphoinositides in Rat Hepatocytes 75 76 Abstract The intracellular site of vasOpressin induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 sec of vaSOpressin treatment of 32Pi' the levels of 32P-labeled hepatocytes prelabeled with phosphatidylinositol 4-phosphate (PI-P) and phosphatidyl- inositol 4.5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%. then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P1PI-P and [32P1PI-P2 were observed in the other subcellular fractions. and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol (PI) in the plasma membrane decreased by 15% after 45 sec of vasOpressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid (PA) levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes. it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphospho- inositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (ZOOnM) of free calcium when assayed at low ionic strength. but the calcium requirement shifted to micromolar concentrations under iso-osmotic. intracellular-like. ionic conditions. Addition 77 of vaSOpressin (ZOOnM) to the [32Plplasma membranes stimulated the breakdown of 20% of the [32P1PI—P present in 2 the plasma membranes in l min when assayed under iso-osmotic conditions in the presence of ZmM MgCl and approximately 2 ZOOnM free calcium. This data suggests that the phosphoinositide-Specific phosphodiesterase is not active under normal cellular conditions. but is activated upon the addition of vasopressin to the intact cell. 78 Footnotes 1The abbreviations used are: PC. phOSphatidyl- choline; PE. phosphatidylethanolamine; PI. phosphatidylinositol; PG. phOSphatidylglycerol: PA. phosphatidic acid: PI—P. phosphatidylinositol 4-phosphate; PI-P phosphotidylinositol 4.5-bisphosphate: I-P. inositol 2. phosphate; I-Pz. inositol bisphosphate; I-P3. inositol trisphosphate; HEPES. N-Z-hydroxyethylpiperazine—N'- 2-ethanesulfonic acid: EDTA. ethylenediaminetetraacetic acid: TRICINE. N-trislhydroxymethyllmethyl glycine; PMSF. phenylmethylsulfonyl flouride; EGTA. ethyleneglycol- bis-(B-aminoethyl ether)-N.N.N'.N'-tetraacetic acid; HPLC. high pressure liquid chromatography. 2Seyfred. M.A.. and Wells. W.W.. manuscript in preparation. 79 Introduction In many tissues. there is an association between stimuli induced changes in phOSphatidylinositol (PI) metabolism and cellular calcium (1.2). Further investigation has shown that the metabolism of the polyphosphoinositides. phoSphatidylinositol 4—phosphate (PI-P) and phOSphatidylinositol 4.5-bisphosphate (PI-P2). is also influenced by the same agents (3-10). In rat hepatocytes. calcium mobilizing hormones such as vasOpressin. angiotensin II and epinephrine enhance PI breakdown and subsequent resynthesis with phosphatidic acid as a key intermediate in this process (ll-l4). These studies have been extended to 32 demonstrate that there is a decrease in P-labeled PI-P and PI-P2 after treatment of prelabeled hepatocytes with calcium mobilizing hormones (ls-17). The correlation between phosphoinositide (PI. PI-P. and PI-Pz) breakdown and increased levels of intracellular calcium has led to speculation that decreases in the plasma membrane concentrations of phosphoinositides could affect calcium channels (1.18. but see 19) or some other calcium transport system such as the plasma membrane (Ca2+- 2+) "9 ATPase (20-22). Alternatively. products of the metabolism of phosphoinositides may serve as calcium mobilizing agents. Inositol trisphoSphate (I-P3). which is released by phoSphodiesterase action on PI-P has been shown to release 2! calcium from non-mitochondrial intracellular stores in 80 permeabilized rat pancreatic acinar cells (23). Deepite the proposed important role of phosphoinositide metabolism in calcium homeostasis. the intracellular site of stimulated phosphoinositide metabolism in response to calcium mobilizing hormones in the hepatocyte has not been fully characterized. Increased PI hydrolysis in response to vasOpressin has been attributed to plasma membranes (11.24). but the plasma membrane fractions were either highly contaminated with other organelles (11) or the reSponse much slower (24) than observed in intact cells. Additional studies using isolated plasma membrane preparations required very high levels of norepinephrine (25) or very long incubation times (30 min) in the presence of detergent to observe significant PI hydrolysis (26). An attempt to localize the intracellular site of PI-P2 hydrolysis in response to vas0pressin was not successful presumably due to the extreme lability of this inositol lipid (15). In a previous report (27). we characterized the incorporation of 32P into phospholipids including polyphosphoinositides in plasma membranes. mitochondria. lysosomes and microsomes isolated from prelabeled rat hepatocytes. It was found that the plasma membrane fraction contained the highest level of 32P-labeled PI-P and PI-P2 per mg protein of all the fractions examined. In the present report. we have extended those studies and have examined the subcellular location of vasopressin induced phosphoinositide breakdown. Also. 32P-labeled plasma 8i membranes isolated from prelabeled hepatocytes were used to investigate the enzymes involved in the degradation of polyphOSphoinositides. 82 EXperimental Procedures ggpatocyte Incubations - Hepatocytes were isolated from fed male CD-Sprague Dawley rats by a modification of the method described by Seglen (28). The preparation procedures and the incubation conditions were described previously (27). After 90 min of incubation with 32Pi' the hepatocytes were treated with ZOOnM vasopressin for 0-10 min. At various times. the hepatocyte suspensions were diluted with 7 volumes of ice cold homogenizing buffer which consisted of ZSOmM sucrose. lmM EDTA. lOmM NaPPi. ZOmM NaP. 75mg/l PMSF and lOnfl TRICINE. pH 8.0. Subcellular Fractionation of Hepatocytes and Phospholip2g Analysis - The hepatocytes were fractionated by dif— ferential centrifugation and Percoll density gradient centrifugation into plasma membranes. mitochondria. lysosomes and microsomes as previously described (27). The phospholipids in the various fractions were isolated by extraction with acidic chloroformxmethanol as described by Schacht (29). The phospholipids were deacylated using the mild alkaline conditions reported by Kates (30) except that two 10 min incubations of the phospholipid fraction with 0.1N NaOH at room temperature were used instead of one 15 min incubation. This led to more consistant and increased recovery of glycero— phosphorylinositol 4-ph08phate and glycerophosphorylinositol 4.5-bisphosphate without risking further breakdown of the 83 more alkali-labile glycerophosphorylcholine and glycerophosphorylinositol. The glycerophosphate esters were separated by high pressure liquid chromatography (HPLC) on a Bio-Rad Aminex A-27 anion exchange column as previously described (27) using a polyphasic gradient beginning with 20mM ammonium borate. lOOmM ammonium formate. pH 9.5 and ending with 20mM ammonium borate. 850mM ammonium formate. pH 9.5. The HPLC system was automated using an ISCO ISIS autosampler and an ISCO Foxy fraction collector. Phospholipid Degradation Studies Using 32gzgabeled Plasma Membranes - 32P—labeled plasma membranes were isolated in the presence of lmM EDTA and in the absence of 20mM NaP from hepatocytes that had been incubated for 90 min with 32P1 as previously described (27). The omission of Na? had no effect on the level of any of the 32P-labeled phospholipids (data not shown). The final pellet was resuspended at a concentration of 1-2 mg protein/ml using 10 strokes of a Teflon-glass Potter-Elvehjem homogenizer in either 20mM Na-HEPES. 0.5" Na-EGTA. pH 7.4 or 120m" KCl. 20mM Na-HBPES. 0.5mM Ma-BGTA. pH 7.4. Typically. 75 ul of plasma membrane suspension was added to 425 ul of assay mix containing 20mM Na or K-MBPES. 0.5mM Na or K-BGTA. pH 7.4 plus other additions which had been preincubated at 37°C for at least 15 min. The suspensions were vortexed and incubated at 37°C in a Dubnoff water bath shaking at 60 oscillations per min. The reactions were stOpped by the addition of 1.5 ml chloroform:methanol (1:2). 84 The phospholipids were isolated. deacylated and analyzed as described above. In addition. the methanol:HCl phases from the phospholipid extractions were saved and evaporated under a N2 stream at 37°C. The residues were dissolved in 3 ml of water and neutralized using 2-3 ul of conc. NH4OH. The solutions were loaded on anion exchange columns containing 0.5 ml of Dowex 1 (x8; 200-400 mesh; formate form) in Pasteur pipets. The columns were washed with 10 ml of 60mM ammonium formate and the radioactive phosphate esters were then sequentially eluted and analyzed as described by Downes and Michell (31). Other Methods - The specific radioactivity of ffl-BZPIATP was determined by the method described by Hawkins et al. (32). Protein was determined by fluorescamine (33) using bovine serum albumin as the standard. 32? radioactivity was determined by Cerenkov radiation. Free calcium levels were established with a calcium-EGTA buffer system using Ka-3.7x107 M-1 to calculate the concentration of free calcium (34). Statistical significance was determined by Student's t-test. Materials - [ArgIVasopressin (grade VI) and Dowex 1 (x8: ZOO-400 mesh; Cl-form) were obtained from Sigma. The sources of all other reagents and materials were described previously (27). 85 Results Effect of Vasopressin Treatment of Rat Hepatocytes on 32P-Labeled Phospholipids Isolated from Various Subcellular Organelles - It was previously shown (27) that after 90 min 32 of incubation with Pi' hepatic intracellular r1-32P1ATP 32 reached isotOpic equilibrium. P-labeled PA. PI-P and PI-P2 in all subcellular fractions analyzed were also at isotopic equilibrium. but the 32? incorporation rate into all other phospholipids was still increasing. Therefore. changes in [32PlPA. PI-P or PI-P represents changes in the 2 total mass of the phospholipid. To investigate the effect of vasOpressin treatment of rat hepatocytes on the phOSphOllpid levels of various subcellular fractions. hepatocytes were incubated with 32P1 for 90 min. and then treated with 200nM vasopressin for various times up to 10 min. The cells were fractionated into plasma membranes. mitochondria. lysosomes and microsomes and the 32P-phospholipid levels in these fractions determined as described in ”Experimental Procedures”. The results are shown in Figures lA—E. The 32 P-phosphatidylcholine (PC) levels in all the various subcellular fractions except mitochondria increased significantly after 2 min of vasOpressin treatment. but returned to near control levels after 10 min (Fig. 1A). Similiar results were also seen with 32P-labeled phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) (data not shown). In contrast. 86 Figure 1. Effect of Vasopressin Treatment of Rat Hepatocytes on 32P—Labeled Phospholipids Isolated from Various Subcellular Fractions. Hepatocytes were incubated with 32F1 for 90 min and then treated with vasopressin for an additional 0-10 min. Partially purified plasma membranes. mitochondria. lysosomes and microsomes were obtained and the levels of 32P-phoSpholipids determined as described in ”Experimental Procedures”. A. Phosphatidyl- choline; B. PhOSphatidylinositol; C. Phosphatidic acid; D. PhoSphatidylinositol 4-phosphate; E. Phosphatidylinositol 4.5—bisph05phate. 0. Cell homogenates; -. Plasma membranes;A. Mitochondria; O. Lysosomes; D. Microsomes. The results shown are the mean percentage changes versus untreated samples obtained from three or four different hepatocyte preparations. *. p< 0.10; '*. p<0.05; ***. p<0.01. 87 '60 @‘ ‘ P'hos'phdtidJIcthin'e I4{) IZO of Control 96 IOO I 80 # l l L l 1 8.0 I0.0 Duration of Vasopressin Treatment (min) 0 2.0 4.0 6.0 Figure 1A 2 00 @ Phosphatidlenosnol _ I l80 I40 *- °/o of Control 60 ' ' 0 2.0 4.0 6.0 8.0 I0.0 Duration of Vasopressin Treatment (min) Figure l B 89 2 2 0 © PhosphatIdIc ACId _ 0‘. t. PM ..., I80 - - _ In... tCell Hom _ 3 Mic 0 g .0 4’ OI” 0 I40 - 6 - ,_ ,—-Lys 0 m .9'\ s - Q”! g/ a lOO 8 ‘vMit -I \‘ 60 - - O 2.0 4.0 6.0 8.0 l0.0 Figure'lC Duration of Vasopressin Treatment (min) 90 |20 ,©' PliosphatidylinosItol '4-phosphcé3tzd - It-Lys - I00 of Control 96 80 60 Figure 10 I l J l l o 2.0 4.0 6.0 8.0 I0.0 Duration of Vasopressin Treatmentimin) 9I IZC)‘ NBC) of Control 36 ..-I I I l I Phosphatidylinositol 4,5-bisphosphate- I /m " PM _ 9. I I l I I I I I l I (D 213 '4CI ISI) 8!) ICHD Figure lE Duration of Vasopressin Treatment (min) 92 the 32P-labeled PI levels decreased 15% (p<0.05) in the plasma membrane fraction after 45 sec of vasopressin treatment (Fig. 13). No significant changes in [32PlPI levels were observed in any other subcellular fraction after 45 sec of vasopressin treatment. however. longer treatment times led to an increase in (32P1PI levels above control in 32P-labeled PA in the all fractions analyzed. The amount of plasma membrane fraction increased 40% after only 45 sec of vasOpressin treatment and continued to increase to 210% of control levels after 5 min of treatment (Fig 1C). The levels of [32 PlPA were also elevated in the cell homogenate and other subcellular fractions in response to vasopressin. but the increases were much smaller and the reSponse much slower than observed in the plasma membrane fraction. The amount of 32P-labeled PI-P in the plasma membrane fraction decreased by 44% after 45 sec of incubation with vasopressin. but returned to near control levels after 10 min of treatment (Fig 1D). A similar response was seen in the cell homogenate. but the decrease in [BZPIPI-P was only approximately one-half as great as seen in the plasma membrane fraction. A significant decrease in [32PlPI-P levels was also observed in the mitochondrial fraction in response to vasopressin. but the response was not maximal until after 2 min of treatment and the decrease was only 20%. This response may simply be a reflection of the plasma membrane contamination of the mitochondrial fraction since 32 the amount of [ PlPI-P per mg of protein in mitochondria 93 was approximately 10% of the [32P1PI-P level in plasma membranes and plasma membranes make up approximately 2% of the mitochondrial fraction protein (27). Analysis of the 32P-labeled PI-P2 levels in the various subcellular fractions showed that they decreased after 45 sec of vasopressin treatment in the plasma membrane. mitochondrial and lysosomal fraction as well as in the cell homogenate (Fig. 13). The decrease in the amount of 3 [ 2PlPI-P2 ranged from 42% in the plasma membrane to only 16% in the lysosomal fraction. After 10 min of vasopressin treatment. the level of [BZPIPI-P in plasma membranes 2 remained depressed by 22% compared with control level while [32P1PI-P2 levels in the other subcellular fractions were not significantly different from control levels. As with 32 [ PlPI—P. the vasOpressin stimulated decrease in [32P1PI-P 2 levels observed in the other subcellular fractions may simply be due to plasma membrane contamination of the other fractions. The plasma membrane contamination ranged from 2-8% of the other fractions protein and the amount of [3 2PJPI-P2 per mg of protein found in the other fractions was only l-4% of the level observed in the plasma membrane fraction (27). There was no change in the cellular [i-BzPlATP specific radioactivity during the course of vasOpressin treatment in three different hepatocyte preparations (data not shown). 94 32 Time Course of the Degradation of P-Labeled Plasma Membrane Phospholipids: Effect of Calcium — Studies with intact hepatocytes indicated that the plasma membrane was the site of vasOpressin stimulated hydrolysis of phosphoinositides. To further characterize phosphoinositide breakdown in plasma membranes. assays using 32P-labeled plasma membranes prepared from hepatocytes which were incubated with 32P1 were used. Figure 2A describes the time course of the breakdown of32P-labeled PA. PI. PI-P. and PI-P2 in the presence or absence of calcium at low ionic strength (20mM Na-HEPES. 0.5mM Na-EGTA. pH 7.4). No 32 significant change in the level of [ P]PI was observed over the 300 sec incubation time in the presence or absence of calcium. The levels of 32P-labeled PC. PE. and PG also did not change. The amount of [32P1PA linearly decreased. independent of calcium. by 13% within 120 sec. In contrast. hydrolysis of [32P1PI-P and PI-P2 was stimulated approximately lO-fold in the presence of llOuM free calcium after 1 min of incubation. Very rapid rates of hydrolysis of [BZPIPI-P and PI—P2 occurred in the first 30 sec of incubation. and within 100 sec. 50% of the amount of 32 [ P]PI-P and PI-P present in the plasma membrane fraction 2 was hydrolyzed in the presence of llOuM free calcium. In the absence of calcium. only 5—10% of the [32P1PI-P and PI-P2 was broken down within the first 120 sec of incubation. but increased to approximately 40% after 300 sec of incubation at 37°C. 95 Figure 2. Time Course of the Degradation of 32P-Labeled Plasma Membrane PhOSpholipids: Effect of Calcium. 32P—labeled plasma membranes were prepared from prelabeled hepatocytes and incubated at 37°C for various times in the presence or absence of llOuM free calcium at low ionic strength. The levels of 32P-labeled PI. PA. PI-P. PI-P . 2 I-P. I-PZ. I-P3 and 32Pi were determined as described in "Experimental Procedures". A. 32P-labeled phOSpholipids- OI. PI: DI. PAtAI‘PI-P: vl' PI-on B. 32P-1abeled inositol phoSphates and 32191- 0.0 I-P and/or Pi: D.. I-Pz: A.‘I-P3. Open symbols— absence of calcium; closed symbols- presence of llOuM free calcium. Typical levels of 32P-labeled compounds (in c.p.m.) obtained at t-O: 1362. PI: 1453. PA; 929. PI-P: 4008. PI-PZ: 11.160. I-P and/or Pi: 1287. I-Pz; 1961. I-P3. The results shown are the mean percentage changes of duplicate samples from two or three different hepatocyte preparations. S.E.M. ranged from S-15%. 96 1 T T I I fl I fij _® . Phospholipids q l/’ tut" e \ 0 Jr |00 ..$___§< ‘ F PA in 7 1‘ 4 B 980* 7 O N 0 .g - ‘ ... '8 °so:- o\ I 4C)~ I- '4 zo- ‘ 1 1 1 1 1 J 1 u L 1 o 30 60 90 I20’ 300 Time of Incubation (sec) Figure 2A 97 @r 300— 250- Zero 200— of °/o TIme I Inositol Phosphates, and'Pi fi‘fi—q A "4 Figure 28 Time of Incubation (sec) 0 ___.A\ “NA — 1 1 1 11% 1 90 I20 300 98 Figure 28 describes the rate of 32P-labeled inositol trisphosphate (I-P3). inositol bisphosphate (I-PZ). inositol phosphate (I-P) and 32Pi released into the water soluble fraction in the presence or absence of calcium. [3 and 32Pi which cannot be separated from one another by the 2911-9 anion exchange method increased by 50% after 300 sec of incubation in the presence or absence of calcium. There are five possible sources for the radioactivity which would co-elute with [32P11—P and 32?1 of 32Pi which was non—covalently bound to membranes present 2 l) non-enzymatic release in the plasma membrane fraction; 2) a calcium independent 32P protein phosphatase releasing i; 3) a calcium independent 32 phospholipid phosphomonoesterase acting on P-labeled PA. PI-P and PI-P2 releasing 32P1; 4) a calcium independent PI-P and PI-P2 specific phosphodiesterase which would release [32911-92 and [32911-93 dephosphorylated to 32P1 and [32Pll-P; and 5) release from membranes of some other 32P-labeled water soluble component which was then rapidly possessing a negative 2 charge. The calcium independent breakdown of 32P-labeled PA. PI-P and PI-P2 supports the presence of a phospholipid phosphomonoesterase in the plasma membrane preparation. The small amount of [32PlI-P2 produced iin the absence of calcium suggests the presence of a phospholipid phosphodiesterase acting on PI-P and/or PI-P2 but not on PI since there was no decrease in the level of [32P1P1. However. the main contributor to the radioactivity recovered in the Pi and I-P fraction was probably 32?1 99 released from non-phospholipid sources since the level of radioactivity which elutes in this region was very high at time-0 and the decrease in radioactivity of the phospholipids in the absence of calcium was only 35% of the amount recovered in the Pi and I-P region (see fig. 2 legend). The addition of lmM Spermine to the assay mix completely inhibited the calcium independent breakdown of 32P-labeled PA. PI-P and PI-P but had no effect on the 2 calcium stimulated breakdown of 32 Spermine caused the level of 32P1 and/or [32PlI-P to be reduced to 120% of t-O after 120 sec of incubation in the 3 P-labeled PI-P and PI-Pz. 2PlI-P2 and I-P3 levels were identical after 120 sec of incubation with lmM spermine presence or absence of calcium. [ in the absence of calcium as seen at t-O (data not shown). Figure 28 also illustrates the calcium dependent release of water soluble 32P-labeled I-P and I-P 2 3 indicating that 32P-labeled PI-P and PI-P2 were hydrolyzed by a calcium dependent phosphodiesterase. A 2.5 to 3 fold increase in the amount of [BZPII-P released into the water 3 soluble fraction was seen within 60 sec of incubation with llOuM free calcium. Release of [BZPII-P2 was also stimulated by calcium but to a lesser extent. Magnesium (0.5mM or 2mM) could not substitute for calcium nor did it affect the calcium dependent phosphodiesterase activity. The addition of 1 mg of cytosol protein had no effect on the calcium dependent phosphodiesterase activity nor did cytosol affect any of the 100 32 . . . P—phospholipid levels when added alone or in the presence of vasopressin or magnesium during a 2 min incubation (data not shown). Figure 28 is somewhat misleading due to the background radioactivity levels of the fractions collected. However. all of the radioactivity lost from 32P-labeled PI-P and PI-P2 by the action of the calcium dependent phOSphodiesterase could be accounted for in the 32P-labeled I-P2 and I-P3 fractions (data not shown). Qose Response of Calcium on Plasma Membrane PolyphoSphoinositide PhOSQhodiesterase Activity: Effect of Ionic Strength - 32P-labeled plasma membranes were incubated for l min at 37°C under various free calcium concentrations at low and normal (iso-osmotic) ionic strength. The activity of the polyphoinositide phOSphodiesterase was monitored by measuring the levels of 32P-labeled PI-P. PI-Pz. I-P2 and I-P3. The results of these experiments are shown in Figures 3A and B. Incubation of the 32P-labeled plasma membranes in 20mM Na-HEPES. 0.5mM Na-EGTA. pH 7.4 (low ionic strength) resulted in maximal 32P-labeled PI-P and PI-P2 breakdown at a free calcium concentration of 8.4uM with half-maximal hydrolysis observed at lOOnM free calcium. Similar results were observed when [32PII-P3 release into the water soluble fraction was measured. There was very little change in phOSphodiesterase activity when assayed over the range of 200nM to lOOuM free calcium. suggesting that the phosphodiesterase activity was dependent on calcium. but not regulated by changes in calcium 101 Figure 3. Dose ReSponse of Calcium on Plasma Membrane Polyphosphoinositide PhoSphodiesterase Activity: Effect of Ionic Strength. 32P-labeled plasma membranes were prepared from prelabeled hepatocytes and incubated for l min at 37°C at various free calcium concentrations under conditions of low (20mM Na-HEPES. 0.5mM Na-EGTA. pH 7.4) and iso-osmotic ionic strength (120mM KCl. 20mM Na-HEPES. 0.5mM Na-EGTA. pH 32 7.4). The levels of P-labeled PI-P. PI-P . I-P and I-P 2 2 3 were determined as described in ”Experimental Procedures”. A. 32P-labeled polyphosphoinositides- D.. PI-P; 0.0 PI-P . 2 B. 32P-labeled inositol phosphates- EJ.II I-Pz; 0.0 I-PB' Open symbols- iso-osmotic ionic strength; closed symbols- low ionic strength. Typical levels of 32P-labeled compounds (in c.p.m.) obtained at zero free calcium: iso-osmotic ionic strength- 1531. PI-P; 7047. PI-PZ; 1963. I-PZ; 1783. 2132. I-P3; low ionic strength- 1485. PI-P; 6416. PI-PZ: I-Pz; 2206. I-P3. The results shown are the mean percentage changes of duplicate samples from three different hepatocytes preparations. S.E.M. ranged from 5-15%. I00 Zero Free Calcium a) to O O of N o °/o 60 50 Figure 3A b be I. L 102 T 1 T I Polyphosphoinositides I;®<:\ \8 FPI-P-t-KCI Y 1 l l Log Molar Free Calcium Cl f'“ ‘ Pl-Pzi-KCI 0 0 FPI-Pz-KCI - PI-P -KCIJ 7 1 ’ .80 1-6.0 1.40 1 I -2.0 103 E T flnosiioI Phosphate: o 340- ~I-P3+ Km . _ O O .1 / I-P:5 - KCI 2; e / ‘9 280*- a O U Q) 0 -1 L: O B N 220 - “5 .\° J |60 . -I Ioo . 0L” -e.o -eo -40 -250 Log Molar Free Calcium Figure 3B 104 concentration. The level of [32PlI-P2 was maximal at llOuM free calcium with half-maximal production observed at Sun free calcium. Higher levels of free calcium (1.5mM) inhibited the phosphodiesterase. Incubation of the 32P-labeled plasma membranes under iso-osmotic conditions established by 120mM KCl. 20mM Na-HEPES. 0.5mM Na-EGTA. pH 7.4 shifted the phosphodiesterase requirement for calcium to the right. hydrolysis and [32911-9 Maximal [32P1PI-P production was 2 3 observed at llOuM free calcium with half-maximal activity 3 observed at SuM free calcium. [ 2P]PI-P hydrolysis and 32 [ PII-P production did not plateau even at 1.5mM free 2 calcium. but did reach the maximum levels of hydrolysis and production observed at low ionic strength at this calcium concentration. No inhibition of phosphodiesterase activity was observed at 1.5mM free calcium under iso-osmotic conditions. Assaying the phosphodiesterase activity under iso-osmotic conditions at llOuM free calcium did not greatly affect the rate of polyphosphoinositide hydrolysis compared with the hydrolysis rate observed at low ionic strength (data not shown). Maintaining the iso-osmotic condition with NaCl instead of KCl and using the K+ salts of HEPES and EGTA also did not change the concentration of calcium required for polyphosphoinositide phosphodiesterase activity (data not shown). 105 32 Effect of Vasopressin on P-Labeled Phosphoinositide Levels 32 in Isolated Plasma Membranes - P-labeled plasma membranes were incubated in 120mM KCl. 20mM Na-HEPES. 0.5mM EGTA. pH 7.4. 2mM MgCl2 and approximately 240nM free calcium for l min at 37°C in the presence or absence of 200nM vasOpressin. Analysis of the 32 level of 32P-labeled PI-P2 decreased 21.5% (p<0.001) in the presence of vasopressin compared with control while no change in 32P-labeled PI. PA or PI-P was observed. The P-labeled phospholipids showed that the corresponding production of [32PII-P and also [32PlI-P was 3 2 observed in response to vasopressin. [32PlI-P2 was produced as a result of phosphatase action on [32Pll-P3 which was stimulated by 2mM MgCl (data not shown).2 Phospho- 2 diesterase activity in the presence of llOuM free calcium was not further stimulated by the addition of vasopressin (data not shown). 106 Discussion In this study. we have examined the intracellular site of phosphoinositide hydrolysis in response to vasOpressin. It was found that the plasma membrane levels of 32P-labeled PI—P and PI-P2 decreased approximately 40% while 32P-PI decreased 15% after a 45 sec incubation of prelabeled hepatocytes with vasOpressin. These results are in good agreement both in terms of time and extent of response with the data reported on whole cell [32Plphosphoinositide changes in response to vasopressin (12.16.17). 32P-labeled PA levels increased rapidly in the plasma membrane probably due to a phosphodiesterase acting on PI. PI-P and/or PI-P2 forming diacylglycerol which was rapidly phosphorylated using endogenous [3-72P1ATP and diglyceride kinase. The increases in 32P-labeled PC. PE. PG. and PI observed in virtually all the subcellular fractions after 2 min of vasOpressin treatment reflected only very small changes in the total mass of these phospholipids (data not shown). Only a small portion (l-3%) of the total hepatocyte cellular pool of these phospholipids becomes labeled after 90 min of incubation with 32P1 (17). so a two—fold increase in the level of 32P-labeled phospholipids represents only a 3-5% increase in mass. The microsome fraction demonstrated the largest increase in [32P1PC. PE. PG. and PI possibly as a result of mobilization of diglyceride and [BZPIPA from the 107 plasma membrane to the endoplasmic reticulum. where the biosynthesis of these phospholipids occurs. The time lag observed between the increase of [32P1PC. PE. PG. and PI levels in the microsome fraction and polyphosphoinositide breakdown in the plasma membrane fraction. along with the transient increase in the level of [BZPIPA in the microsome fraction after 2 min of vasopressin treatment supports this 32 conclusion. [ P]PC. PE. PG. and PI could then be mobilized to other parts of the cell. 32 Using P-labeled plasma membranes isolated from prelabeled hepatocytes. PI-P and PI-P2 were broken down by a membrane bound polyphosphoinositide specific phOSphodiesterase to form 32P-labeled I-P2 and I—P3. The 32 reaction was rapid with 15% of the P-labeled PI-P and PI-P2 broken down within 15 sec under Optimal conditions. The enzyme was specific for polyphosphoinositides since no breakdown of [BZPIPI was observed under any condition. Very little 32P-labeled PI-P and PI-P2 hydrolysis and 32P-labeled I-P2 and I-P3 release was observed in the absence of calcium. but the phosphodiesterase activity was stimulated lO-fold in the presence of micromolar concentrations of free calcium. The enzyme appears to prefer PI-P2 to PI-P as evidenced by the lower calcium requirement for hydrolysis of PI-P2 under iso—osmotic conditions. Although the requirement for calcium in the breakdown of PI-P and PI-P2 in rat hepatocytes contradicts many of the reports of vasopressin stimulated PI-P and PI-P2 breakdown in cells 108 depleted of calcium by EGTA. and the lack of polyphosphoinositide breakdown induced by the calcium ionOphore A23187 (11.13.16). there is sUpporting evidence that at least a minimal amount of calcium is required for polyphosphoinositide breakdown in reSponse to vaSOpressin (14.15.17). Hydrolysis of polyphosphoinositides by a calcium stimulated phosphodiesterase has been demonstrated in brain (35). platelets (36). and erythrocyte membranes (37). The studies on the dose response of calcium on the polyphosphoinositide phosphodiesterase demonstrated that half-maximal hydrolysis of PI-P and PI-P2 could be obtained at lOOnM free calcium when assayed at low ionic strength. However. under conditions which simulate intracellular K... and Na... concentrations. the calcium requirement for phosphodiesterase activity shifted markedly to the right. Half-maximal phosphodiesterase activity on PI-P2 was then observed at SuM free calcium. Similar effects of ionic strength on the erythrocyte membrane polyphosphoinositide phosphodiesterase have been reported (38). Since hepatocyte intracellular calcium concentrations range from 0.2 - 0.6uM (39.40). it is questionable whether the polyphosphoinositide phosphodiesterase is active under normal intracellular conditions. Basal turnover of polyphosphoinositides may occur instead via a phosphomonoesterase whose presence is suggested by the data in Figure 2. A metal-independent phospholipid phosphomonoesterase has been described in 109 erythrocyte membranes (41) and rat liver nuclear envelopes (42). However. perturbations in the lipid and/or protein micro-environment surrounding the phOSphodiesterase. may result in shifting the phosphodiesterase requirement for calcium toward normal intracellular concentrations. Trauble (43) reported that when a lipid membrane is fully ionized (pH>7). the membrane's transition temperature is increased upon increasing the ionic strength of the surrounding environment. Therefore. in-vitro. decreasing the ionic strength surrounding the membrane would probably make it more fluid. and may result in less constraints on the phosphodiesterase. thus lowering it's requirement for calcium. It has also been shown that norepinephrine and angiotensin II can increase the lipid fluidity. as measured by diphenylhexatriene fluorescence polarization. of plasma membranes isolated from rat liver (44). We have shown that the addition of vasOpressin to plasma membranes incubated under ionic conditions similar to the 2n-vivo intracellular environment. stimulated the breakdown of 20% of the 32 [ P]PI-P present in the plasma membrane fraction in l min. 2 The amount of [32P1PI-P2 breakdown was less than observed during a 1 min incubation of intact cells with vasopressin suggesting that some other factor may have been lost during isolation of the plasma membranes. Alternatively. an intact cytoskeleton connnected to the plasma membrane may be necessary to invoke the maximum changes in the plasma 110 membrane structure which occurs during hormone-receptor binding and subsequent phOSphodiesterase activity. This study is the first demonstration that the plasma membrane is the location of polyphosphoinositide breakdown in response to vasopressin treatment of rat hepatocytes. The breakdown proceeds by the action of a calcium-dependent. polyphosphoinositide-specific phosphodiesterase. This enzyme can be activated by incubation of isolated plasma membranes with vasOpressin under intracellular-like ionic conditions. The increase in intracellular calcium may then procede by inhibiting the calcium pumping activity of the 2+ plasma membrane (Ca -Mg2+)ATPase (20—22) and/or by releasing I-P3 from PI-P2 which acts to mobilize endOplasmic reticulum calcium stores (23). 10. 11. 12. Ill References Michell. R.H. (1975) Biochim. BiOphys. Acta 122. 81-147. Michell. R.H. (1979) Trends Biochem. Sci. 1. 128-131. Akhtar. R.A.. and Abdel-Latiff. A.A. (1978) J. Pharmacol. Exp. Ther. _91. 655-668. Farese. R.V.. Saber. M.A.. and Vandor. S.L. (1979) J. Biol. Chem. 221. 6842-6844. Kirk. C.J.. Creba. J.A.. Downes. 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Smith. C.D.. and Wells. W.W. (1984) Submitted to Arch. Biochem Biophys for publication. Trauble. H. (1976) in Structure of Biological Membranes. (Abrahamsson. s.. and Pascher. 1.. eds.) pp. 509-550. Plenum Press. New York. Burgess. G.M.. Giraud. F.. Poggioli. J.. and Claret. M. (1983) Biochim. BiOphys. Acta 731. 387-396. CHAPTER I I I Characterization of myg-Inositol Trisphosphate PhoSphatase in Rat Liver Plasma Membranes lI5 116 Abstract myg-Inositol triSphosphate (I-P3) has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study. the breakdown of I-P3 by a phosphatase activity was characterized. Using partially purified subcellular fractions. it was found that Eye-inositol trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction. while myg-inositol biSphosphate phosphatase Specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P ase was 3 Mg2+-dependent with Optimal activity observed at 0.5-1.5 mM free MgZ+. The enzyme had a neutral pH optimum suggesting that it was neither an acid or alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However. both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine inhibited the I-P ase activity by 3 50%. while putrescine and spermidine had litle or no effect. These data suggest that cellular I-P levels may not be 3 regulated by compartmentalization within the cell. but rather by some other agents affecting the enzyme and/or the substrate such as spermine. II7 Footnotes 1The abbreviations used are: PI. phOSphatidyl- inositol; PI-P. phoSphatidylinositol 4-phoSphate; PI-PZ. phosphatidylinositol 4.5-bi5ph05phate; I-P. myg-inositol phosphate; I-P . myg—inositol biSphOSphate; I-P . 2 3 myQ-inositol triSphOSphate; I-Pzase. Egg-inositol biSphOSphate phosphatase; I-P ase. myg-inositol 3 triSphosphate phosphatase; HEPES. N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid; MES. 2-[N-Morpholinolethanesulfonic acid; BICINE. N.N-bis[2-Hydroxyethyllglycine; EDTA. ethylenediaminetetraacetic acid; EGTA. ethyleneglycol- bis-(B-aminoethyl ether)-N.N.N'-tetraacetic acid; TCA. trichloroacetic acid. 118 Introduction In rat hepatocytes. calcium mobilizing hormones such as vasopressin. angiotensin II and epinephrine enhance phOSphatidylinositol (PI)1 breakdown (1-4) as well as decrease the level of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4.5-bisphosphate (PI-P2) in prelabeled rat hepatocytes (5-8). Previous studies in this laboratory (8) demonstrated that the plasma membrane is the site of increased polyphosphoinositide (PI-P and PI-Pz) breakdown in reSponse to vasopressin. A calcium-dependent polyphosphoinositide-specific phosphodiesterase activity in partially purified plasma membranes was described. which was responsible for the agonist stimulated breakdown of polyphosphoinositides. The correlation between phosphoinositide (PI. PI-P. and PI-PZ) breakdown and increased levels of intracellular calcium. suggests that phosphoinositides or products of the metabolism of phosphoinositides may somehow influence the various mechanisms which act to modulate intracellular calcium. Indeed. it has been postulated that decreases in the concentration of plasma membrane phosphoinositides could affect calcium channels (9.10. but see 11) or some other calcium transport system such as the plasma membrane (Ca2+-MgZ+)ATPase (12-14). Alternatively. myg-inositol trisphosphate (I-P3)' the water soluble product released from PI-P2 by action of the calcium-dependent II9 polyphOSphoinositide phosphodiesterase. releases calcium from non-mitochondrial intracellular stores in saponin-permeabilized rat hepatocytes (15) and pancreatic acinar cells (16). I-P3 is rapidly degraded in rat hepatocytes (15). blowfly salivary glands (l7) and human erythrocyte membranes (18). presumably by a phosphatase-type reaction. However. only the erythrocyte membrane myo-inositol triSphosphatase (I-P3ase) has been characterized. Therefore. in light of the possible important role of I-P as a second messenger 3 for the hormonal mobilization of intracellular calcium in rat liver. we have further characterized the I-P3ase activity in rat liver. 120 EXperimental Procedures Hepatocyte Incubations and 32P-Labeled Plasma Membrane Isolation - Hepatocytes were isolated from fed male CD-Sprague Dawley rats by a modification of the method described by Seglen (19). The preparation procedures. incubation conditions and the isolation of 32P-labeled plasma membranes were previously described (8.20). Isolation of Subcellular Organelles - Plasma membranes were prepared from rat liver as previously described (21). with the following modifications: 10 mM KHZPO4. pH 7.5 was used instead of 1 mM NaHCO3; the lSOOxg pellets were resu5pended to 200 m1; and the lOOxg/lOOOxg layer was resUSpended to 70 ml. Mitochondria. microsomes and cytosol were prepared from a single rat liver by standard differential centrifugation. Preparation of 32P-labeled I-P2 and I-P3 - Utilizing the presence of an endogenous Ca2+-dependent polyphosphoinositide-specific phosphodiesterase in rat 32P-labeled I-P2 and I-P3 P-labeled plasma membranes hepatocyte plasma membranes (20). were isolated by incubating 32 (4-5 mg protein) with 20 mM Na-HEPES. 0.5 mM Na-EGTA. pH 7.4 and 110 uM free Ca2+ in a final volume of 10 ml for 10 min at 37°C. The reactions were terminated by the additon of 1 ml of 25 mM Na-EGTA. pH 7.4 and placed on ice. The sample was centrifuged at 4°C for 20 min at 15.000xg to pellet the plasma membrane fragments. The supernatant was removed and loaded onto an anion-exchange column containing 2 ml of 121 Dowex 1 (x8; 200-400 mesh; formate form). The column was washed with 15 ml of 60 mM ammonium formate and the radioactive phOSphate esters sequentially eluted as described by Downes and Michell (22) using 20 m1 of 0.1 M formic acid. 0.2 M ammonium formate to elute Pi and Exp-inositol phosphate (I-P). 35 ml of 0.1 M formic acid. 0.4 M ammonium formate to elute Exp-inositol bisphosphate (I-Pz). and 25 m1 of 0.1 M formic acid. 1.0 M ammonium formate to elute I-P . The fractions corresponding to I-P 3 2 and I-P3 were pooled and acidified by batchwise treatment with Dowex 50 (H+). The resins were pelleted and the sUpernatants lyophilized to remove the formic acid. The residues were dissolved in l-l.5 ml of water. To further characterize the isolated [BZPII-P2 and I-PB' the products were examined by paper chromatography for 20 hrs in l-prOpanol:conc. NH4OH:water (5:4:1) (23.24) and the chromatogram exposed to x-ray film. The [32Pll-P product 2 separated into two radioactive components. One component contained 33% of the radioactivity and had a Rf relative to 329. (a 1 P ) of 1.29. The second component had a R of 0.79. 1 Pi which corresponds identically to inositol bisphosphate (24). The [32PlI-P3 product also separated into two radioactive components. One component had a RP of 0.80. analogous to i inositol bisphosphate. but comprised only 10% of the total radioactivity. The other component had a RP of 0.57. which i is very close to the published RP for inositol i trisphosphate of 0.59 (24). There was not enough [32PlI-P2 122 or I—P3 isolated. in terms of mass. for any further chemical analysis. Assay for the Degradation of [BZPII-P2 and I-P3 - The standard assay to determine the rate of [BZPII-P2 and I-P3 breakdown consisted of preincubating 50-100 ug of protein from either the purified plasma membrane fraction or some other fraction in 250 ul of 50 mM Na-HEPES. 1.0 mM Na-EDTA. pH 7.4 for 2 min at 37°C. Various additions were made (metals. effectors) and the incubations continued for an 32 PlI-P2 or I-P3 cpm) to give a final volume of 500 ul and the reactions additional 10 min. [ was added (3000-4000 continued for various times at 37°C in an oscillating water bath. The reactions were terminated by adding 500 01 of ice-cold 20% trichloroacetic acid (TCA). The precipitates were pelleted by centrifugation and the supernatants extracted four times with 1 m1 diethyl ether to remove the TCA and then neutralized with 2 ul of cone. NH4OH. The phosphate esters were separated on 0.4 m1 Dowex 1 (x8; 200-400 mesh; formate form) columns as described by Downes and Michell (22) into Pi/I-P. I-P2 and I-P3 fractions. Other Methods - Marker enzyme analysis on the subcellular fractions were performed using alkaline phosphodiesterase I as a marker for plasma membranes (25). fumarase for mitochondria (26). and glucose 6-phosphatase in the presence of 40mM L-tartrate for microsomes (27.28). Protein was determined by fluorescamine (29) using bovine serum albumin as the standard. 32P radioactivity was determined by 123 Cerenkov radiation. Free magnesium concentrations were calculated according to the method described by Bartfai (30). Materials - The sources of all reagents and materials were previously described (8.20). 124 Results Breakdown of Endogenously Produced [32PlI-P3by Partially Purified Rat Hepatocyte Plasma Membranes - As previously 32 described (8). when P-labeled plasma membranes isolated from rat hepatocytes were incubated with micromolar concentrations of free calcium. [32P1PI-P and PI-P2 were cleaved by a phoSphodiesterase producing [32PlI-P2 and I-P3. reSpectively. If 2 mM MgCl2 was present in the reaction mix. there was no change in the rate of [32PlPI-P2 or PI-P breakdown. but there was a decrease in the level of 3 [ 2PlI-P produced. concurrent with an increase in [32PlI-P 3 2 and [BZPII-P/Pi. This observation is more adequately described in Figure 1. Adding llO uM free calcium to 32 from [32P1PI-P . Plplasma membranes released [JZPJI-P 2 I 3 Na-EGTA (0.5 mM) was added to block further [BZPJI-P3 release. When 2 mM MgCl was added to the same sample. the 2 level of [BZPII-P was reduced by 50% within 2 min with 3 corresponding increases in [32PlI-P and [BZPJI-P/Pi. Thus. 2 it appears that endogenously produced [BZPII-P is broken 3 down by a magnesium-dependent phosphatase. However. due to the contamination of the [32Plplasma membranes with other organelles. primarily microsomes (20). the subcellular location of I-P phosphatase (I-P ase) could not be 3 3 established. Also. there was a very high background of 32P radioactivity in the [32PlI-P/Pi fraction making quantitation difficult. For these reasons. it was decided 125 Figure l. Endogenous Mg2+-dependent I-P ase Activity in 3 Partially Purified Plasma Membranes Isolated from Rat Hepatocytes. 32 P-Labeled plasma membranes (100 ug protein) were preincubated in 20 mM Na-HEPES. 0.5 mM Na-EGTA. pH 7.4 for 5 min at 37°C. At time-0. CaCl2 was added to give a final concentration of 110 uM free calcium in a total volume of 500 ul. The reaction was allowed to continue for 5 min. and then 10 ul of 25 mM Na-EGTA. pH 7.4 was added to stop the phOSphodiesterase activity. The samples were further incubated for 3 min at 37°C in order to observe any non-metal dependent breakdown of [32PlI-P2 or I-P3. (2 mM final concentration) was added and the samples MgCl2 incubated at 37°C for 2 min. The reactions were stOpped by the addition of 1.5 ml of methanol:chloroform (2:1) and the samples processed as previously described (8). The water soluble phosphate esters were separated by anion exchange chromatography (22). The results shown are the mean values of dUplicate samples from one of three similar [32Pl-labeled hepatocyte plasma membrane preparations. 126 \\ 360- 4 EGTA Mg” ' 320 - I l/ - 0 O 280~ - Rad-RP, 2401 - 4 200v - 2 :- 1 . IOO: ‘ = Q 0 80 .. lad-PB .1 60- - A 40l._...—————"'-—“———s - I t-LPZ 20- - o I I l I I 2 4 6 8 IO Time (min) Figure l 127 to synthesize [32PII-P3 and I-P2 from 32P—labeled plasma membranes and use the labeled inositol polyphoSphates as substrates to determine the subcellular distribution of I-Pzase and I-P3ase using purified subcellular fractions and also further characterize the phosphatase activities. Subcellular Distribution of I—Pzase and I-P3ase Activities — Partially purified plasma membranes, mitochondria and microsomes were prepared as described in "Experimental Procedures.‘ and assayed for various marker enzymes along with an aliquot of cell homogenate and cytosol. The results are shown in Table I. The plasma membrane fraction was enriched ll-fold in alkaline phoSphodiesterase activity, while the mitochondrial and microsomal fractions were enriched 2-fold in their reSpective marker enzymes, fumarase and glucose 6-phosphatase. There was little organelle marker enzyme activity in the cytosolic fraction suggesting good integrity of the isolated organelles. These fractions were assayed. as described in "Experi— mental Procedures." for their ability to degrade [BZPJI—P 2 and I-P3. and the results are described in Table II. The I-P3ase activity was found almost exclusively in the plasma membrane fraction. while I-Pzase activity was found to be more ubiquitous with the highest Specific activity observed in the cytosolic and microsomal fractions. Since the plasma membrane exhibited the highest Specific activity for I-P3ase. all further eXperiments were done using 128 Avh.ov woo. + vmo.o Ao.dv Nmo. + NQN.O aho.ov mac. + mNH.o ANm.ov Noo. + AOA.O Aoo.av 000. + owm.o Ao.av Aoo. H va.o mE\cwEVmGHOE: omnunEDh Awh.ov mno. H HNm.o AO.AV mmoo. H mN.H Amo.ov doc. H 000.0 AmO.Av N00. H hm.a Adm.ov mno. H mhh.o Ao.av Nnd. H vmm.o MEVCAEVu0aOEc ouc.mlo amouoao AH.HAV ANN. + tm.H AO.AV mmNo. H and.o Aho.ov NHOO. H ovoo.o Aoo.av oaoo. H moa.o and.ov Ndoo. + bwoo.o Ao.av Nooo. H Nooo.o mE\cwEVwOHOE: H omuuoquHflozamOLm .xH4 .mdnosucouca Cw co>uo ma OCGHDEOZ QEOQH Q ouncomOEO: AAoU «HH ucoeuuonxm ufiaomou>o HoEOwouuw: Hnwuflc0:00uwz ouocomOEO: AHOU «H ucoewuonxm couuuouh .ouocomOEO: aaou ocu cw 0£>Nco on» no >um>wuuo Daumuonm\cowuuouu any Cw Oaxaca any uo xum>wuuo ufiuauoam on» no vacuuov .oE>sco uoxuue zoo. no moguu>fluuo ufiufluoau o>wunaou och, .ucmewuoaxo cuoo u0u acowuuuonoun accumuufiw o3» EOuu «Canaan ououwaaov uo mcowuofi>ov vuovcoum H c005 ozu Ono c305u uuasnou 0:9 :.mouov000um doucoeduoaxu: cw flonwuumofl no uOE>Nco uuxuoe uOu flosxaoco ouo3 acowuuouu nodsaaounom mum>doc< oE>Ncm uoxuo: H canoe 129 onmn on H Nmnn o NH H «Nu Nana mnow no H mow” . no~ H m~o~ mun uaHOmOuxu ahmn mo H moon m H on on.H mmv Mala omen Hma H hood NNH H mad” mun moEOnOuua: comm cod H onnn o - H emu Menu nHHn m~ H m~o~ mod H mm. an” Hoauvc0500uaz when mm H onma ~ H.vao ha H nmna Mann onnn cod H unmN no H amp mun ocuunsoz osooam o~on o~ H nnom e H hm ma H owm Nana onnm a H onofl om H mafia mun ouocovoso: Haou Ill AI ‘ IN ”ouch .muH rmaH .m .mnH ououumnam coauooum .EQUV xuw>uuuoouuomlm~n .mcowuouoaoua ucououufiv o3» EOuu qunEum ououwanov no cowuofi>ov fluoflcoum H come ecu mun muasmom :.mouov000um «oucoewuoaxm: cu wonwuunon no vomHOUOHQ «cause» 0:» van (wk wow cud: fiMQnOum one: mcofluuoou one .u0nn no cas oH uOu toacaucoo can mun—m _ no mun—m _ «0 coauauvo 0:» >3 wouowuficw one: mcowuuoon 0:8 . m: oouu ZENm.H nco amouan comuuouu no 0: ooH mean: mowuw>wuuo ououozan:Q «mum was NmIH uOu fichoauu vac :.mou:v000um Houcoewuoaxu: cu wonwuum0fl mo wouoaoua aha: acouuuuuu uoaaadounam >ufi>wuu< mmouocamOcm nmIH flcu NQIH mo cowuanuuumwo hoasaaounom HH Odnoh 130 purified plasma membranes as the source of I-P3ase activity. Metal Requirement for Plasma Membrane I—P ase Activity - As 3 shown in Figure l. Mg2+ was required in the assay mix to observe the endogenous breakdown of [BZPlI-P3. Using purified plasma membranes as the source of I-F ase activity. 3 the metal requirement for I-P breakdown was investigated. 3 It was found that calcium and manganese at concentrations of either 1.0 or 5.0 mM (approximately 0.5 and 4.5 mM free metal, respectively) gave only 30% of the activity observed using magnesium at the same concentrations (data not shown). No breakdown of [32911-9 was observed using the polyamines 3 putrescine. spermidine or Spermine at 1.0 or 5.0 mM (data not shown). The dose response of magnesium on I-P ase activity is 3 shown in Figure 2. I-P3ase activity was maximal between 0.5-1.5 mM free Mg2+ with inhibition of I-P3ase activity observed at higher free M92+ concentrations. Time Course of the Breakdown of [32911-9 by Plasma Membranes 3 The rate of [32911-9 breakdown by 50 ug of purified plasma 3 membrane protein is shown in Figure 3. A somewhat linear 32 rate of [ PlI-P3 breakdown was observed over the first 20 . . . o . . min of incubation at 37 C with concurrent production of [32911—92 and [BZPJI—P/Pi. Approximately 65% of the 32911-93 was degraded after 60 min of incubation. Effect of pH on Plasma Membrane I—P ase Activity - The 3 effect of pH on the breakdown of [32?]I-P by plasma 3 membranes was investigated to determine if the I-P3ase 131 Figure 2. Dose ReSponse of Magnesium on Plasma Membrane I-P3ase Activity. I-P3ase activity of purified plasma membranes was assayed as described in ”Experimental Procedures.” using 50 ug of plasma membrane protein and incubating for 20 min at 37°C at various free Mg2+ concentrations. The results shown are the mean values of triplicate samples. The standard error of the mean ranged from S-lO%. 132 ‘1 v . q . . . . 4 ¢ 1 J U 2800 2000 / \ \ \ cpm fi I l200 400' e""”' Of T 1. p- ,- Log (Mmiree Figure 2 133 Figure 3. Time Course of I-P3 Breakdown. I-P3 breakdown was assayed as described in "Experimental Procedures,” using 50 ug of plasma membrane protein and a free magnesium concentration of 1.5 mM. The results shown are the mean values of triplicate samples. The standard error of the mean ranged from S-lO%. 134 2700' 4 \ A 2|00 "' \A _. I'\..|-F’3 A g woo~ I - 9< I 900 ’ Ll-F’, Pi A I * //// ..————-*" /. 1 . 0 I-P I ./ 2 300- ,z 0 0 20 40 60 time (min) Figure 3 135 activity was the result of either an alkaline or acid phOSphatase activity: or a separate enzyme activity. As shown in Figure 4. very litle activity was observed below a pH of 6.0 or above a pH of 8.5. Although the amount of free Mg2+ varies depending upon the pH of the environment. the pH range used in this study would only vary the free Mg2+ concentration between 1.0 mM and 1.5 mM. Very little change in I-PBase activity was observed over the range of 0.5—1.5 mM free M92+ (see Fig. 2). Maximal activity was observed at a pH of 7.5 suggesting that I-P ase is a unique enzyme 3 activity and not the result of a general acid or alkaline phosphatase acting on I-P3. Effect of Various Agents on Plasma Membrane I-P ase Activity 3 Various agents were preincubated for 10 min at 37°C with isolated plasma membranes. The I-PBase activity was then assayed as described in ”EXperimental Procedures.” The results are shown in Table 3. NaF. an inhibitor of acid phosphatase (31): protein phosphatase (32,33) and glucose 6—pho5phatase (34) had no effect on the I-P ase activity at 3 either 0.5 or 5.0 mM. LiCl. which inhibits myo-inositol l-phosphatase in rat brain (35) and rat mammary gland (36). had no effeCt on I-P3ase activity at 1.0 mM concentration and slightly increased the activity at 10 mM. Both L-cysteine and dithiothreitol (0.5 mM) stimulated the I-P3ase activity approximately 2-fold. Only slightly more stimulation was observed using either compound at 5.0 mM. Putrescine and spermidine. at 0.2 and 2.0 mM. either had no 136 Figure 4. Effect of pH on the Plasma Membrane I-P ase 3 Activity. I-P3 ase activity was assayed as described in "Experimental Procedures." using 50 ug of plasma membrane protein and a free magnesium concentration which ranged from 1.0 mM at pH 9.5 to 1.5 mM at pH 4.5. The buffers used to extablish the various pH's were: Na-acetate. pH 4.5—5.5; Na-MES: pH 6.0,6.S; Na-HEPES, pH 7.0-8.0: and Na-BICINE, pH 8.5-9.5. The samples were incubated for 20 min at 37°C. The results shown are the mean values of triplicate determinations. The standard error of the mean ranged from 5-10%. 137 I 4000 .000 \ cpm T /?— do 2000' .A \ A _ ‘\ / .a’dh“I / «xi-P, Pi HDOC)’ ’//f . I '- \\\ / ..v-C/ .\ . .z’. 16/4;4-Fé \\.‘\\I .:.— /1 l J l ?\. l 91.0 5.0 6 0 7.0 8.0 9.0 l0.0 pH Figure 4 138 Table III Effect of Various Agents on I-P PhOSphatase ActiVity 3 Agent was preincubated for 10 min at 37°C with the assay mix described in ”EXperimental Procedurgg" which contains 50 ug of plasma membrane protein and31.0 mM free Mg . The reactions were initiated by the addition of 1 PlI-P and continued for 10 min. The reactions were stopped with TCA and tee samples were processed as described in "Experimental Procedures." The results are expressed as the mean 1 standard deViation of duplicate samples from two different experiments. 32 P~Radioactiv1ty (Cpm) Agent Conc.(mM) I-P. P I—P2 I—P3 None - 894 1 33 297 1 14 2017 1 24 L-Cysteine 0.5 1782 1 18 S31 1 14 1029 1 0 5.0 1862 1 28 616 1 79 796 1 98 Dithiothreitol 0.5 1744 1 127 487 1 36 1135 1 143 5.0 1893 1 116 $01 1 25 884 1 44 NaF 0.5 1040 1 37 272 1 4 1834 1 47 5.0 1042 1 9 316 1 10 1817 1 S3 LiCl 1.0 943 1 71 269 1 2 2052 1 56 10.0 1230 1 221 277 1 12 1823 1 173 Putrescine 0.2 907 1 S4 287 1 17 1934 1 47 2.0 1143 1 33 322 1 24 1679 1 76 Spermidine 0.2 1163 1 34 350 1 2 1773 1 22 2.0 1102 1 6 233 1 25 1917 1 29 Spermine 0.2 911 1 38 273 1 27 1949 1 34 2.0 435 1 S 73 1 S 2727 1 S 2.3-BiSpho5pho- 0.2 675 1 4 174 1 20 2340 1 60 glycerate 2.0 292 1 7 73 1 14 2850 1 83 139 effect or only slightly stimulated the I-P ase activity. 3 Spermine. at 0.2 mM. had no effect on the activity, but 2.0 mM spermine inhibited I-P ase approximately 50%. 3 2:3—Bi3ph05phoglycerate, which acts as a competitive inhibitor of I-P3ase in human erythrocyte membranes (18), also inhibited the I-P3ase of rat hepatocyte plasma membranes by 24% and 67% at 0.2 and 2.0 mM, reSpectively. 140 Discussion The recent reports of Egg-inositol trisphosphate acting as the second messenger for hormonal mobilization of cellular calcium (15.16) has increased the need for understanding the enzymes involved in polyphosphoinositide metabolism. In this study. we have characterized the prOperties of a myg-inositol trisphosphate phosphatase in rat liver. [BZPII-P2 and I-P3 were obtained from partially purified plasma membranes containing 32P-labeled PI-P and PI-P2 by activating the Ca2+-dependent polyphosphoinositide-specific phosphodiesterase (8) with 110 uM free calcium. and then isolating the phoSphate esters released into the aqueous media by anion exchange chromatography. The radiochemical purity of the isolated 3 [ 2PII—P and I-P was 66% and 90%. respectively. Using 2 3 these substrates. it was found that the I-P3ase activity is located almost entirely in the plasma membrane. while I-Pzase activity is located primarily in the cytosolic and microsomal fractions. I—P3ase has a pH Optimum of 7.5 and is not inhibited by other phosphatase inhibitors such as NaF or LiCl. This evidence. along with the difference in subcellular distribution of the I—Pzase and I-Paase. suggest that the I-Paase activity is the result of a specific enzyme and not a general alkaline or acid phosphatase. nor a non-specific protein phosphatase or myg-inositol l-phosphatase acting on I-P3. 141 The I-P3ase requires magnesium for activity and is maximally activated at 0.5-1.5 mM. which is well within the range of 1333139 intracellular magnesium levels (37). I—P3ase is stimulated by L-cysteine and dithiothreitol suggesting a role for sulfhydryl groups in the catalytic properties of the enzyme. Downes et a1. (18) demonstrated that 2.3-bisphosphoglycerate acted as a competitive inhibitor of the I—P3ase activity in erythrocyte membranes. A similar level of inhibition by 2.3-bisphosphoglycerate was seen when tested on I-P3ase in rat liver plasma membranes. This suggest that the enzyme may have a particular affinity for pairs of vicinal monoester phosphates. The presence of an I-P ase activity in rat liver 3 invokes a second level of control between the calcium-mobilizing agonist binding to the receptor and the increased level of intracellular calcium. Release of I-P3 from PI-P2 is regulated through a calcium-dependent polyphosphoinositide-specific phosphodiesterase. which appears to be linked either directly or indirectly to the hormone binding to the receptor (8). However. regulation of I-P3ase activity. and thus cellular I-P3 levels. remains undefined. Subcellular compartmentalization does not directly regulate I-P levels since the production of I-P 3 3 and the I-Pzase activity reside in the plasma membrane. Thus for I-P3 to act as a second messenger for the hormonal mobilization of intracellular calcium. the 1232139 rate of I-P3 synthesis must exceed the rate of its breakdown at the 142 plasma membrane. Figure 1 demonstrates that I-P breakdown 3 is actually faster than its synthesis in isolated plasma membrane fragmentts. Therefore. one must postulate that inhibitors of the I-P3ase activity located at or near the plasma membrane may inactivate the I-P ase during 3 calcium-mobilizing agonist activation of the phoSphodisterase. in-vivo. Data in this report suggest that spermine. at near physiological levels (38). inhibits I-P3ase activity. The mode of this inhibition is not clear. Yip and Balis (37) demonstrated that polyamine-polyphosphate complexes can act as enzyme inhibitors. Furthermore. it was shown that 2.3-diphosphoglycerate-spermine complex acts as an uncompetitive inhibitor of 2.3-diphosphoglyceric acid phosphatase. Wells and Smith (39) have shown that spermine has the highest affinity of all the polyamines for PI-Pz. and three times higher affinity for PI-P2 than calcium. Studies on the regulation of intracellular polyamine levels have focussed primarily on examining the effects on ornithine decarboxylase. the rate limiting enzyme in the polyamine biosynthetic pathway (40). Ornithine decarboxlyase activity is regulated by cAMP (41.42). presumably through a cAMP-dependent protein kinase. These studies show stimulation of ornithine decarboxylase 1-4 hrs following the elevation of cAMP. Therefore. a cAMP-type mechanism for increasing intracellular spermine levels would be too slow to affect calcium mobilization. which occurs only seconds after agonist binding. Thus. the in-vivo 143 regulation of myo-inositol triSphosphate phosphatase and the possible role of spermine remains unknown. 10. 11. 12. 13. 144 References Kirk. C.J.. Michell. R.H.. and Hems. D.A. (1981) Biochem. J. 123. 155-165. Litosch. 1.. Lin. S.H.. and Fain. J.N. (1983) J. Biol. Chem. ggg. 13727-13732. Billah. M.M.. and Michell. R.H. (1979) Biochem. J. 1gg. 661-668. Pripic. V.. Blackmore. P.F.. and Exton. J.H. (1982) J. Biol. Chem. ggz. 11323-11331. Rhodes. D.. Pripic. V.. Exton. J.H.. and Blackmore. P.F. (1983) J. Biol. Chem. 3§§. 2770-2773. Creba. J.A.. Downes. C.P.. Hawkins. P.T.. Brewster. 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C.V.. and Russell. D.H. 650-652. Natl. Acade SCi e 22' (1975) Science 1;]. SUMMARY The plasma membrane is believed to be the subcellular site of vasopressin-induced polyphosphoinositide breakdown. however. it has not been adequately documented. In Chapter II of this thesis. it was demonstrated that the plasma membrane is the subcellular site of polyphosphoinositide breakdown in reSponse to vasOpressin. Using plasma membrane fragments. it was found that the breakdown was catalyzed by a calcium-dependent. polyphOSphoinositids-specific phosphodiesterase. At low ionic strength. the phosphodiesterase was maximally activated at 200 nM free calcium. while at physiological ionic strength. the calcium requirement increased to 110 uM free calcium for maximal activity. This would suggest that under normal in-vivo conditions. the phosphodiesterase is inactive and normal turnover of polyphosphoinositides would probably proceed by a kinase-phosphomonoesterase pathway. However. addition of vasOpressin to plasma membrane fragments incubated in buffer containing 200 nM free calcium at physiological ionic strength. activated the phosphodiesterase. Under these conditions. only phosphatidylinositol 4.5-bisphosphate (PI-P2) was broken down. suggesting that in-vivo PI-P is 2 the preferred substrate for vasopressin-induced 148 149 phosphodiesterase activity. Although these studies were conducted using only vasopressin. the result could be extended to include other calcium-mobilizing hormones. It is reported in the literature that vasOpressin acts similarly in the rat hepatocyte as other calcium-mobilizing hormones such as angiotensin II. epinephrine and phenylephrine. The mechanism by which vasOpressin activates the phoSphodiesterase is unknown. One could postulate that the binding of hormone to receptor induces a change in the membrane structure allowing calcium to have easier access to the enzyme. A more reasonable mechanism would involve a conformational change in the enzyme Upon hormone binding to the receptor. which would then decrease the amount of calcium needed to activate the enzyme. Obviously. more information concerning the nature of the hormone-receptor and receptor-enzyme interactions is necessary. This could be obtained by purifying the enzyme and the vasOpressin receptor. and studying their physical prOperties. The purified proteins could then be used to prepare antibodies. which could be used to probe for the tOpical relationship of the enzyme and the receptor in the plasma membrane. The final studies presented in this thesis are perhaps the most interesting and important in terms of the role of phosphoinositides in calcium homeostasis. As was described in Chapter II of this thesis. plasma membranes contain a calcium-dependent. polyphosphoinositide-specific 150 phosphodiesterase. This enzyme is activated under physiological conditions of calcium and ionic strength. by the calcium-mobilizing hormone. vasopressin. The product of the phoSphodiesterase activity acting on PI-P myo-inositol 2! triSphOSphate (I-P3). has been demonstrated in saponin-permeabilized cells to release calcium from non-mitochondrial stores. These two pieces of evidence strongly suggest that I-P may act as a second messenger for 3 the hormone-induced mobilization of intracellular calcium. Therefore. the metabolism of I-P3 may be very important in regulating intracellular calcium levels. In Chapter III of this thesis. an I-P3-specific phosphatase (I-P3ase) was described in purified plasma membranes. This phOSphatase required physiological levels of magnesium for optimum activity. displayed a neutral pH Optimum for activity. and was not inhibited by phosphatase inhibitors such as NaF or LiCl. The I-Pzase activity was confined almost exclusively to plasma membranes. whereas 322-inositol bisphosphate phosphatase was more ubiquitous. with its highest specific activity in the microsomal and cytosolic fractions. The I-P3ase activity was found to be inhibited by physiological levels of spermine. but not by spermidine or putrescine. The regulation of I-P3ase is very important in light of the proposed role of I-P3 as the second messenger for hormone-induced mobilization of intracellular calcium. The level of I-P3 does not appear to be regulated by subcellular 151 I compartmentalization. In fact. the plasma membrane contains by far the highest level of I-P synthetic and degradative 3 activity. This would suggest that an inhibitor of the I-P3ase activity must be localized at or near the plasma membrane in order for adequate levels of I-P3 to be released. Spermine. at physiological concentrations. was found to inhibit I-P3ase. Therefore. the regulation of intracellular spermine levels may be linked to I-P ase 3 activity and intracellular calcium levels. Alternatively. the calcium mobilizing ability of I-P may be an artifact of 3 the saponin-permeabilized cell system. Further work on the role of phosphoinositides in calciumm homeostasis and cell differendiation will undoubtedly prove to be both controversial and exciting.