Journal of Narrochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture Maria del Carmen Garcia, *Mamela G. Lbpez, *Antonio G. Garcia, and Mariano Sinchez Crespo Departamento de Bioquimica y Fisiologia-CSIC, Facdtad de Medicina, Valladolid; and *Depariamento de Farmacologia, Facultad de Medicina. Universidad Autbnoma, Madrid, Spain

Abstract: Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation ofchromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with ['H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerolin chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in ['Hloleate molecu-

lar species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor. Key Words: Diacylglycerol-Phospholipid-Signaling. Garcia M. C. et al. Muscarinic acetylcholine receptor enhances phosphatidylcholine hydrolysis via phospholipase D in bovine chromaffin cells in culture. J. Neurochem. 59, 2244-2250 (1992).

Many hormones and neurotransmitters act through the hydrolysis of phosphatidylinositol (PI) bisphosphate to inositol trisphosphate and 1,2-sn-diacylglycerol (DAG) (for review, see Fisher et a]., 1992). In accord with this view, PI turnover in chromaffin cells has been shown to be activated by different agonists (Fisher et al., 1981; Bunn et al., 1988; Sasakawa et al., 1989; Stauderman and Pruss, 1990). Whereas nicotinic agonists produce a well-characterized secretory response, muscarinic receptor stimulation causes different secretory and biochemical responses in bovine adrenal medullary chromaffin cells. Bovine chromaffin cells show a poor (O'Sullivan and Bur-

goyne, 1988; Kim and Westhead, 1989) or nonexistent (Fisher et al., 1981; Almazin et al., 1984; Livett and Boksa, 1984;Cheek and Burgoyne, 1985; Ballesta et al., 1989)secretory response on stimulation of their muscarinic receptors and clear biochemical changes such as an increase in cyclic GMP levels (Yanagihara et al., 1979; Derome et al., 1981) and enhanced turnover of inositol-containing lipids (Fisher et al., 1981; Mohd-Adnan and Hawthorne, 1981; Forsberg et al., 1986). The breakdown of phosphatidylcholine (PC) has emerged as an alternative pathway for phospholipid turnover linked to signal transduction in both neural

Received February 21, 1992; revised manuscript received May 10, 1992; accepted May 29, 1992. Address correspondence and reprint requests to Dr. M. Snchez Crespo at Departamento de Bioquimica y Fisiologia, Facultad de Medicina, Universidad de Valladolid, 47005-Valladolid, Spain.

Abbreviafions used: CDP-choline, cytidine 5'-diphosphocholine; DAG, 1,2-sn-diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol; PLD, phospholipase D.

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PC CYCLE IN CHROMAFFIN CELLS and nonneural tissues (Pelech and Vance, 1989; Billah and Anthes, 1990; Exton, 1990). In keeping with this view is the description of a muscarinic receptor coupled to phospholipase D (PLD) acting on PC in canine brain (Quian and Drewes, 1989), the activation of PC hydrolysis via muscarinic receptors in 1321N1 astrocytoma cells (Martinson et al., 1989), and the coupling of muscarinic receptor subtypes to PLD, as shown by transfection of human embryonic kidney cells with the genes for four muscarinic acetylcholine receptors (m 1, m2, m3, and m4) (Sandmann et al., 1991). Moreover, stimulation of perfused hearts in vitro and of rat brains in vivo with muscarinic agonists causes the release of choline, and this appears to be related to PC hydrolysis (Brehm et al., 1987;Lindmar and Loffelholz, 1988). However, this pathway has received little attention in chromaffin cells (Purkiss et al., 1991; Rivera et al., 1991), and different mechanisms of PC hydrolysis have been reported in bovine adrenal cells as compared with rat pheochromocytoma-derived PC12 cells (Purkiss et al., 1991). In this article we show (a) the activation by acetylcholine of PC breakdown, (b) the possible involvement of PLD in PC breakdown, and (c) the association of acetylcholine-induced PC turnover with the activation of muscarinic receptors. MATERIALS AND METHODS

Materials Phosphorylcholine chloride, cytidine S’-diphosphocholine (CDP-choline), L-PC, L-phosphatidic acid (PA), Lphosphatidylethanolamine, L-PI, L-phosphatidylserine, DAG, sodium oleate, acetylcholine, atropine, and d-tubocurarine were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Calbiochem (La Jolla, CA, U.S.A.) supplied choline chloride. d-Propranolol was from ICI-Spain. [methyl-3H]Choline chloride (76 Ci/mmol), 1-palmitoyl-2[ l-14C]oleoyl-sn-glycero-3-phosphocholine (52 Ci/mmol) and [14C]oleicacid (53.9 mCi/mmol) were purchased from New England Nuclear (Boston, MA, U.S.A.). [2-,H]Glycerol (15 Ci/mmol) was supplied by Amersham (Bucks, U.K.).

Isolation and culture of bovine chromaffin cells Bovine adrenal chromaffin cells were isolated as described (Moro et al., 1990, 1991). In brief, the glands were washed three times with Ca2’, Mg2+-freeLocke’s medium (154 mMNaCI, 0.56 M K C I , 3.5 mMNaHCO,, 5.6 mM glucose, and 10 mM HEPES, pH 7.4). Medullae digestion was carried out by injecting with a syringe 5 ml of a solution containing 0.25% collagenase, 0.5% bovine serum albumin, and 0.01% soybean trypsin inhibitor in Ca2+, Mg*+-free Locke’s buffer. The glands were kept at 37°C for 15 min, and the procedure was repeated three times. The cells were filtered first through a 217-pm and then through a 82-pm nylon mesh. The collagenase was washed out with a large volume of Caz+, M$’-free Locke’s buffer. The cells were resuspended in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum at a density of 1 X lo6 cells/ml and incubated in suspension in glass bottles under continuous magnetic stimng at 37°C in a water-saturated

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5% c o 2 / 9 5 % air atmosphere. Cells were used for experiments after a period of -24 h in culture, and each experiment was carried out with a different batch of cells. Fibroblasts made up 4%of total cells in the batches.

Cell radiolabeling and agonist treatment Labeling conditions were adapted as described below, after checking that the times selected allowed incorporation of >SO% of the label in phospholipid pools. Labeling of cells with [’Hlcholine. Cells were incubated for 2 h a t 37°C with [rnethyl-3H]cholinechloride (1 pCi/ml) in a Ca2+-free buffer and then washed twice. After these washings, the incubation was started by addition of 1 mM acetylcholine or control solutions and then stopped at different times by the extraction procedure of Bligh and Dyer ( 1959).Aqueous [3H]choline-labeledmetabolites were separated and quantified by the procedure described by Vance et al. ( 1 980). In brief, the aqueous phase was dissolved in water and applied to TLC plates, which were developed to the top in a solvent composed of 0.5% NaCl/ethanol/methanol/ concentrated NH40H (50:30:20:5 by volume). The areas of the plates coincidental with the ratio of fronts of choline, phosphocholine, and CDP-choline standards were scraped off and counted for radioactivity. Phospholipids were separated using TLC plates, developed in the solvent system of chloroform/methanol/acetic acid/water (50:25:8:6 by volume). Areas corresponding to the phospholipid standards were scraped off, and the associated radioactivity was measured. Labeling of cells with [’HJglycerol. Cells were radiolabeled with 5 pCi/ml of [3H]glycerol for 20 min at 37°C before treatment with either 1 mMacetylcholine or control solution. The reaction was stopped at different times as above. Radiolabeled neutral lipids were resolved on TLC plates in a solvent system containing hexane/diethyl ether/ acetic acid (60/40/ 1 by volume). Phospholipids were chromatographed as above. In experiments with muscarinic and nicotinic antagonists, 5 pMatropine and 10 pMd-tubocurarine were incubated for 10 rnin before stimulation by acetylcholine. In experiments performed with propranolol, this inhibitor of PA:phosphohydrolase was added 5 rnin before stimulation of the cells at the concentration of 0.3 mM. Lubeling ofcells with f “CJoleate. Cells were labeled with 1 pCi/ml of [‘4C]sodium oleate for 2 h a t 37°C. After washing, cells were stimulated in the presence of 1 mM acetylcholine. Phospholipids and neutral lipids were separated as above.

PLD assay PLD activity was measured according to the procedure of Kobayashi and Kanfer (1987). This method takes advantage of the transphosphatidylase activity of the enzyme, which in the presence of ethanol allows the transfer of the phosphatidyl moiety of the phospholipid substrate to ethanol and produces phosphatidylethanol. In brief, [“C]phosphatidylethanol formation was measured in a medium containing 2.5 mM microdispersed [I4C]PC, 10 mM EDTA, 25 mM potassium fluoride, 4 m M sodium oleate, and 400 mM ethanol, pH 6.5. The reaction was started by addition of the sonicate of 5 X lo6 cells. Incubations were carried out for 90 min at 30°C and stopped as above. Lipids were separated by TLC with a solvent system consisting of chloroform/methanol/acetone/acetic acid/water (50: 15: 15: 105 by volume) and visualized by iodine vapor. Radioactivities cochromatographing with standards of PA. phosphaJ. Neurocliem.. VoL 59. No. 6 , 1992

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tidylethanol, and neutral lipids were quantified by scintillation counting. 3500

Statistical analyses Data are expressed as mean f SEM values. For companson of two groups of samples normally distributed, Student's two-tailed t test was used to analyze differences for significance. A p value of ~ 0 . 0 5was considered significant.

RESULTS Generation of [3H]cholineby chromaffin cells Because acetylcholine is the physiological agonist of chromaffin cells and can act through either muscarinic or nicotinic receptors, we selected this agonist for this study. Chromaffin cells labeled with ['HIcholine showed a predominant incorporation into PC (>SO%)of total radioactivity associated with phospholipids. In experiments aimed at assessing the time course of the changes in the distribution of ['HIcholine label after exposure to 1 mM acetylcholine, an increase in ['Hlcholine content was detected within the 5-10 min following addition of the agonist (Fig. l), whereas [3H]CDP-choline content showed a reduction of < 15% and ['H]phosphocholine content Accordingly, these results suggested increased 4%. that the dominant effect of acetylcholine on the distribution of 'H radioactivity in water-solublecholine metabolites was an increase in level of intracellular free ['Hlcholine at later times. Coincidental with this change, the 'H radioactivity associated with the PC fraction showed a decrease of -25% at 5- 10 rnin after acetylcholine challenge. Production of 13H]DAGin response to acetylcholine A further appraisal of PC breakdown was carried out in cells labeled with ['Hlglycerol. Under the label-

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FIG. 2. Effect of stimulation by acetylcholineon formation of r3H]DAG by chromaffin cells. Chromaffin cells were labeled with [3H]glycerol and then incubated in the presence (0)or absence (0)of 1 mM acetylcholine. See the legend to Fig. 1 for further details. Data are mean f SEM (bars) values from five separate experiments performed in duplicate. *p < 0.05.

ing conditions used, >75% of the radioactivity associated with phospholipids was incorporated into PC, and the remainder was distributed among PI and phosphatidylethanolamine. As shown in Fig. 2, treatment with I mMacetylcholine produced an increased formation of ['HIDAG as compared with control cells. This increase was maximal at 5-10 rnin after stimulation with acetylcholine, i.e., an interval longer than that required for acetylcholine to induce catecholamine secretion. In a control experiment, acetylcholine induced a reduction in the amount of ['HIglycerol label in the PC fraction that reached up to 20% of prechallenge label at 10 min, which again is consistent with the occurrence of PC hydrolysis. It should be noted that control cells also exhibited some formation of DAG at these times, but the stimulation consistently produced a two- to threefold increase above control values. This strongly indicates the existence of PC breakdown because the accumulation of ['HIDAG shows a time course different from the reported accumulation of inositol trisphosphate (from PI hydrolysis),which peaks at very early times (30 s- 1 min) after stimulation (Plevin and Boarder, 1988; Sasakawa et al., 1989; Stauderman and Pruss, 1990). The lack of PA accumulation during these experimental conditions could be explained by the rapid degradation of PA to DAG, when PA:phosphohydrolase is not inhibited.

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FIG. 1. Time course and effect of acetylcholine on formation of [3H]choline in chromaffin cells. Cells were labeled with [3H]choline for 2 h at 37°C as described in Materials and Methods. At the end of this period 1 mM acetylcholine (0)or control solution (0)was added, and the reaction was stopped at different times. Data are mean f SEM (bars) values from three experiments performed in duplicate. *p < 0.05.

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Cellular source of [3H]DAG The increased rate of ['HIDAG formation in stimulated cells pointed to PC hydrolysis. However, even though most ['Hlglycerol is incorporated into PC, we required further evidence to ascertain whether the ['HIDAG produced in chromaffin cells on stimulation was derived from PC breakdown. That the fatty

PC CYCLE IN CHROMAFFIN CELLS acid composition of PC in mammalian tissues is different from that of PI is well-known (Cockcroft and Allan, 1984; Cabot et al., 1988; Martin and Michaelis, 1988). PC is enriched in oleate-containing molecular species, whereas PI mostly contains stearate and arachidonate molecular species. As shown in Fig. 3, an increased formation of [ ''C]~leate-containing DAG was observed at 5- 10 min after stimulation with acetylcholine, i.e., a temporal course analogous to that detected with ['Hlglycerol labeling. Taken together, these results allow us to conclude that the origin ofthe increased DAG production during stimulation of chromaffin cells by acetylcholine is PC breakdown rather than PI turnover. However, an increase in content of [ ''C]~leoyl-containing DAG larger than that observed when quantifying total DAG would be expected. Because this has not been observed, we may explain this finding on the basis of differences in the labeling turnover of neurotransmitter-sensitive glycerol and oleate pools or, alternatively, on the metabolic routes of DAG species.

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PC breakdown and P A accumulation These studies were carried out taking advantage of the inhibitory effect of d-1-propranolol on PA:phosphohydrolase (Koul and Hauser, 1987), which allows the accumulation of the products of PLD-catalyzed PC hydrolysis. Chromaffin cells were incubated in the presence of 0.3 mMpropranolo1 for 5 min, after labeling with ['H]glycerol and before stimulation with acetylcholine. Both ['HIPA and ['HIDAG were separated and assayed by liquid scintillation. Figure 4A shows that in the presence of propranolol, acetylcholine produces a timedependent accumulation of ['HIPA, whereas this is not observed in the absence of propranolol (Fig. 4B). These results indicate that the hydrolysis of PC is via PLD, which produces a tran-

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FIG. 3. Formation of ['4C]oleoyl-containing DAG by chromaffin cells in response to acetylcholine. Cells were labeled with [I4C]oleate and then incubated with 1 mM acetylcholine (0)or control solution (0).Data are mean f SEM (bars) values from three experiments performed in duplicate. ' p < 0.05.

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FIG. 4. Formation of (A) [3H]PA and (B) [3H]DAGin response to acetylcholine. Cells were labeled with [3H]glyceroland then incubated in the presence (solid symbols) or absence (open symbols) of 0.3 mM propranolol for 5 min before addition of 1 mM acetylcholine (squares) or control solution (circles). Results are expressed after subtraction of control values. Data are mean f SEM (bars) values from three experiments with duplicate samples. ' p < 0.05.

sient formation of PA, which is immediately converted into DAG by PA:phosphohydrolase.

PLD enzymatic activity PLD activity (1,280 2 18 pmol/h/ 10' cells, n = 4 different cell batches) could be detected in resting control cells, which is in keeping with a recent report focusing on different bovine tissues (Wang et al., 199 1). However, we failed to show an increased activity in chromaffin cells stimulated with acetylcholine (1,332 k 158 and 1,158 k 196 pmol/h/107 cells at 1 and 10 min after challenge, respectively).This is probably a consequence of the dependence of this activity on Ca2+,which makes the enzyme assayable on cell disruption (Pai et al., 1988a,b). Propranolol did not modify PLD activity when added to cell homogenates, but as shown in Fig. 4A, it was able to produce an accumulation of ['HIPA in resting cells, which is in keeping with the presence of significant PLD activity in resting chromaffin cells.

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Relation between muscarinic receptor and acetylcholine stimulation Because acetylcholine is an agonist of chromaffin cells that may act via either muscarinic or nicotinic receptors, we designed experiments with selective anJ. Neurochem.. Vol. 59, No. 6. 1992

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FIG. 5. Effect of either nicotinic and muscarinic receptor antage nists on ['HIDAG accumulation after stimulation with acetylcholine. Cells were prelabeled with ['H]glycerol in the presence of medium (0). 10 @Id d-tubocurarine0,or 5 @Id atropine (H)for 10 rnin before stimulation with 1 mM acetylcholine. The incubation was stopped at 0, 10. or 30 min. Data are mean f SEM (bars) values from three experiments performed in duplicate. ' p c 0.05.

tagonists of those receptors to check the possible relationship between the activation of the receptor and the PC cycle evidenced above. Atropine (5 p M ) was used as the muscarinic antagonist, with 10 pA4 d-tubocurarine as the nicotinic antagonist. Figure 5 shows that the ['HIDAG accumulation increased by acetylcholine was inhibited in the presence of atropine, whereas no effect was observed when cells were preincubated with d-tubocurarine. We conclude that acetylcholine-induced stimulation of PC turnover in chromaffin cells occurs via the muscarinic receptor.

DISCUSSION It is established that in many cell types, including a host of bovine tissues, the hydrolysis of the phosphodiester bond of PC is triggered by various agonists. Of particular relevance are recent studies demonstrating that P,-purinergic agonists and vasopressin (known to activate phosphoinositide-specific phospholipase C and mobilize Ca2+)also stimulate phosphodiestearic cleavage of PC (Bocckino et al., 1987; Irving and Exton, 1987; Cabot et al., 1988). In addition, there are some reports of PC breakdown without a concomitant change in inositol lipids (Rosoff et al., 1988; Wright et al., 1988). According to these reports, many cells produce DAG in a biphasic manner on stimulation with agonists. The early phase is quantitatively small, peaks within 30 s of stimulation, and coincides with PI hydrolysis, whereas the delayed phase is large, reaches a maximum within 2- 15 min of stimulation, and is sustained (Dennis et al., 1991). This second phase shows a kinetic pattern similar to what we have measured and can be associated with PC turnover based on the results obtained by labeling the different J. Neurochem.. Vol. 59. No.6, I992

ET AL. moieties of the PC molecule. PC breakdown may be produced by either phospholipase (Grillone et al., 1988; Wright et al., 1988) or PLD (Bocckino et al., 1987; Ragab-Thomas et al., 1987) activities. In most cell types the activation of PLD is more rapid, and this explains why PA mass increases before DAG accumulates through the action of PA:phosphohydrolase (Martin, 1988; Martin and Michaelis, 1988; Pai et al., 1988a,b). PLD activity has been detected in most bovine tissues thus far analyzed (Wang et al., 1991). Rivera et al. ( 199 1) have shown, using NMR spectroscopicanalysis, that a major fraction of labeled PC is on the external membrane surface and can be released by PLD. On the other hand, it has been reported (Purkiss et al., 199 1) that PLD activity is absent from bovine chromaffin cells on the basis of the failure to detect phosphatidylbutanol formation, and there is no evidence of an enhanced formation of water-soluble products of PC hydrolysis on stimulation with either bradykinin or phorbol ester. Our data strongly support the existence of a PLD route for PC hydrolysis in bovine chromaffin cells coupled to the muscarinic receptor, as judged from the net increase of ['Hlcholine content in acetylcholine-stimulated cells, the accumulation of PA in cells stimulated in the presence of propranolol, and the detection of PLD activity in the cell-free system. A question that requires further investigation is the ultimate role of the PC cycle and DAG accumulation in chromaffin cells following activation of muscarinic-type receptors. The primary role of DAG is to activate protein kinase C, although DAG may also directly affect other enzymes (Pelech and Vance, 1984; Maroney and Macara, 1989). Because activation of protein kinase C linked to PC hydrolysis is likely to be more prolonged than that due to PI bisphosphate breakdown, it is reasonable to propose that PC breakdown is involved in cellular control mechanisms that require prolonged activation of protein kinase C, e.g., control of ion channel activity and events related to cell growth and differentiation. With the discovery of multiple forms of protein kinase C exhibiting different tissue and subcellular distribution, substrate specificity, translocation, and regulation (Nishizuka, 1988), it is possible that different molecular species of DAG may exert differential effects on the various forms of protein kinase C. If this was the case, DAG derived from PC breakdown could be involved in cellular effects different from those resulting from PI bisphosphate hydrolysis. It has been proposed that another function of DAG is to act as a source of arachidonate for eicosanoid production. This would be an alternative to the pathway of arachidonate liberation via a Ca2+-dependent phospholipase A,. In fact, our studies in bovine chromaffin cells have failed to show both assayable phospholipase A, activity and production of platelet-activating factor, a phospholipid autacoid produced via

PC CYCLE IN CHROMAFFIN CELLS phospholipase A, in several cell types (data not shown). Our data agree with those obtained by Morgan and Burgoyne (1990) and Zahler et al. (1 986), who concluded that arachidonic acid is not essential for Ca2+dependentexocytosis in adrenal chromaffin cells and who were also unable to detect phospholipase A, activity in membrane fractions from these cells. In keeping with these views would be the finding that DAG breakdown in chromaffin cells is a two-step mechanism mediated by a DAG and a monoacylglycerol lipase (Rindlisbacher et al., 1987). In addition, aside from any involvement in agonist stimulation, PLD alone or linked to PA:phosphohydrolase could function to mobilize PC for synthesis of other phospholipids or triacylglycerols via PA or DAG intermediates. Although further experiments are required to determine the exact mechanism that couples m u m rinic receptor and PC breakdown and the possible effects of DAG and PA as second messengers, it is evident that the PC-specific PLD described here could initiate a pathway of DAG formation that is nearly as specific for PC as the PI-PLC is for phosphoinositides. Acknowledgment: This study has been supported by grants PM88-0010 and PB87-93-C03-01 from DGCICYT.

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Dennis E. A., Rhee S. G., Billah M. M., and Hannum Y.A. (1991) Role of phospholipases in generating lipid second messengers in signal transduction. FASEB J. 5, 2068-2077. Derome G., Tseng R., Mercier P., Lemaue J., and Lemaire S. (198 I ) Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells. Biochem. Pharmacol. 30,855-860. Exton J. H. (1990) Signaling through phosphatidylcholine breakdown. J. Biol. Chem. 265, 1-4. Fisher S. K., Holz R. W., and Agranoff B. W. (1981) Muscarinic receptors in chromaffin cell cultures mediate enhanced phospholipid labeling but not catecholamine secretion. J. Neurochem. 37,49 1-497. Fisher S. K., Heacock A. M., and Agranoff B. W. (1992) Inositol lipids and signal transduction in the nervous system: an u p date. J. Neurochem. 58, 18-38. Forsberg E. J., Rojas E., and Pollard H. B. (1986) Muscarinic recep tor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells. J. Biol. Chem. 261,49 154920. Grillone L. R.,Clark M. A., Godfrey R. W., Stassen F., and Crooke S. T. (1988) Vasopressin induces V1 receptorsto activate phosphatidylinositol- and phosphatidylcholine-specificphospholipase C and stimulates the release ofarachidonic acid by at least two pathways in the smooth muscle cell line, A-10. J. Biol. Chem. 263,2658-2663. Irving H. R. and Exton J. H. (1987) Phosphatidylcholine breakdown in rat liver plasma membranes. J. Biol. Chem. 262, 3440-3443. Kim K. T. and Westhead E. W. (1989) Cellular responses to Ca2+ from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic Ca2+and secretion from bovine chromaffin cells. Proc. Natl. Acad. Sci. USA 86,9881-9885. Kobayashi M. and Kanfer J. N. ( 1 987) Phosphatidylethanolformation via transphosphatidylation by rat brain synaptosomal phospholipase D. J. Neurochem. 48, 1597-1603. Koul 0. and Hauser G. (1987) Modulation of rat brain cytosol phosphatidate phosphohydrolase: effect of cationic amphiphilic drugs and divalent cations. Arch. Biochem. Biophys. 253, 453-46 1. Lindmar R. and LBffelholz K. (1988) On the mechanism ofmuscarinic hydrolysis of cboline phospholipids in the heart. Biochem. Pharmacol. 37,4689-4695. Livett B. G. and Boksa P. (1984) Receptors and receptor modulation in cultured chromaffin cells. Can. J. Physiol. Pharmacol. 62,467-476. Maroney A. C. and Macara I. C. (1989) Translocation ofdiacylglycerol kinase from the cytosol to the membrane in phorbol estertreated Swiss 3T3 fibroblasts. J. Cell. Biochem. 40, 165-172. Martin T. W. (1988) Formation of diacylglycerol by a phospholip a x Dphosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells. Biochim. Biophys. Acta 962,282-296. Martin T. W. and Michaelis K. C. (1988) Bradykinin stimulates phosphodiesteariccleavage of phosphatidylcholinein cultured endothelial cells. Biochem. Biophys. Res. Commun. 157, 1271-1279. Martinson E. A., Goldstein D., and Brown J. H. (1989) Muscarinic receptor activation of phosphatidylcholine hydrolysis. J. Biol. Chem. 264, 14748- 14754. Mohd-Adnan N. A. and Hawthorne J. N. (1981) Phosphatidylinositol labeling in response to activation of muscarinic receptors in bovine adrenal medulla. J. Neurochem. 36, 1858-1860. Morgan A. and Burgoyne R. D. (1990) Relationship between arachidonic acid release and Ca2+dependentexocytosisin digitonin-permeabilized bovine adrenal chromaffin cells. Biochem. J. 271,571-574. Moro M. A., Lbpez M. G., Gandia L., Michelena P., and Garcia A. G. (1 990) Separation and culture of living adrenaline- and noradrenaline-containingcells from bovine adrenal medullae. Anal. Biochem. 185,243-248.

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