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Ca2+ Ionophore and Phorbol Ester Stimulate Diacylglycerol Formation and Phosphatidylcholine Hydrolysis in Rat Parotid Acinar Cells
Ca 2+ lonophore
and
Formation
Phorbol
and in Rat
Takao
Komabayashi,
Ester
Stimulate
Phosphatidylcholine Parotid
Atsushi Kazuhiro
Yakata,
Acinar Tetsuya
Suda and Minoru
Diacylglycerol Hydrolysis
Cells
Izawa',
Masamichi
Noguchi,
Tsuboi
Department of Physiology and Pharmacology, and 'Laboratory for Exercise and Applied Physiology, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received
December
13, 1991
Accepted
February
13, 1992
ABSTRACT-We investigated the effects of A23187 and phorbol 12,13-dibutyrate (PDBu) on sn-1,2 diacylglycerol (DAG) accumulation and phosphatidylcholine (PC) hydrolysis in rat parotid acinar cells. Both A23187 and PDBu, in concentration ranges of 0.001-0.1 uM, stimulated DAG accumulation and PC hydrolysis in a time and concentration-dependent manner. Treatment with A23187 and PDBu stimulated the release of [3H]choline and [3H]phosphocholine into the medium, indicating [3H]PC hy drolysis is due to the activation of phospholipases C and D; however, [3H]phosphatidylethanolamine hydrolysis was not indicated. These releases were unaffected by the addition of glucose 6-phosphate, a phosphatase inhibitor. Staurosporine, a protein kinase C inhibitor, significantly inhibited the DAG accumulation and the PC hydrolysis stimulated by these agents. Combinations of A23187 and PDBu potentiated the stimulatory effect which each of these agents alone had on DAG accumulation and PC hydrolysis. This mode of action was additive but not synergistic. These results suggest that DAG accu mulation induced by A23187 and PDBu is related to the PC hydrolysis mediated via the activation of phospholipases C and D, and that it is not related to phosphatidylethanolamine hydrolysis. Keywords: Parotid Phosphatidylcholine
acinar cells, hydrolysis
A23187,
Phorbol
Agonist binding to its receptor leads to phosphatidyl inositol 4,5-bisphosphate breakdown into sn-1,2-diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (1,4,5 IP3) via activation of inositol phospholipid-specific phospholipase C coupled with a GTP binding protein; DAG activates protein kinase C and 1,4,5-IP3 mobilizes Ca 2+ from the endoplasmic reticulum (1-4). These events are thought to play an important role in stimulus-secretion coupling (5 7). DAG was shown to be derived from phosphoino sitides, phosphatidylcholine (PC), and other lipids (7 10), but the relationship between DAG generation and their source during agonist stimulation is not fully understood. This may be due to the complexity of the kinetics of DAG formation; e.g., the kinetics of DAG formation varies with the agonist and the cell type. Re cently, PC hydrolysis has gained much attention, be cause DAG generated by protein kinase C-dependent
12,13-dibutyrate,
sn-1,2-Diacylglycerol
accumulation,
PC-specific phospholipase C can provide a sustained activation of protein kinase C (11). In many cell types, CA2+ ionophore or phorbol ester stimulates PC hy drolysis through phospholipase C and/or D and results in DAG accumulation. However, the mode (phospho lipase C, phospholipase D) by which these agents elicit DAG formation from PC varies with the cell type (12). As reviewed by Billah and Anthes (12), there are many reports concerning PC hydrolysis induced by Ca2+ ionophore and phorbol esters, but the mechanism of PC hydrolysis in this system remains unknown. We therefore examined the effects of A23187 and phorbol 12,13-dibutyrate (PDBu) on DAG accumula tion and PC hydrolysis in rat parotid acinar cells. These results provide evidence that a protein kinase C-depend ent PC hydrolysis is closely related to DAG accumula tion and that the hydrolysis is mediated through phos pholipases C and D.
MATERIALS AND METHODS Materials The following chemicals were obtained from the indi cated firms: [ y-32P]ATP (> 4000 Ci/mmol, ICN Radio chemicals), [meth yl-3H]choline chloride (75 85 Ci/ mmol) and [1-3H]ethan-l-ol-2-amine hydrochloride (5 30 Ci/mmol, Amersham), DAG kinase (Lipidex), PDBu and staurosporine (Sigma), and A23187 (Calbi nochem). All other reagents were of analytical grade. Preparation of parotid acinar cells Parotid glands were removed from sodium pentobar bital (50 mg/kg)-anesthetized male Wistar rats (180 200g). Acinar cells were prepared by enzymatic diges tion with collagenase, as described previously (13). These cells (106cells/ml) were incubated in a HEPES buffered Krebs-Henseleit medium containing: 98 mM NaCl, 5 mM KCI, 1.3 mM CaC12, 1.2 mM MgSO4, 2.4 mM NaHCO3, 10 mM Na-HEPES, 11 mM dextrose, and 1% (w/v) bovine serum albumin. The medium also contained essential amino acids and was maintained at pH 7.4 under an atmosphere of 95% oxygen and 5% carbon dioxide. The number of cells was determined microscopically in a hemacytometer. All experiments were carried out at 37°C. DAG assay DAG levels in the lipid extracts of acinar cells were measured by a highly sensitive assay capable of quanti tating the mass of DAG in crude lipid extracts of cells, as previously described by Preiss et al. (14). This assay depends on the measurement of the DAG mass con verted from DAG to [32P]phosphatidic acid ([32P]PA) by DAG kinase in the presence of [ y-32P]ATP. [32P]PA was separated by thin layer chromatography on a Silica Gel 60 plate. The radioactive spot corresponding to 32p]PA was scraped off and counted by liquid scintilla tion spectrometry. The DAG content of cells was deter mined from the sample volume and the specific activity of the ATP. PC and phosphatidylethanolamine hydrolysis assay Acinar cells were prelabeled with [3H]choline chlo ride (10 ,uCi/ml) or [3H]ethanolamine hydrochloride (10 ,uCi/ml) for 90 min at 37°C, as described previously by Matozaki and Williams (15). The percentage of tritium incorporated into the cellular PC and phosphatidyletha nolamine fractions in the phospholipids was 84% and 99%, respectively, when acinar cells were labeled with [3H]choline or [3H]ethanolamine. For the hydrolysis assay, acinar cells were incubated with agonists for the indicated time. After centrifuga
tion at 10000 X g for 15 sec, each supernatant (0.8 ml) was extracted with 3 ml of a chloroform/methanol mix ture (1 : 2, v/v), and then 1 ml of chloroform and 1 ml of water were added. The aqueous phase was counted to determine the release of [3H]choline or [3H] ethanolamine metabolites. For the analysis of the released radioactivity, [3H]phosphocholine, [3H]glycerophosphocholine, [3H] choline, and [3H]betaine were separated by thin layer chromatography in a solvent system containing water (0.9% NaCl)/methanol/concentrated NH4OH (50:50:5, v/v). Each radioactive spot was scraped off and counted by liquid scintillation spectrometry. Data analyses All data are expressed as the mean ± S.E. Signifi cance of difference between means was determined us ing Student's t-test. P values < 0.05 were considered significant. In each figure where S.E. bars are not shown, they are within the symbols. RESULTS Action of A23187 on DAG accumulation induced by PDBu Figure 1 shows the time course of DAG accumula tion in response to A23187 and PDBu. DAG accumula tion induced by A23187 and PDBu increased linearly with time. The percentages of DAG accumulation at 30 min were 40% and 38% for stimulation by A23187 and PDBu, respectively. 4 a-Phorbol 12,13-didecanoate, an inactive phorbol ester, did not cause in DAG accumula tion (data not shown). As seen in Fig. 2, DAG accu mulation induced by A23187 or PDBu alone was dose dependent. These data also show that combinations of A23187 and PDBu significantly potentiated the stimula tory effect of each agent acting alone; the combinations produced an additive increase. Analysis of [3H]choline metabolites released into the medium We evaluated [3H]choline metabolites released into the medium (Fig. 3). Both A23187 and PDBu caused the release of [3H]choline and [3H]phosphocholine into the medium; more [3H]phosphocholine than [3H]choline was released. In contrast, the effects of A23187 and PDBu on the release of [3H]glycerophosphocholine were barely discernible. In order to study whether the formation of [3H]choline by A23187 and PDBu was due to the hy drolysis of [3H]phosphocholine released into the medium by extracellular phosphatases, the phosphatase inhibitor (16) glucose 6-phosphate was added with these
Fig. 1. Time course of DAG formation stimulated with A23187 and PDBu. Acinar cells were incubated with A23187 or PDBu. Each point represents the mean ± S.E. of quadruplicate determinations from two separate experiments. Basal (.A , 0.1,uM A23187 (o), 0.1,uM PDBu (0).
Fig.
2.
Effect
of A23187
and
PDBu
alone
cubation solution and incubated with various mean ± S.E. of quadruplicate determinations
and
in combination
on
DAG
accumulation.
concentrations of A23187 or from two separate experiments.
agents for 30 min. Glucose 6-phosphate (20 mM) did not modify the release of [3H]phosphocholine and that of [3H]choline induced by 0.1 ,uM A23187 and 0.1 ,uM PDBu: for the basal, basal plus glucose 6-phosphate, A23187, A23187 plus glucose 6-phosphate, PDBu, and PDBu plus glucose 6-phosphate, the levels of [3H] phosphocholine assayed in the medium were 300 ± 58, 320 ± 41, 509 ± 44, 501 ± 36, 515 ± 33, and 502 ± 44, respectively; and the levels of [3H]choline assayed in the medium were 125 ± 17, 120 ± 16, 237 ± 32, 230 ± 31, 187 ± 28, and 175 ± 11, respectively (dpm/106 cells, n = 5). The sum of the radioactivities of [3H]phosphQcholine,
PDBu
for
Acinar 30 min.
cells Each
were column
suspended represents
in in the
[3H]choline, and [3H]glycerophosphocholine released into the medium at 30 min was less than the total activ ity estimated (Figs. 3 and 5). Since the [3H]choline uti lized in this study was labeled in the methyl position, additional labeled metabolites might be generated in the cells, in particular betaine through the action of be taine aldehyde dehydrogenase. We further examined the release of [3H]betaine into the medium. Stimulation by these agents for 30 min had a significant (P < 0.05) effect on the radioactivity associated with this metabo lite (dpm/106 cells, n = 5, basal, 50 ± 6; 0.1,uM A23187, 188 ± 42; 0.1 pM PDBu, 163 ± 31).
Fig. 3. Analysis of [3H]cholinemetabolites released into the medium. Acinar cells were stimulated with 0.1 ,UMA23187 or 0.1 uM PDBu for 30 min. The amounts of each [3H]choline metabolite observed at 0 time were subtracted from each value. Each column represents the mean ± S.E. of quadruplicate determinations from two separate experiments. *P < 0.05, **P < 0.01 vs. basal value. [3H]phosphocholine(L), [3H]choline(•), [3H]glycerophosphocholine(®).
Effects of A23187 and PDBu on the release of [3HJcholine or [3H]ethanolamine metabolites Both A23187 (0.1,uM) and PDBu (0.1,uM) stimu lated the release of [3H]choline metabolites with time and these effects were dose-dependent (Figs. 4 and 5). Combinations of A23187 and PDBu also significantly potentiated the stimulatory effect of each agent acting alone; the combinations produced an additive increase. In several cell types, phosphatidylethanolamine hy
drolysis was shown to be related to DAG formation (17). To further investigate this possibility in the . pres ent study, we next examined the effect of 0.1 ,uM A23187 or 0.1,uM PDBu on the release of [3H] ethanolamine metabolites into the medium at 30 min. These agents had no effect on the release of such metabolites (dpm/106 cells, n = 5, basal, 897 ± 25; 0.1 ,uM A23187, 885 ± 30; 0.1,aM PDBu, 900 ± 21).
Fig. 4. Time course of the release of [3H]choline metabolites induced by A23187 or PDBu. Acinar cells were stimulated with 0.1 uM A23187 or 0.1 uM PDBu for the indicated time. The radioactivity observed at 0 time was subtracted from each value. Each point represents the mean ± S.E. of quadruplicate determinations from two separate experiments. Basal (0), 0.1 pM A23187(0), 0.1 pM PDBu (0).
Fig. 5. Effect of A23187and PDBu alone and in combination on the release of [3H]cholinemetabolites. Acinar cells were suspended in incubation solution and incubated with various concentrations of A23187or PDBu for 30 min. The radioactiv ity observed at time 0 was subtracted from each value. Each column represents the mean ± S.E. of quadruplicate deter minations from two separate experiments.
Effects of staurosporine on DAG accumulation and the release of f3HJcholine metabolites induced by A23187 or PDBu We next evaluated the effect of staurosporine (18), a putative protein kinase C inhibitor, on DAG formation and the release of [3H]choline metabolites induced by A23187 or PDBu. The data given in Table 1 revealed that staurosporine significantly inhibited DAG accu mulation and the release of [3H]choline metabolites in duced by A23187 or PDBu; however, it did not alter basal responses in control cells. In addition, the inhibi tory effect of staurosporine on DAG formation and the
release of [3H]choline metabolites was observed 5 min after the addition and continued for at least 30 min (data not shown). DISCUSSION Activation important
of protein in various
kinase C has been physiological
shown
processes.
In
to be exo
crine glands, protein kinase C activation is involved in the secretory response and seems to modulate the ac tion of various substances. As already indicated, car bachol,
a Ca t+-mobilizing
receptor
agonist,
stimulates
Table 1. Effects of staurosporine on DAG accumulation and the release of [3H]choline metabolites induced by A23187 or PDBu
DAG formation in part through a protein kinase C in rat parotid acinar cells (13). In many cell types, Ca2+ ionophores and phorbol esters are now known to in crease DAG levels through PC, but not phosphoinosi tide, hydrolysis (12, 15, 19-21). In this study, we showed that both A23187 and PDBu stimulated DAG generation (Figs. 1 and 2) and the release of [3H]choline metabolites into the medium (Figs. 4 and 5), and that their effects were significantly inhibited by staurosporine, a protein kinase C inhibitor, which is equivalent to H-7 in inhibiting DAG accumulation in duced by carbachol (13) (Table 1). The dose-depend ence of the inhibitory influence of staurosporine was similar for both responses. These results show that PC metabolism in parotid acinar cells is regulated by a pro tein kinase C, and are in agreement with those reported by others in different cell types. Preliminary experi ments had shown that neither A23187 nor PDBu stimu lated IP3 production in rat parotid acinar cells. The pres ent study also shows that neither A23187 nor PDBu stimulated phosphatidylethanolamine hydrolysis. These observations indicate that DAG generated by A23187 and PDBu is derived from PC. Staurosporine also inhibits cyclic AMP and cyclic GMP-dependent kinases, although with much less potency. McAtee and Dawson (22) have reported that activation of protein kinase A decreases DAG genera tion through the inhibition of phospholipase C in the neurotumor cell line NCB-20. If staurosporine had an effect on protein kinase A, DAG generation should in crease. Therefore, it does not seem that the inhibitory effect of staurosporine is related to protein kinase A. PC hydrolysis by a phospholipase C produces phos phocholine and DAG. In contrast, a phospholipase D would catalyze the formation of choline and phosphati date from PC. Phosphatidate releases DAG by the sub sequent action of phosphatidate phosphohydrolase (23). Thus, PC hydrolysis is mediated through phospholipase C and/or D (12); however, no conclusions regarding phospholipase stimulation were reached in this study on parotid acinar cells. We have observed that carbachol stimulated PC hydrolysis is related to the activation of phospholipase C in this system (24). In the present study, both A23187 and PDBu stimulated the release of [3H]phosphocholine and [3H]choline into the medium, indicating the activation of phospholipases C and D (Fig. 3). The contribution of a phospholipase D to PC hydrolysis stimulated by these agents is further sup ported by evidence that [3H]choline released into the medium is not due to the degradation of [3H]phos phocholine by phosphatase. These results are similar to those shown by Huang and Cabot (25) in rat aorta smooth muscle cells, but is not in agreement with re
sults that PC hydrolysis induced by phorbol ester, 12-0 tetradecanoylphorbol 13-acetate, is mediated through a phospholipase D in REF52 cells (23) and a phospho lipase C in myoblast cells (26), respectively. From these observations it seems likely that the mode of PC hy drolysis varies among cell and agonist types. The synergistic action of phorbol ester and Ca2+ ionophore has been observed in several cell types (27 31). This action has been ascribed to the interaction of the Ca2+ /calmodulin-dependent pathway and the pro tein kinase C-dependent pathway, but the detailed mechanism underlying the synergistic action is unclear. Takuma and Ichida (32) reported that the combination of A23187 (I /_M) and phorbol 12-myristate 13-acetate (1 ,uM) induced a weak synergistic effect on amylase re lease (about 5%). This synergistic effect is much small er than that seen in other cell types (27-31). Our pre vious report (33) showed that an increase in DAG accumulation was involved in the potentiating effect of A23187 on carbachol-stimulated amylase release in parotid acinar cells and that this effect was additive. Furthermore, the present study shows that the com bination of A23187 and PDBu evoked an additive in crease in DAG accumulation and PC hydrolysis (Figs. 2 and 5). These data can not completely account for the synergistic effect of Ca2+ ionophore and phorbol ester on amylase release, but DAG, generated from PC, may be closely related to the increase in amylase release in duced by the combination of Ca2+ ionophore and phor bol ester. In summary, the present results demonstrate that A23187 and PDBu-stimulated DAG accumulations are associated with PC hydrolysis mediated through phos pholipases C and D, and it extends the hypothesis of a role of DAG in stimulus-response coupling to the en zyme secretion process in parotid glands. REFERENCES
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A23187 induced release of arachidonic acid from rabbit poly morphonuclear leucocytes. Biochem. J. 238, 425-436 (1986) Muir, J.G. and Murray, A.W.: Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C. J. Cell. Physiol. 130, 382 391 (1987) McAtee, P. and Dawson, G.: Rapid dephosphorylation of
phatidylcholine cycles. Trends Biochem. Sci. 14, 28-30 (1989) 10 Rana, R.S. and Hokin, L.E.: Role of phosphoinositides in transmembrane signaling. Pharmacol. Rev. 70, 115 -164
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protein kinase C substrates by protein kinase A activators re sults from inhibition of diacylglycerol release. J. Biol. Chem. 264, 11193-11199 (1989) Cabot, M.C., Welsh, C.J., Cao, H-T. and Chabbott, H.: The
(1990) 11 Besterman, J.M., Duronio, V. and Cuatrecasas, P.: Rapid formation of diacylglycerol from phosphatidylcholine: a path way for generation of a second messender. Proc. Natl. Acad. Sci. U.S.A. 83, 6785 (1986) 12 Billah, M.M. and Anthes, J.C.: The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem. J. 269, 281 291 (1990) 13 Komabayashi, T., Yakata, A., Izawa, T., Suda, K., Rubin, R.P. and Tsuboi, M.: Protein kinase C-dependent diacyl
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Nishizuka, Y.: Phospholipid degradation and signal trans lation for protein phosphorylation. Trends Biochem. Sci. 8, 13-16 (1983) Bruzzone, R.: The molecular basis of enzyme secretion. Gas troenterology 99, 1157 -1176 (1990) Augert, G., Bocckino, S.B., Blackmore, P.F. and Exton, J.H.: Hormonal stimulation of diacylglycerol formation in hepatocytes: evidence for phosphatidylcholine breakdown. J. Biol. Chem. 264, 21689-21698 (1989) Pelech, S.L. and Vance, D.E.: Signal transduction via phos
glycerol formation is mediated via Cat+-calmodulin in parotid cells. Eur. J. Pharmacol. Mol. Pharmacol. 207, 175-181 (1991) 14 Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedle, J.E. and Bell, R.M.: Quantitative measurement of sn-1,2 diacylglycerol present in platelets, hepatocytes, and ras and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597 8600 (1986) 15 Matozaki, T. and Williams, J.A.: Multiple sources of 1,2 diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin: involvement of phosphatidylinositol bisphos
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29
phospholipid/Ca2+ dependent protein kinase. Biochem. Bio phys. Res. Commun. 135, 397-402 (1986) 19 Bocckino, S.B., Blackmore, P.F. and Exton, J.H.: Stimula tion of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J. Biol. Chem. 260, 14201-14207 (1985) 20 Meade, C., Turner, G.A. and Bateman, P.E.: The role of polyphosphoinositides and their breakdown products in
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phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation. FEBS Lett. 233, 153 157 (1988) Komabayashi, T., Yakata, A., Izawa, T., Fujinami, H., Suda, K. and Tsuboi M.: Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells. Eur. J. Pharmacol. Mol. Pharmacol. 225, 209 216 (1992) Huang, C. and Cabot, M.C.: Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types: evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacyl glycerol by a phospholipase C pathway. J. Biol. Chem. 265, 14858 14863 (1990) Grove, R.I. and Schimmel, S.D.: Effects of 12-O-tetradeca noylphorbol 13-acetate on glycerolipid metabolism in cultured myoblasts. Biochim. Biophys. Acta 711, 272-280 (1982) Katakami, Y., Kaibuchi, K., Sawamura, M., Takai, Y. and Nishizuka, Y.: Synergistic action of protein kinase C and cal cium for histamine release from rat peritoneal mast cells. Biochem. Biophys. Res. Commun. 121, 573-578 (1984) de Pont, J.J.H.H.M. and Fleuren-Jakobs, A.M.M.: Synergis tic effect of A23187 and a phorbol ester on amylase secretion from rabbit pancreatic acini. FEBS Lett. 170, 64-68 (1984) Tanaka, C., Taniyama, K. and Kusunoki, M.: A phorbol ester and A23187 act synergistically to release acetylcholine from the guinea pig ileum. FEBS Lett. 175, 165-169 (1984) Merritt, J.E. and Rubin, R.P.: Pancreatic amylase secretion and cytoplasmic free calcium: effects of ionomycin, phorbol dibutyrate and diacylglycerols alone and in combination. Biochem. J. 230, 151-159 (1985) Kinoshita, Y., Fukasa, M., Yamatani, T., Hishikawa, R. and Fujita, T.: Phorbol esters stimulate phosphate accumulation synergistically with A23187 in cultured renal tubular cells. Biochem. Biophys. Res. Commun. 136, 177-182 (1986) Takuma, T. and Ichida, T.: Phorbol ester stimulates amylase secretion from rat parotid cells. FEBS Lett. 199, 53 56 (1986) Komabayashi, T., Yakata, A., Izawa, T., Suda, K. and Tsuboi, M.: Diacylglycerol accumulation is involved in the potentiating effect of A23187 on carbachol-stimulated amyl ase secretion from parotid gland. Res. Commun. Chem. Pathol. Pharmacol. 70, 297-305 (1990)
and
Formation
Phorbol
and in Rat
Takao
Komabayashi,
Ester
Stimulate
Phosphatidylcholine Parotid
Atsushi Kazuhiro
Yakata,
Acinar Tetsuya
Suda and Minoru
Diacylglycerol Hydrolysis
Cells
Izawa',
Masamichi
Noguchi,
Tsuboi
Department of Physiology and Pharmacology, and 'Laboratory for Exercise and Applied Physiology, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received
December
13, 1991
Accepted
February
13, 1992
ABSTRACT-We investigated the effects of A23187 and phorbol 12,13-dibutyrate (PDBu) on sn-1,2 diacylglycerol (DAG) accumulation and phosphatidylcholine (PC) hydrolysis in rat parotid acinar cells. Both A23187 and PDBu, in concentration ranges of 0.001-0.1 uM, stimulated DAG accumulation and PC hydrolysis in a time and concentration-dependent manner. Treatment with A23187 and PDBu stimulated the release of [3H]choline and [3H]phosphocholine into the medium, indicating [3H]PC hy drolysis is due to the activation of phospholipases C and D; however, [3H]phosphatidylethanolamine hydrolysis was not indicated. These releases were unaffected by the addition of glucose 6-phosphate, a phosphatase inhibitor. Staurosporine, a protein kinase C inhibitor, significantly inhibited the DAG accumulation and the PC hydrolysis stimulated by these agents. Combinations of A23187 and PDBu potentiated the stimulatory effect which each of these agents alone had on DAG accumulation and PC hydrolysis. This mode of action was additive but not synergistic. These results suggest that DAG accu mulation induced by A23187 and PDBu is related to the PC hydrolysis mediated via the activation of phospholipases C and D, and that it is not related to phosphatidylethanolamine hydrolysis. Keywords: Parotid Phosphatidylcholine
acinar cells, hydrolysis
A23187,
Phorbol
Agonist binding to its receptor leads to phosphatidyl inositol 4,5-bisphosphate breakdown into sn-1,2-diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (1,4,5 IP3) via activation of inositol phospholipid-specific phospholipase C coupled with a GTP binding protein; DAG activates protein kinase C and 1,4,5-IP3 mobilizes Ca 2+ from the endoplasmic reticulum (1-4). These events are thought to play an important role in stimulus-secretion coupling (5 7). DAG was shown to be derived from phosphoino sitides, phosphatidylcholine (PC), and other lipids (7 10), but the relationship between DAG generation and their source during agonist stimulation is not fully understood. This may be due to the complexity of the kinetics of DAG formation; e.g., the kinetics of DAG formation varies with the agonist and the cell type. Re cently, PC hydrolysis has gained much attention, be cause DAG generated by protein kinase C-dependent
12,13-dibutyrate,
sn-1,2-Diacylglycerol
accumulation,
PC-specific phospholipase C can provide a sustained activation of protein kinase C (11). In many cell types, CA2+ ionophore or phorbol ester stimulates PC hy drolysis through phospholipase C and/or D and results in DAG accumulation. However, the mode (phospho lipase C, phospholipase D) by which these agents elicit DAG formation from PC varies with the cell type (12). As reviewed by Billah and Anthes (12), there are many reports concerning PC hydrolysis induced by Ca2+ ionophore and phorbol esters, but the mechanism of PC hydrolysis in this system remains unknown. We therefore examined the effects of A23187 and phorbol 12,13-dibutyrate (PDBu) on DAG accumula tion and PC hydrolysis in rat parotid acinar cells. These results provide evidence that a protein kinase C-depend ent PC hydrolysis is closely related to DAG accumula tion and that the hydrolysis is mediated through phos pholipases C and D.
MATERIALS AND METHODS Materials The following chemicals were obtained from the indi cated firms: [ y-32P]ATP (> 4000 Ci/mmol, ICN Radio chemicals), [meth yl-3H]choline chloride (75 85 Ci/ mmol) and [1-3H]ethan-l-ol-2-amine hydrochloride (5 30 Ci/mmol, Amersham), DAG kinase (Lipidex), PDBu and staurosporine (Sigma), and A23187 (Calbi nochem). All other reagents were of analytical grade. Preparation of parotid acinar cells Parotid glands were removed from sodium pentobar bital (50 mg/kg)-anesthetized male Wistar rats (180 200g). Acinar cells were prepared by enzymatic diges tion with collagenase, as described previously (13). These cells (106cells/ml) were incubated in a HEPES buffered Krebs-Henseleit medium containing: 98 mM NaCl, 5 mM KCI, 1.3 mM CaC12, 1.2 mM MgSO4, 2.4 mM NaHCO3, 10 mM Na-HEPES, 11 mM dextrose, and 1% (w/v) bovine serum albumin. The medium also contained essential amino acids and was maintained at pH 7.4 under an atmosphere of 95% oxygen and 5% carbon dioxide. The number of cells was determined microscopically in a hemacytometer. All experiments were carried out at 37°C. DAG assay DAG levels in the lipid extracts of acinar cells were measured by a highly sensitive assay capable of quanti tating the mass of DAG in crude lipid extracts of cells, as previously described by Preiss et al. (14). This assay depends on the measurement of the DAG mass con verted from DAG to [32P]phosphatidic acid ([32P]PA) by DAG kinase in the presence of [ y-32P]ATP. [32P]PA was separated by thin layer chromatography on a Silica Gel 60 plate. The radioactive spot corresponding to 32p]PA was scraped off and counted by liquid scintilla tion spectrometry. The DAG content of cells was deter mined from the sample volume and the specific activity of the ATP. PC and phosphatidylethanolamine hydrolysis assay Acinar cells were prelabeled with [3H]choline chlo ride (10 ,uCi/ml) or [3H]ethanolamine hydrochloride (10 ,uCi/ml) for 90 min at 37°C, as described previously by Matozaki and Williams (15). The percentage of tritium incorporated into the cellular PC and phosphatidyletha nolamine fractions in the phospholipids was 84% and 99%, respectively, when acinar cells were labeled with [3H]choline or [3H]ethanolamine. For the hydrolysis assay, acinar cells were incubated with agonists for the indicated time. After centrifuga
tion at 10000 X g for 15 sec, each supernatant (0.8 ml) was extracted with 3 ml of a chloroform/methanol mix ture (1 : 2, v/v), and then 1 ml of chloroform and 1 ml of water were added. The aqueous phase was counted to determine the release of [3H]choline or [3H] ethanolamine metabolites. For the analysis of the released radioactivity, [3H]phosphocholine, [3H]glycerophosphocholine, [3H] choline, and [3H]betaine were separated by thin layer chromatography in a solvent system containing water (0.9% NaCl)/methanol/concentrated NH4OH (50:50:5, v/v). Each radioactive spot was scraped off and counted by liquid scintillation spectrometry. Data analyses All data are expressed as the mean ± S.E. Signifi cance of difference between means was determined us ing Student's t-test. P values < 0.05 were considered significant. In each figure where S.E. bars are not shown, they are within the symbols. RESULTS Action of A23187 on DAG accumulation induced by PDBu Figure 1 shows the time course of DAG accumula tion in response to A23187 and PDBu. DAG accumula tion induced by A23187 and PDBu increased linearly with time. The percentages of DAG accumulation at 30 min were 40% and 38% for stimulation by A23187 and PDBu, respectively. 4 a-Phorbol 12,13-didecanoate, an inactive phorbol ester, did not cause in DAG accumula tion (data not shown). As seen in Fig. 2, DAG accu mulation induced by A23187 or PDBu alone was dose dependent. These data also show that combinations of A23187 and PDBu significantly potentiated the stimula tory effect of each agent acting alone; the combinations produced an additive increase. Analysis of [3H]choline metabolites released into the medium We evaluated [3H]choline metabolites released into the medium (Fig. 3). Both A23187 and PDBu caused the release of [3H]choline and [3H]phosphocholine into the medium; more [3H]phosphocholine than [3H]choline was released. In contrast, the effects of A23187 and PDBu on the release of [3H]glycerophosphocholine were barely discernible. In order to study whether the formation of [3H]choline by A23187 and PDBu was due to the hy drolysis of [3H]phosphocholine released into the medium by extracellular phosphatases, the phosphatase inhibitor (16) glucose 6-phosphate was added with these
Fig. 1. Time course of DAG formation stimulated with A23187 and PDBu. Acinar cells were incubated with A23187 or PDBu. Each point represents the mean ± S.E. of quadruplicate determinations from two separate experiments. Basal (.A , 0.1,uM A23187 (o), 0.1,uM PDBu (0).
Fig.
2.
Effect
of A23187
and
PDBu
alone
cubation solution and incubated with various mean ± S.E. of quadruplicate determinations
and
in combination
on
DAG
accumulation.
concentrations of A23187 or from two separate experiments.
agents for 30 min. Glucose 6-phosphate (20 mM) did not modify the release of [3H]phosphocholine and that of [3H]choline induced by 0.1 ,uM A23187 and 0.1 ,uM PDBu: for the basal, basal plus glucose 6-phosphate, A23187, A23187 plus glucose 6-phosphate, PDBu, and PDBu plus glucose 6-phosphate, the levels of [3H] phosphocholine assayed in the medium were 300 ± 58, 320 ± 41, 509 ± 44, 501 ± 36, 515 ± 33, and 502 ± 44, respectively; and the levels of [3H]choline assayed in the medium were 125 ± 17, 120 ± 16, 237 ± 32, 230 ± 31, 187 ± 28, and 175 ± 11, respectively (dpm/106 cells, n = 5). The sum of the radioactivities of [3H]phosphQcholine,
PDBu
for
Acinar 30 min.
cells Each
were column
suspended represents
in in the
[3H]choline, and [3H]glycerophosphocholine released into the medium at 30 min was less than the total activ ity estimated (Figs. 3 and 5). Since the [3H]choline uti lized in this study was labeled in the methyl position, additional labeled metabolites might be generated in the cells, in particular betaine through the action of be taine aldehyde dehydrogenase. We further examined the release of [3H]betaine into the medium. Stimulation by these agents for 30 min had a significant (P < 0.05) effect on the radioactivity associated with this metabo lite (dpm/106 cells, n = 5, basal, 50 ± 6; 0.1,uM A23187, 188 ± 42; 0.1 pM PDBu, 163 ± 31).
Fig. 3. Analysis of [3H]cholinemetabolites released into the medium. Acinar cells were stimulated with 0.1 ,UMA23187 or 0.1 uM PDBu for 30 min. The amounts of each [3H]choline metabolite observed at 0 time were subtracted from each value. Each column represents the mean ± S.E. of quadruplicate determinations from two separate experiments. *P < 0.05, **P < 0.01 vs. basal value. [3H]phosphocholine(L), [3H]choline(•), [3H]glycerophosphocholine(®).
Effects of A23187 and PDBu on the release of [3HJcholine or [3H]ethanolamine metabolites Both A23187 (0.1,uM) and PDBu (0.1,uM) stimu lated the release of [3H]choline metabolites with time and these effects were dose-dependent (Figs. 4 and 5). Combinations of A23187 and PDBu also significantly potentiated the stimulatory effect of each agent acting alone; the combinations produced an additive increase. In several cell types, phosphatidylethanolamine hy
drolysis was shown to be related to DAG formation (17). To further investigate this possibility in the . pres ent study, we next examined the effect of 0.1 ,uM A23187 or 0.1,uM PDBu on the release of [3H] ethanolamine metabolites into the medium at 30 min. These agents had no effect on the release of such metabolites (dpm/106 cells, n = 5, basal, 897 ± 25; 0.1 ,uM A23187, 885 ± 30; 0.1,aM PDBu, 900 ± 21).
Fig. 4. Time course of the release of [3H]choline metabolites induced by A23187 or PDBu. Acinar cells were stimulated with 0.1 uM A23187 or 0.1 uM PDBu for the indicated time. The radioactivity observed at 0 time was subtracted from each value. Each point represents the mean ± S.E. of quadruplicate determinations from two separate experiments. Basal (0), 0.1 pM A23187(0), 0.1 pM PDBu (0).
Fig. 5. Effect of A23187and PDBu alone and in combination on the release of [3H]cholinemetabolites. Acinar cells were suspended in incubation solution and incubated with various concentrations of A23187or PDBu for 30 min. The radioactiv ity observed at time 0 was subtracted from each value. Each column represents the mean ± S.E. of quadruplicate deter minations from two separate experiments.
Effects of staurosporine on DAG accumulation and the release of f3HJcholine metabolites induced by A23187 or PDBu We next evaluated the effect of staurosporine (18), a putative protein kinase C inhibitor, on DAG formation and the release of [3H]choline metabolites induced by A23187 or PDBu. The data given in Table 1 revealed that staurosporine significantly inhibited DAG accu mulation and the release of [3H]choline metabolites in duced by A23187 or PDBu; however, it did not alter basal responses in control cells. In addition, the inhibi tory effect of staurosporine on DAG formation and the
release of [3H]choline metabolites was observed 5 min after the addition and continued for at least 30 min (data not shown). DISCUSSION Activation important
of protein in various
kinase C has been physiological
shown
processes.
In
to be exo
crine glands, protein kinase C activation is involved in the secretory response and seems to modulate the ac tion of various substances. As already indicated, car bachol,
a Ca t+-mobilizing
receptor
agonist,
stimulates
Table 1. Effects of staurosporine on DAG accumulation and the release of [3H]choline metabolites induced by A23187 or PDBu
DAG formation in part through a protein kinase C in rat parotid acinar cells (13). In many cell types, Ca2+ ionophores and phorbol esters are now known to in crease DAG levels through PC, but not phosphoinosi tide, hydrolysis (12, 15, 19-21). In this study, we showed that both A23187 and PDBu stimulated DAG generation (Figs. 1 and 2) and the release of [3H]choline metabolites into the medium (Figs. 4 and 5), and that their effects were significantly inhibited by staurosporine, a protein kinase C inhibitor, which is equivalent to H-7 in inhibiting DAG accumulation in duced by carbachol (13) (Table 1). The dose-depend ence of the inhibitory influence of staurosporine was similar for both responses. These results show that PC metabolism in parotid acinar cells is regulated by a pro tein kinase C, and are in agreement with those reported by others in different cell types. Preliminary experi ments had shown that neither A23187 nor PDBu stimu lated IP3 production in rat parotid acinar cells. The pres ent study also shows that neither A23187 nor PDBu stimulated phosphatidylethanolamine hydrolysis. These observations indicate that DAG generated by A23187 and PDBu is derived from PC. Staurosporine also inhibits cyclic AMP and cyclic GMP-dependent kinases, although with much less potency. McAtee and Dawson (22) have reported that activation of protein kinase A decreases DAG genera tion through the inhibition of phospholipase C in the neurotumor cell line NCB-20. If staurosporine had an effect on protein kinase A, DAG generation should in crease. Therefore, it does not seem that the inhibitory effect of staurosporine is related to protein kinase A. PC hydrolysis by a phospholipase C produces phos phocholine and DAG. In contrast, a phospholipase D would catalyze the formation of choline and phosphati date from PC. Phosphatidate releases DAG by the sub sequent action of phosphatidate phosphohydrolase (23). Thus, PC hydrolysis is mediated through phospholipase C and/or D (12); however, no conclusions regarding phospholipase stimulation were reached in this study on parotid acinar cells. We have observed that carbachol stimulated PC hydrolysis is related to the activation of phospholipase C in this system (24). In the present study, both A23187 and PDBu stimulated the release of [3H]phosphocholine and [3H]choline into the medium, indicating the activation of phospholipases C and D (Fig. 3). The contribution of a phospholipase D to PC hydrolysis stimulated by these agents is further sup ported by evidence that [3H]choline released into the medium is not due to the degradation of [3H]phos phocholine by phosphatase. These results are similar to those shown by Huang and Cabot (25) in rat aorta smooth muscle cells, but is not in agreement with re
sults that PC hydrolysis induced by phorbol ester, 12-0 tetradecanoylphorbol 13-acetate, is mediated through a phospholipase D in REF52 cells (23) and a phospho lipase C in myoblast cells (26), respectively. From these observations it seems likely that the mode of PC hy drolysis varies among cell and agonist types. The synergistic action of phorbol ester and Ca2+ ionophore has been observed in several cell types (27 31). This action has been ascribed to the interaction of the Ca2+ /calmodulin-dependent pathway and the pro tein kinase C-dependent pathway, but the detailed mechanism underlying the synergistic action is unclear. Takuma and Ichida (32) reported that the combination of A23187 (I /_M) and phorbol 12-myristate 13-acetate (1 ,uM) induced a weak synergistic effect on amylase re lease (about 5%). This synergistic effect is much small er than that seen in other cell types (27-31). Our pre vious report (33) showed that an increase in DAG accumulation was involved in the potentiating effect of A23187 on carbachol-stimulated amylase release in parotid acinar cells and that this effect was additive. Furthermore, the present study shows that the com bination of A23187 and PDBu evoked an additive in crease in DAG accumulation and PC hydrolysis (Figs. 2 and 5). These data can not completely account for the synergistic effect of Ca2+ ionophore and phorbol ester on amylase release, but DAG, generated from PC, may be closely related to the increase in amylase release in duced by the combination of Ca2+ ionophore and phor bol ester. In summary, the present results demonstrate that A23187 and PDBu-stimulated DAG accumulations are associated with PC hydrolysis mediated through phos pholipases C and D, and it extends the hypothesis of a role of DAG in stimulus-response coupling to the en zyme secretion process in parotid glands. REFERENCES
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