PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Prostaglandins Leukornenes and Essential Fatty Acid, 0 Longman Group UK Ltd 1992
C1992) 47. ?lS-21X
Inhibition by Protein Kinase C Activation of Melittin-induced Acid Release in PC12 Pheochromocytoma Cells
Arachidonic
0. Kozawa, M. Miwa* and H. Tokuda* Department of Biochemistry. Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03. Japan and *First Department of Internal Medicine, Nagoya University School of Medicine. Nugoya 466, Japan (Reprint requests to OK) ABSTRACT. In rat PC12 pheochromocytoma cells, melittin, a phospholipase A2 activator, stimulated the release of arachidonic acid in a dose-dependent manner in the range between 0.1 and 1 FM. 12-U-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, inhibited the melittin-induced release of arachidonic acid dose-dependently in the range between 0.1 nM and 0.1 PM, whereas 4a-phorbol 12, 13-didecanoate, which is inactive for protein kinase C, was ineffective in this capacity. Staurosporine, a protein kinase C inhibitor, recovered the inhibitory effect of TPA on the melittin-induced release of arachidonic acid. These results suggest that the activation of protein kinase C inhibits phospholipase A, activity in PC12 pheochromocytoma cells.
that protein kinase C activation suppresses the AA release have been obtained in rabbit neutrophils and rabbit platelets (5, 7). Accumulating evidence indicates that AA and its metabolites such as 12-hydroxyeicosatetraenoic acid (12-HETE) and PGE? might act as messengers in nerve cells, and AA acts as a retrograde messenger in longterm potentiation in the vertebrate hippocampus (8-11). It has been reported that several neurotransmitters stimulate the release of AA in nervous tissue (9-11). In Aplysia neurons, it has been shown that PLA,-stimulating activity is enhanced by the activation of protein kinase C (12). However, the effect of protein kinase C on PLA? activity has not yet been elucidated in mammalian neural cells. In this study, we examined the effect of protein kinase C activation on melittin-, a PLAz activator (13, 14), induced release of AA in rat PC12 pheochromocytoma cells. Herein, we show that the activation of protein kinase C inhibits the melittin-induced release of AA in rat PC12 pheochromocytoma cells.
INTRODUCTION Upon stimulation, esterified arachidonic acid (AA) can be released through the activation of cellular phospholipases, and then metabolized to a variety of bioactive products such as prostaglandins (PGs). It is now considered that AA release is a rate-limiting step of the AA cascade. Two major pathways of AA release are generally accepted (1). One is the activation of phospholipase A, (PLA,) which causes liberation of AA directly from esterified stores of phospholipids, and the other is the sequential phosphoinositide hydrolysis by phospholipase C and glycerol lipases. Although the precise regulatory mechanism of AA release remains unclear, evidence is accumulating that activation of protein kinase C, which is considered to be activated physiologically by diacylglycerol resulting from phosphoinositide hydrolysis (2). can modulate AA release in several kinds of cells. It has been reported that the activation of protein kinase C enhances AA release stimulated by exogenous stimuli in Madin-Darby canine kidney cells, human platelets and rabbit platelets (3-5). In a previous report (6), we have also demonstrated that the activation of protein kinase C amplifies PGF,,-induced AA release through the potentiation of phospholipase AZ activity in mouse osteoblast-like cells. Opposite results showing
MATERIALS
AND METHODS
Materials [5, 6, 8, 9, 11, 12, 14, 15-3H]Arachidonic acid (212.1 Ci/ mmol) and myo-[2-“Hlinositol (8 1.5 Cifmmol) were purchased from Amersham International. Melittin (sequencing grade), 12-O-tetradecanoylphorbol13-acetate
Date received 23 March 1992 Date accepted 14 May 1992 215
216
Prostaglandins Leukotrienes and Essential Fatty Acids
(TPA). 4a-phorbol 12, 13-didecanoate (4ct-PDD), staurosporine and poly-L-lysine were purchased from Sigma Chemical. Other materials and chemicals were obtained from commercial sources. Cell culture PC12 pheochromocytoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% horse serum and 5% fetal calf serum at 37 “C in a humidified atmosphere of 5% CO,/95% air. The cells (4 x IO’) were seeded into poly-t.-lysinecoated 35mm diameter dishes in 2 ml of DMEM containing 10% horse serum and 5% fetal calf serum and fed every 2 days. The cells were used for experiments after 6 days. Assay for AA release The cells were labeled with [jH] AA (0.5 pCi/dish) for 24 h. Then, the medium was removed and the cells were washed with I ml of an assay buffer (5 mM 4-(2hydroxyethyl)-I-piperazineethanesulfonic acid. pH 7.4, 150 mM NaCl, 5 mM KCI, 5.5 mM glucose, 0.8 mM MgSO, and I mM CaCI,) four times. The cells were preincubated with I ml of the assay buffer containing 0. I% essentially fatty acid-free bovine serum albumin at 37 “C for 20 min. The cells were then stimulated by various does of melittin. After the indicated periods, the medium was collected and the radioactivity of the medium was determined. When indicated, the cells were pretreated with TPA. ~CX-PDD or staurosporine for 20 min prior to the stimulation by melittin.
1
10
Time
I
I
20
30
(min)
Fig. I Tnne-dependent cl’lrct of melittin on the rclcaw of AA III PCI? cells. The [jHj AA labeled cells were stimulated by 0.5 ,t~uM melittin (0) or vehicle (’ ) t’or the indicated periods. The radioactivity in the medium was determmed. Each value represents the mean + SD of triplicate determinations. Similar results were obtained with two additional and different cell preparation\.
I
1
I
1
Determination The radioactivity of 3H-samples was determined with an Aloka LSC 3 100 liquid scintillation spectrometer. Statistical
analysis
All data are presented as the mean f SD of triplicate determinations. The data were analyzed by Student’s t-test.
RESULTS Time-dependent PC12 cells
effect of melittin of AA release in
Melittin. which is known to be a PLA, activator (I 3, 14). stimulated AA release in PC I2 pheochromocytoma cells (Fig. 1). The release of AA increased until 30 min. whereas the control. which was stimulated by vehicle. had little changed until 30 min. Dose-dependent effect of melittin on AA release in PC12 cells: effect of TPA The melittin-induced
release of AA was dose-dependent
0 0.1
0.3
0.5
Melittin
_
1.0 (PM)
Fig. 2 Dowdepcndent cl’(L.ct ol~mcl~ttin on the relax ol AA u, PC I2 cells: TPA-cffcct. The [‘H 1AA-labeled cells were pretreatctl with 0. I PM TPA (0) or vehicle (’ ) lor 20 min. and then the ccII\ wcrc stimulated by various doses 01 mciittm for 30 min. I’hc radioactivity in the medium was determined. Each value rcprescnt\ the mc:m i- SD oI trlpliate dckrminations. Similar results wcrc obtained with two additionul and dit’t’erent cell preparation\.
Effect of Protein Kinase C on AA Release
0
10
- Log
9
8
(Phorbol ester),
7
Cont.
Cont.
TPA
M
Fig. 3 Effects of phorbol esters on the melittin-induced release of AA in PC12 cells. The [‘HI AA-labeled cells were pretreated with TPA (0) or 4a-PDD (c:) for 20 min. and then the cells were stimulated by 0.5 ,uM melittin for 30 min. The radioactivity in the medium was determined. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
in the range between 0.1 and 1 FM (Fig. 2). TPA (0.1 PM), a protein kinase C-activating phorbol ester (2), which by itself had little effect on AA release (data not shown), inhibited the melittin-induced release of AA (Fig. 2). Effects of phorbol esters on the melittin-induced release of AA in PC12 cells The effect of TPA was dose-dependent in the range between 0.1 nM and 0.1 PM (Fig. 3). TPA (0.1 PM) led the melittin-induced release of AA to about 40% reduction. Whereas 4a-PDD, which is inactive for protein kinase C (2), had little effect on the melittin-induced release of AA (Fig. 3). Effect of staurosporine on the inhibition by TPA in the melittin-induced release of AA in PC12 cells Staurosporine (0.3 FM), a protein kinase C inhibitor (15), which by itself had little effect on the melittin (0.5 PM)-induced release of AA. recovered the inhibitory effect of TPA (0.1 ,uM) on the melittin-induced AA release in PC 12 cells (Fig. 4).
Stauros;Jorine Fig. 4 Effect of staurosporine on the inhibition by TPA in the melittin-induced release of AA in PC12 cells. The [?H] AA-labeled cells were pretreated with 0.3 FM staurosporine or vehicle in the presence of 0.1 PM TPA for 20 min. and then the cells were stimulated by 0.5 ,uM melittin for 30 min. The radioactivity in the medium was determined. Each value represents the mean k SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *Significantly different from the value stimulated by melittin with TPA pretreatment (~~0.05).
inhibited the melittin-induced release of AA, but 4aPDD was ineffective in this capacity in PC 12 pheochromocytoma cells. It is generally accepted that TPA activates protein kinase C, whereas 4cx-PDD is known to be inactive for protein kinase C (2). Thus, it seems certain that the inhibitory effect of TPA on the melittininduced release of AA is mediated through the activation of protein kinase C in PC 12 cells. Moreover, we demonstrated that staurosporine. a protein kinase C inhibitor (15), recovered the inhibitory effect of TPA on the melittin-induced release of AA in these cells. Therefore, these results suggest that the activation of protein kinase C inhibited the melittin-induced release of AA in PC 12 pheochromocytoma cells. In addition, it is well known that melittin, which is purified form bee venom, stimulates the release of AA through the direct activation of PLA, (13, 14). Taken together, our present results strongly suggest that the activation of protein kinase C inhibits the release of arachidonic acid through the modulation of phospholipase A2 activity in rat PC 12 pheochromocytoma cells.
DISCUSSION
Acknowledgment
In the present study, we showed that TPA significantly
The authors assistance.
C
TPA
2 I7
are very
grateful
to Jun Kotoyori
for his technical
218
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Reference I. Smith W L. The eicosanoids and their biochemical mechanisms of action Biochem 1989; 259: 315-33-l 2. Nishizuka Y. Studies and perspectives of protein kinasc C. Science 1986: 233: 30.5-3 12 3. Slivka S R, Insel P A. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in MadinDarby canine kidney cells: Evidence that bradykinin mediates noninterdependent activition of phospholipaae A, and C. .I Biol Chem 1988: 263: 14640-14647 4. Halenda S P. Banga H S. Zavioco G B. Lau F L. Feinstein M B. Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca’ionophores: Evidence for the role of protein phosphorylation in the activation of phospholipase A, and independence form the Nat/H+ exchanger. Biochemistry 1989; 28: 7356-7363 5. Murayama T. Kajiyama Y. Nomura Y. Histaminestimulated and GTP -binding proteins-mediated phospholipase A2 activation in rabbit platelets. J Biol Chem 1990; 265: 4290-4295 6. Tokuda H, Oiso Y, Kozawa 0. Protein kinase C activation amplifies prostaglandin F,,-induced prostaglandin E? synthesis in osteoblast-like cells. J Cell Biochem 1992; 48: 262-268 7. Tao W, Molski T F P. Sha’afi R. Arachidonic acid release in rabbit neutrophils. Biochem J 1989: 257: 633-637
8. ShimiLu T. Wolfe L S. Arachidonic acid cascade and +nal transduction. .I Neurochem 1990: S5: I-15 Y. Piomelli D. Shapiro E. Feinmark S J, Schusrtf J H. Metabolites of arachidonic acid in the nervous by\tem of Aplysia: Possible mediators of synaptic modulation. J Neurosci 1987: 367.5-3686 10. Dumuis A. Stebben M. Haynea L. Pin J-P. Bochacrl J. NMDA trceptw activate the arvchidonic acid cascade \yhtem in \triatal neuron\. Nature 1988: 336: 68-70 I I. Gammon C M. .41len A C. Morel1 P. Bradykinin stimulate\ phosphoinowide hydroly& and mobilization of arachidonic acid and dorsal root ganglion neuron,. J Neurochem IYXY:53: 9.5~101 17. Culignano A. Piomrlli D. Sacktor T C, Schwartz J H. A phospholipase A,-stimulating protein regulated by protell hinase C in Aplyhia newon
C1992) 47. ?lS-21X
Inhibition by Protein Kinase C Activation of Melittin-induced Acid Release in PC12 Pheochromocytoma Cells
Arachidonic
0. Kozawa, M. Miwa* and H. Tokuda* Department of Biochemistry. Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03. Japan and *First Department of Internal Medicine, Nagoya University School of Medicine. Nugoya 466, Japan (Reprint requests to OK) ABSTRACT. In rat PC12 pheochromocytoma cells, melittin, a phospholipase A2 activator, stimulated the release of arachidonic acid in a dose-dependent manner in the range between 0.1 and 1 FM. 12-U-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, inhibited the melittin-induced release of arachidonic acid dose-dependently in the range between 0.1 nM and 0.1 PM, whereas 4a-phorbol 12, 13-didecanoate, which is inactive for protein kinase C, was ineffective in this capacity. Staurosporine, a protein kinase C inhibitor, recovered the inhibitory effect of TPA on the melittin-induced release of arachidonic acid. These results suggest that the activation of protein kinase C inhibits phospholipase A, activity in PC12 pheochromocytoma cells.
that protein kinase C activation suppresses the AA release have been obtained in rabbit neutrophils and rabbit platelets (5, 7). Accumulating evidence indicates that AA and its metabolites such as 12-hydroxyeicosatetraenoic acid (12-HETE) and PGE? might act as messengers in nerve cells, and AA acts as a retrograde messenger in longterm potentiation in the vertebrate hippocampus (8-11). It has been reported that several neurotransmitters stimulate the release of AA in nervous tissue (9-11). In Aplysia neurons, it has been shown that PLA,-stimulating activity is enhanced by the activation of protein kinase C (12). However, the effect of protein kinase C on PLA? activity has not yet been elucidated in mammalian neural cells. In this study, we examined the effect of protein kinase C activation on melittin-, a PLAz activator (13, 14), induced release of AA in rat PC12 pheochromocytoma cells. Herein, we show that the activation of protein kinase C inhibits the melittin-induced release of AA in rat PC12 pheochromocytoma cells.
INTRODUCTION Upon stimulation, esterified arachidonic acid (AA) can be released through the activation of cellular phospholipases, and then metabolized to a variety of bioactive products such as prostaglandins (PGs). It is now considered that AA release is a rate-limiting step of the AA cascade. Two major pathways of AA release are generally accepted (1). One is the activation of phospholipase A, (PLA,) which causes liberation of AA directly from esterified stores of phospholipids, and the other is the sequential phosphoinositide hydrolysis by phospholipase C and glycerol lipases. Although the precise regulatory mechanism of AA release remains unclear, evidence is accumulating that activation of protein kinase C, which is considered to be activated physiologically by diacylglycerol resulting from phosphoinositide hydrolysis (2). can modulate AA release in several kinds of cells. It has been reported that the activation of protein kinase C enhances AA release stimulated by exogenous stimuli in Madin-Darby canine kidney cells, human platelets and rabbit platelets (3-5). In a previous report (6), we have also demonstrated that the activation of protein kinase C amplifies PGF,,-induced AA release through the potentiation of phospholipase AZ activity in mouse osteoblast-like cells. Opposite results showing
MATERIALS
AND METHODS
Materials [5, 6, 8, 9, 11, 12, 14, 15-3H]Arachidonic acid (212.1 Ci/ mmol) and myo-[2-“Hlinositol (8 1.5 Cifmmol) were purchased from Amersham International. Melittin (sequencing grade), 12-O-tetradecanoylphorbol13-acetate
Date received 23 March 1992 Date accepted 14 May 1992 215
216
Prostaglandins Leukotrienes and Essential Fatty Acids
(TPA). 4a-phorbol 12, 13-didecanoate (4ct-PDD), staurosporine and poly-L-lysine were purchased from Sigma Chemical. Other materials and chemicals were obtained from commercial sources. Cell culture PC12 pheochromocytoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% horse serum and 5% fetal calf serum at 37 “C in a humidified atmosphere of 5% CO,/95% air. The cells (4 x IO’) were seeded into poly-t.-lysinecoated 35mm diameter dishes in 2 ml of DMEM containing 10% horse serum and 5% fetal calf serum and fed every 2 days. The cells were used for experiments after 6 days. Assay for AA release The cells were labeled with [jH] AA (0.5 pCi/dish) for 24 h. Then, the medium was removed and the cells were washed with I ml of an assay buffer (5 mM 4-(2hydroxyethyl)-I-piperazineethanesulfonic acid. pH 7.4, 150 mM NaCl, 5 mM KCI, 5.5 mM glucose, 0.8 mM MgSO, and I mM CaCI,) four times. The cells were preincubated with I ml of the assay buffer containing 0. I% essentially fatty acid-free bovine serum albumin at 37 “C for 20 min. The cells were then stimulated by various does of melittin. After the indicated periods, the medium was collected and the radioactivity of the medium was determined. When indicated, the cells were pretreated with TPA. ~CX-PDD or staurosporine for 20 min prior to the stimulation by melittin.
1
10
Time
I
I
20
30
(min)
Fig. I Tnne-dependent cl’lrct of melittin on the rclcaw of AA III PCI? cells. The [jHj AA labeled cells were stimulated by 0.5 ,t~uM melittin (0) or vehicle (’ ) t’or the indicated periods. The radioactivity in the medium was determmed. Each value represents the mean + SD of triplicate determinations. Similar results were obtained with two additional and different cell preparation\.
I
1
I
1
Determination The radioactivity of 3H-samples was determined with an Aloka LSC 3 100 liquid scintillation spectrometer. Statistical
analysis
All data are presented as the mean f SD of triplicate determinations. The data were analyzed by Student’s t-test.
RESULTS Time-dependent PC12 cells
effect of melittin of AA release in
Melittin. which is known to be a PLA, activator (I 3, 14). stimulated AA release in PC I2 pheochromocytoma cells (Fig. 1). The release of AA increased until 30 min. whereas the control. which was stimulated by vehicle. had little changed until 30 min. Dose-dependent effect of melittin on AA release in PC12 cells: effect of TPA The melittin-induced
release of AA was dose-dependent
0 0.1
0.3
0.5
Melittin
_
1.0 (PM)
Fig. 2 Dowdepcndent cl’(L.ct ol~mcl~ttin on the relax ol AA u, PC I2 cells: TPA-cffcct. The [‘H 1AA-labeled cells were pretreatctl with 0. I PM TPA (0) or vehicle (’ ) lor 20 min. and then the ccII\ wcrc stimulated by various doses 01 mciittm for 30 min. I’hc radioactivity in the medium was determined. Each value rcprescnt\ the mc:m i- SD oI trlpliate dckrminations. Similar results wcrc obtained with two additionul and dit’t’erent cell preparation\.
Effect of Protein Kinase C on AA Release
0
10
- Log
9
8
(Phorbol ester),
7
Cont.
Cont.
TPA
M
Fig. 3 Effects of phorbol esters on the melittin-induced release of AA in PC12 cells. The [‘HI AA-labeled cells were pretreated with TPA (0) or 4a-PDD (c:) for 20 min. and then the cells were stimulated by 0.5 ,uM melittin for 30 min. The radioactivity in the medium was determined. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
in the range between 0.1 and 1 FM (Fig. 2). TPA (0.1 PM), a protein kinase C-activating phorbol ester (2), which by itself had little effect on AA release (data not shown), inhibited the melittin-induced release of AA (Fig. 2). Effects of phorbol esters on the melittin-induced release of AA in PC12 cells The effect of TPA was dose-dependent in the range between 0.1 nM and 0.1 PM (Fig. 3). TPA (0.1 PM) led the melittin-induced release of AA to about 40% reduction. Whereas 4a-PDD, which is inactive for protein kinase C (2), had little effect on the melittin-induced release of AA (Fig. 3). Effect of staurosporine on the inhibition by TPA in the melittin-induced release of AA in PC12 cells Staurosporine (0.3 FM), a protein kinase C inhibitor (15), which by itself had little effect on the melittin (0.5 PM)-induced release of AA. recovered the inhibitory effect of TPA (0.1 ,uM) on the melittin-induced AA release in PC 12 cells (Fig. 4).
Stauros;Jorine Fig. 4 Effect of staurosporine on the inhibition by TPA in the melittin-induced release of AA in PC12 cells. The [?H] AA-labeled cells were pretreated with 0.3 FM staurosporine or vehicle in the presence of 0.1 PM TPA for 20 min. and then the cells were stimulated by 0.5 ,uM melittin for 30 min. The radioactivity in the medium was determined. Each value represents the mean k SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *Significantly different from the value stimulated by melittin with TPA pretreatment (~~0.05).
inhibited the melittin-induced release of AA, but 4aPDD was ineffective in this capacity in PC 12 pheochromocytoma cells. It is generally accepted that TPA activates protein kinase C, whereas 4cx-PDD is known to be inactive for protein kinase C (2). Thus, it seems certain that the inhibitory effect of TPA on the melittininduced release of AA is mediated through the activation of protein kinase C in PC 12 cells. Moreover, we demonstrated that staurosporine. a protein kinase C inhibitor (15), recovered the inhibitory effect of TPA on the melittin-induced release of AA in these cells. Therefore, these results suggest that the activation of protein kinase C inhibited the melittin-induced release of AA in PC 12 pheochromocytoma cells. In addition, it is well known that melittin, which is purified form bee venom, stimulates the release of AA through the direct activation of PLA, (13, 14). Taken together, our present results strongly suggest that the activation of protein kinase C inhibits the release of arachidonic acid through the modulation of phospholipase A2 activity in rat PC 12 pheochromocytoma cells.
DISCUSSION
Acknowledgment
In the present study, we showed that TPA significantly
The authors assistance.
C
TPA
2 I7
are very
grateful
to Jun Kotoyori
for his technical
218
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Reference I. Smith W L. The eicosanoids and their biochemical mechanisms of action Biochem 1989; 259: 315-33-l 2. Nishizuka Y. Studies and perspectives of protein kinasc C. Science 1986: 233: 30.5-3 12 3. Slivka S R, Insel P A. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in MadinDarby canine kidney cells: Evidence that bradykinin mediates noninterdependent activition of phospholipaae A, and C. .I Biol Chem 1988: 263: 14640-14647 4. Halenda S P. Banga H S. Zavioco G B. Lau F L. Feinstein M B. Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca’ionophores: Evidence for the role of protein phosphorylation in the activation of phospholipase A, and independence form the Nat/H+ exchanger. Biochemistry 1989; 28: 7356-7363 5. Murayama T. Kajiyama Y. Nomura Y. Histaminestimulated and GTP -binding proteins-mediated phospholipase A2 activation in rabbit platelets. J Biol Chem 1990; 265: 4290-4295 6. Tokuda H, Oiso Y, Kozawa 0. Protein kinase C activation amplifies prostaglandin F,,-induced prostaglandin E? synthesis in osteoblast-like cells. J Cell Biochem 1992; 48: 262-268 7. Tao W, Molski T F P. Sha’afi R. Arachidonic acid release in rabbit neutrophils. Biochem J 1989: 257: 633-637
8. ShimiLu T. Wolfe L S. Arachidonic acid cascade and +nal transduction. .I Neurochem 1990: S5: I-15 Y. Piomelli D. Shapiro E. Feinmark S J, Schusrtf J H. Metabolites of arachidonic acid in the nervous by\tem of Aplysia: Possible mediators of synaptic modulation. J Neurosci 1987: 367.5-3686 10. Dumuis A. Stebben M. Haynea L. Pin J-P. Bochacrl J. NMDA trceptw activate the arvchidonic acid cascade \yhtem in \triatal neuron\. Nature 1988: 336: 68-70 I I. Gammon C M. .41len A C. Morel1 P. Bradykinin stimulate\ phosphoinowide hydroly& and mobilization of arachidonic acid and dorsal root ganglion neuron,. J Neurochem IYXY:53: 9.5~101 17. Culignano A. Piomrlli D. Sacktor T C, Schwartz J H. A phospholipase A,-stimulating protein regulated by protell hinase C in Aplyhia newon
