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

Rapid Communication

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis Immaculada Herrero, Mafia Teresa Miras-Portugal, and Josk Shchez-Prieto Departamento de Bioquimica, Facultad de Veterinaria, Universidad Comphtense, Madrid, Spain

hances the release oftransmitter glutamate induced by depolarization with 4-aminopyndine (4-AP) (Bame et al., 1991). cis-Unsaturated fatty acids can also activate PKC (Murakami and Routtenberg, 1985; Murakami et al., 1986; Shearman et al., 1991a). However, we have recently shown that low concentrations of exogenous arachidonic acid inhibit glutamate exocytosis (Herrero et al., 1991a,b) by a mechanism that is independent of PKC activation (Herrero et al., 1992). That diacylglycerol or phorbol esters and certain unsaturated fatty acids synergistically activate PKC in vitro and in vivo has recently been described (Verkest et al., 1988; Lester et al., 1991; Shinomura et al., 1991). The purpose of this article was to investigate whether the activation of PKC by both phorbol esters and arachidonic acid has any effect on glutamate release. The results indicate that arachidonic acid dramatically reduced the concentration of 4P-phorbol dibutyrate (4P-PDBu) necessary to potentiate glutamate exocytosis and that both lipids activate a PKC-dependent mechanism for the optimal potentiation of glutamate exocytosis in cerebrocortical synaptosomes.

Abstract: The effects of arachidonic acid and phorbol esters in the Ca"-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations ( 5 0 0 n M ) of 48phorbol dibutyrate (48-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 48-PDBu (1-50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis. Key Words: Synaptosomes-Glutamate exocytosis-Arachidonic acid-Phorbol esters-Protein kinase C-Calcium-Membrane potential. Herrero I. et al. Activation of protein kinase C by phorbol esters and arachidonic acid required for the optimal potentiation of glutamate exocytosis. J. Neurochem. 59, 1574- 1577 (1992).

Protein kinase C (PKC) is a family of enzymes that play an important role in several neuronal functions, such as transmitter release, long-term potentiation, and modulation of ionic channels (Nishizuka, 1988). The activation of PKC with phorbol esters, such as 12-0-tetradecanoylphorbol 13-acetate (TPA), that mimic the physiological activator diacylglycerol (Castagna et al., 1982) enhances the release of various neurotransmitters (Nichols et al., 1987; Shu and Selmanoff, 1988; Shuntoh et al., 1988; Oda et al., 1991), including glutamate (Lynch and Bliss, 1986; Diaz-Guerra et al., 1988; Banie et al., 1991). In isolated nerve terminals (synaptosomes) from cerebral cortex the activation of PKC with high concentrations of phorbol esters (0.1-1 p M ) en-

Resubmitted manuscript received July 6, 1992; accepted July 6, 1992. Address correspondence and reprint requests to Dr. J . SanchezPrieto at Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, Madrid 28040, Spain.

MATERIALS AND METHODS Methyl arachidonate, ~P-PDBu,and TPA were prepared in dimethyl sulfoxide. Arachidonic acid was stored under a nitrogen atmosphere at -70°C as a 15 mM stock suspension in water and occasionally in dimethyl sulfoxide. The P, fraction of synaptosomes was prepared from the cerebral cortices of male Wistar rats (Nicholls, 1978). Synaptosomes (I-mg pellets) were resuspended into 1.5 ml of incubation medium [ 122 mM NaCI, 3.1 m M KCI, 0.4 mM KH,P04, 5 mM NaHCO,, I .2 m M MgS04, 10 mM glucose, and 20 mM N-tris(hydroxymethyl)methyl-2-amino-

Abbrevialions used: 4-AP, 4-aminopyridine; BSA, bovine serum albumin; [Ca2'],, cytosolic free Caz' concentration; ~P-PDBu,48phorbol dibutyrate; PKC, protein kinaseC TPA, 12-0-tetradecanoylphorbol 13-acetate.

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POTENTIATION OF GLUTAMATE RELEASE ethanesulfonie acid buffer, pH 7.41 and incubated for 1 h at 37°C in the presence of 16 pA4 bovine serum albumin (BSA), essentially fatty acid free (Sigma), to prevent the effects of free fatty acids released from synaptosomes during the incubation (Herrero et al., 1991~). Glutamate release was determined as previously described (Nicholls et al., 1987; Rubio et al., 1991). After preincubation for 1 h in the presence of BSA, the synaptosomes were pelleted and resuspended in fresh incubation medium without albumin. An aliquot (1 ml) was transferred to a stirred cuvette containing 1 m M NADP, 50 U of glutamate dehydrogenase, and 1.33 mM CaCI, or 200 nM free [Ca”] [ S O p M EGTA and 38 pMCaCI, (Verhage et al., 1989)]. The fluorescence was measured using a PerkinElmer model LS-50 luminescence spectrometer at 340 and 460 nm. Data were collected at 2-s intervals. The Ca*+-dependent release was calculated by subtracting release at 200 n M frce [Ca”] from release at I .33 mM CaCI,. TPA-preincubated synaptosomes were exposed to 1 piM TPA for 30 min, and the incubation was stopped by placing the tubes in ice. Samples were washed by centrifugation at 17,000 g for 10 min. After preincubation for 30 rnin in the presence of BSA, the synaptosomes were pelleted, resuspended in fresh incubation medium without albumin, and assayed for glutamate release. The cytosolie free Ca” concentration ([Ca”],) was dctermined with fura-2 (Grynkiewicz ct al., 1985). Synaptosomes (2 mg/ml) were resuspended in incubation medium with BSA. At 5 min, 1.33 miM CaCI, and 5 pM fura 2-acetoxymethyl ester were added; at 35 rnin the synaptosomes were pelleted and resuspended into fresh medium without albumin. CaCI, was added at 1.33 mM, and the fluorescence was monitored at 340 and 510 nm. Data were collected at 2-s intervals. The plasma membrane potential was estimated with 3,3‘diethylthiadiearbocyanine iodide [DiSC,(5)] (Sims et al., 1974). Synaptosomes in incubation medium with BSA were preincubated at 37°C for 1 h, pelleted, and resuspended ( 1

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mg/ml) in incubation medium without BSA but containing 5 p M 3,3’-diethylthiadiearbocyanineiodide and 1.33 mM CaCI,. After 5 rnin to allow for equilibration, the fluorescence was determined at 643 and 680 nm. Data were eollected at 1 -s intervals.

RESULI’S AND DISCUSSION In the absence of arachidonic acid, high concentrations of the phorbol ester 4p-PDBu (0.5 p M ) enhanced the 4-APevoked release of glutamate (Fig. IA). Arachidonic acid alone inhibited the 4-AP-evoked glutamate exocytosis. However, in the presence of arachidonie acid, low coneentrations of 4b-PDBu ( 1-50 nM) were effective in increasing glutamate exocytosis (Fig. 1A). Similar results were obtained when the diacylglycerol diolein, instead of ~P-PDBu, was used (data not shown). These results show that arachidonic acid increases the sensitivity of glutamate exoeytosis to the potentiation by phorbol esters and that the optimal potentiation of glutamate release requires both phorbol esters and arachidonic acid. The synergistic activation of PKC by unsaturated fatty acids and diaeylglycerol or phorbol esters (Verkest et al., 1988; Lester et al., 1991; Shinomura et al., 1991) has been reported. To determine whether the increase of glutamate release observed in the presence of both phorbol esters and arachidonic acid was mediated by the activation of PKC, we prcineubated the synaptosomes with the protein kinase inhibitor staurosporine. It can be seen in Fig. 1 B that the increase in glutamate release by arachidonie acid and 4pPDBu was prevented in staurosporine-treated synaptosomes. The preincubation of synaptosomes with 1 pMTPA for 30 min, to down-regulate the PKC activity (Diaz-Guerra et al., 1988; Oda et al., I99 1 ), also abolished the potentiating effect by arachidonic acid and 48-PDBu (data not shown). Because staurosporine also inhibits other protcin kinases, apart from PKC (Linden and Routtenberg, 1989; Yanagihara et al., 1991), an alternative strategy in establishing

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T FIG. 1. Low concentrations of 4P-PDBu enhance glutamate exocytosis in the presence of arachidonic acid (AA) by a PKCdependent mechanism. AA (2 pM) or methyl arachidonate (MAA; 2 pbf) was added 1 min beforedepolarization of synaptosomes with 1 mM 4-AP. The phorbol ester 4P-PDBu was added 30 s before depolarization. In experiments with staurosporine (St), synaptosomes were preincubated with the PKC inhibitor at 100 nM for 30 min. The Ca2’-dependent release was calculated as described in Materials and Methods. Results are mean f SEM (bars) values of three independent experiments. The values under the line were compared for significance of differences by the Student’s t test: ‘p < 0.05; “p < 0.01; NS. not significant.

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I. HERRERO ET AL.

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with high concentrations of phorbol ester in glutamate release (Bame et al., 1991) is consistent with the synergistic activation of PKC by both lipids. A synergistic action by diacylglycerol or phorbol esters and &unsaturated fatty acids has been reported for the activation of the a,/3, and y subspecies ofPKC (Shinomura et al., 1991), all ofwhich are present in cerebrocortical synaptosomes (Shearman et al., I991 b).

The results shown in this article indicate that pathways for the generation of diacylglycerol and arachidonic acid in a signal-dependent manner may play an important role in the modulation of glutamate releasc.

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FIG. 2. Effects of phorbol esters and arachidonic acid (AA) on the increase in [Ca2+Ic (A-D) and in plasma membrane depolarization (E-H) evoked by 4-AP. 4P-PDBu and AA were added 60 and 30 s. respectively. before ciepolarization of synapiosomes with i mivi 4-AP. Results are t h e means of three independent expenments. The SEMs (bars) are shown at 30-sintervals.

whether the potentiation of glutamate release by arachidonic acid and phorbol esters was mediated by the activation of PKC is to use methyl arachidonate, which docs not activate PKC (Murakami and Routtenberg, 1985), instead of arachidonic acid. It is also shown in Fig. 1B that methyl arachidonate alone was as effective as arachidonic acid in inhibiting glutamate exocytosis; however, the methyl ester in combination with 4P-PDBu was not effcctivc in potentiating glutamate exocytosis. These results indicate that the potentiation of glutamate exocytosis observed in the presence of arachidonic acid and phorbol esters is mediated by the activation of PKC. The increase in [Ca2+],and the plasma membrane depolarization evoked by 4-AP were estimated in parallel to investigate the mechanism by which the addition of both arachidonic acid and phorbol esters potentiates glutamate exocytosis. The plasma membrane depolanzation and the fura 2 response evoked by 4-AP in control synaptosomes (Fig. 2A and E, respectively) were reduced in the presence of arachidonic acid (Fig. 2B and F, respectively). Consistent with release experiments, only a small change in these parameters was observed with the addition of 0.5 phi’ 4PPDBu (Fig. 2C and G , respectively). However, in the presence of both arachidonic acid and ~ P - P D B ua, further increase in the 4-AP-evoked depolarization and fura 2 response was observed (Fig. 2D and H, respectively). The enhanced time-averaged depolarization of the synaptosoma1 population by phorbol esters and arachidonic acid is consistent with the PKC-mediated inhibition of the K+ channels involved in the control of the duration of the action potentials initiated by 4-AP (Bame et al., 1991). The increase in depolarization in the presence of both arachidonic acid and phorbol esters would enhance Ca2+entry through the noninactivating Ca2+channels (McMahon and Nicholls, 1991 ), thereby increasing glutamate release. The fact that low concentrations of phorbol esters and arachidonic acid mimic the effects of the activation of PKC

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Acknowledgment: This work was supported by grants from DGICYT (PM89/0045) and from the Universidad Complutense de Madrid (Spain). I. Herrero was also supported by a fellowship from the Universidad Complutense. We also thank Erik Lundin for his help in the preparation of this manuscript.

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