- Email: [email protected]
Protein kinase C modulates ischemia-induced amino acids release in the striatum of hypertensive rats
Brain Research 782 Ž1998. 290–296
Research report
Protein kinase C modulates ischemia-induced amino acids release in the striatum of hypertensive rats Hiroshi Nakane a
a,b,)
, Hiroshi Yao a,b , Setsuro Ibayashi a , Takanari Kitazono a , Hiroaki Ooboshi a , Hideyuki Uchimura b , Masatoshi Fujishima a
Second Department of Internal Medicine, Faculty of Medicine, Kyushu UniÕersity, Fukuoka, Japan b Center for Emotional and BehaÕioral Disorders, Hizen National Mental Hospital, Saga, Japan Accepted 21 October 1997
Abstract The role of protein kinase C ŽPKC. in mediating the ischemia-induced release of amino acids in the striatum was studied using an in vivo brain dialysis technique in the striatum of spontaneously hypertensive rats ŽSHRs.. Using HPLC combined with fluorescence detection methods, we investigated the concentrations of amino acids in the dialysates produced by 20 min of transient forebrain ischemia. We studied the effects of an inhibitor of PKC, 1-Ž5-isoquinolinesulfonyl.-2-methylpiperazine dihydrochloride ŽH7. and another isoquinoline analog ŽHA1004. with less inhibitory effect on the C kinase in ischemia-induced amino acids release. Bilateral carotid artery occlusion caused a marked reduction in the striatal blood flow by 91 " 6%. The extent of the cerebral blood flow ŽCBF. reduction were essentially the same among H7-, HA1004-, and the vehicle-treated groups. Forebrain ischemia produced a marked increase in glutamate Ž21-fold of the basal concentration., aspartate Ž19-fold. and taurine Ž16-fold.. Pretreatment with H7 markedly attenuated the ischemia-induced release of these three amino acids to 3, 3 and 4-fold of the basal values, respectively. Increase of g-aminobutyric acid ŽGABA. was also attenuated by H7 Žvehicle; 2.46 " 1.26 m M, H7; 0.62 " 0.75 mM.. HA1004 did not affect the release of glutamate, aspartate or GABA during ischemia. The ischemia-induced release of taurine was significantly inhibited by HA1004 but the effect was much smaller than that of H7. These results thus indicate that PKC plays a major role in the ischemia-induced release of amino acids in the striatum of SHR. q 1998 Elsevier Science B.V. Keywords: Cerebral ischemia; Protein kinase C; Glutamate; Aspartate; Taurine; GABA
1. Introduction A massive release of glutamate occurs during cerebral ischemia w3x, which produces an accumulation of intracellular Ca2q, and thereby causes neuronal cell death w5x. It was recently reported that staurosporine, a potent PKC inhibitor w21x and fasudil ŽHA1077., a novel protein kinase inhibitor w39x were effective in protecting hippocampal CA1 neurons against ischemic damage and thus significantly improved the neurological function in both rats and gerbils. Other PKC inhibitors, 1-Ž5- isoquinolinesulfonyl.-2-methylpiperazine dihydrochloride ŽH7. and calphostin C, were also reported to prevent glutamate toxicity in primary neuronal cultures w11x. The protective mechanism for the PKC inhibitor is not clear, but one
possibility is PKC may be involved in the ischemia-induced massive release of excitatory amino acids from presynaptic nerve terminals, glial cells, and postsynaptic elements. Although several reports have shown that activated PKC enhances the release of neurotransmitters under nonischemic conditions w2,10,17,28,29,33x, there are a few reports about the involvement of PKC on ischemia-induced neurotransmitter release w27,38x. To clarify the involvement of PKC in ischemic amino acids release, we evaluated the effects of two inhibitors of PKC, H7 and an isoquinoline sulfonamid, HA1004, on the amino acids release in transient forebrain ischemia in rats. 2. Materials and methods 2.1. Animals
)
Corresponding author. Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan. Telex: q81-92-632-2551; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 3 1 - 0
Male spontaneously hypertensive rats ŽSHRs., aged 7–9 months and weighing 350–450 g, were used in the present
H. Nakane et al.r Brain Research 782 (1998) 290–296
study. As hypertension is one of the most important risk factors of cerebral infarction, we have used SHRs to examine the pathophysiology of cerebral ischemia. The rats were maintained in the Kyushu University Animal Center and fed a standard chow diet and tap water ad libitum. 2.2. Brain ischemia The rats were anesthetized with amobarbital Ž100 mgrkg, i.p.., and allowed to breathe spontaneously. Forebrain ischemia was produced by bilateral carotid artery occlusion according to the original method w13x as described previously w31,36x. In our previous studies, bilateral carotid artery occlusion caused an extremely high mortality w15x, a marked increase in anaerobic glycolytic metabolites of the brain w13x, and diffuse-extensive cerebral infarcts in SHR w35x. Although the configuration of the circle of Willis is essentially identical in both normotensive rat and SHR w35x, SHR shows a marked fall of carotid back pressure to below the critical perfusion pressure of cerebral autoregulation w14x. Thus this hemodynamic change is thought to be a major causative factor of diffuse and extensive cerebral infarction following bilateral carotid artery occlusion. Their body temperature was maintained at 378C using a heat pad. The femoral arteries were cannulated with PE 50 tubing for the anaerobic sampling of blood and the continuous recording of the arterial blood pressure with an AP 601 G electromanometer ŽNihon Koden, Tokyo, Japan.. Arterial blood gases and pH were determined with an IL meter model 1304 ŽInstrumentation Laboratories, Lexington, KY, USA.. Both common carotid arteries were exposed through a ventral midline incision in
291
the neck, carefully separated from the vagosympathetic trunks, and loosely encircled with sutures for the later ligation. 2.3. In ÕiÕo brain dialysis and CBF measurement Extracellular amino acids Žglutamate, aspartate, GABA, taurine., and regional CBF in the striatum were simultaneously determined using an in vivo brain dialysis technique and a hydrogen clearance method, respectively, as described previously w45x. The rats were fixed in a head holder, and a small burr hole was made in the skull. A dialysis probe, measuring 500 m m in outer diameter Ž3-mm dialysis membrane; molecular cut off approximately 20,000 Da; Carnegie Medicine, Stockholm, Sweden. and a Teflon-coated platinum CBF electrode attached to a thermometer, 200 m m in diameter, were placed stereotaxically into the right striatum Ž0.5 mm anterior and 3.0 mm lateral to the bregma and 4.5 mm in depth from the brain surface. according to the atlas of Paxinos and Watson w37x. The distance between the microdialysis membrane and the hydrogen clearance electrode was about 1 mm. The striatum was perfused with Ringer solution through a dialysis probe at a rate of 3.0 m lrmin with a syringe pump Žmodel EP-60, Eicom, Kyoto, Japan. and the perfusates were collected every 10 min into a plastic tube. 2.4. Biochemical assay The concentrations of amino acids were determined using HPLC combined with fluorescence detection after precolum derivatization with 4 mM o-phthalaldehyde w26x. Each 24 m l of perfusate and 8 m l of o-phthalaldehyde
Table 1 Physiological parameters Ž1. Rest
Drug
pH Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
7.42 " 0.01 7.42 " 0.03 7.43 " 0.02
7.42 " 0.02 7.42 " 0.02 7.44 " 0.02
7.52 " 0.06 7.57 " 0.06 7.53 " 0.08
7.42 " 0.03 7.43 " 0.03 7.44 " 0.02
pa CO2 Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
38.2 " 1.0 37.7 " 2.3 38.6 " 0.8
40.5 " 1.8 38.6 " 2.6 37.5 " 3.1
31.2 " 5.2 25.6 " 5.0 27.0 " 10.2
39.6 " 3.0 38.4 " 1.5 34.4 " 5.9
pa O2 Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
87.3 " 2.8 89.5 " 4.6 90.5 " 2.9
85.6 " 4.6 89.0 " 3.3 93.5 " 3.1a
98.3 " 6.4 102.7 " 5.5 94.3 " 12.0
83.8 " 5.5 89.6 " 4.5 85.8 " 21.5
Ht (%) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
41 " 5 43 " 3 42 " 3
43 " 3 42 " 5 43 " 1
44 " 3 43 " 4 45 " 3
42 " 1 44 " 4 46 " 2
Values are mean " S.D. ) p - 0.05 vs. Vehicle.
Ischemia
Recirculation
H. Nakane et al.r Brain Research 782 (1998) 290–296
292 Table 2 Physiological parameters Ž2. Rest
Drug
Ischemia
Recirculation 10 min
60 min
MABP (mmHg) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
194 " 14 193 " 26 184 " 22
191 " 13 187 " 15 184 " 18
208 " 35 211 " 25 198 " 29
174 " 24 168 " 11 160 " 14
180 " 24 181 " 11 151 " 9
CBF (% of rest) Vehicle 100 H7 5 = 10y4 M HA1004 5 = 10y4 M
100 100 100
119 " 19 137 " 27 101 " 19
9.1 " 5.5 7.0 " 3.2 7.0 " 6.2
168 " 74 119 " 80 95 " 34
54 " 17 114 " 23 79 " 46
Brain temparature (8C) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
35.7 " 0.2 35.7 " 0.2 35.8 " 0.3
35.8 " 0.2 35.8 " 0.2 36.0 " 0.3
34.3 " 0.6 34.7 " 0.4 34.8 " 0.8
36.4 " 0.3 35.7 " 0.4 ) 36.2 " 0.5
35.8 " 0.5 35.8 " 0.3 35.6 " 0.4
Values are mean " S.D. ) p - 0.05 vs. Vehicle.
solution was reacted for 2 min, and then 30 m l of the mixed solution were injected into the chromatography using an autoinjector Žmodel 231-401, Gilson Medical Electronics, Villiers le Bel, France.. The chromatographic system consisted of an LC-6A pump ŽShimadzu, Kyoto, Japan. at a flow rate of 1.0 mlrmin, a reverse-phase column ŽEicompac MA-5ODS, 4.6 = 150 mm, Eicom, Kyoto, Japan. and a fluorescent detector ŽRF-535, Shimadzu, Tokyo, Japan.. The mobile phase was 0.1 M sodium phosphate ŽpH 6.0. containing 30% Žvol.rvol.. methanol.
2.5. Experimental protocol We divided the rats into three groups, consisting of the H7-treated group Ž n s 6., the HA1004-treated group Ž n s 4., and the vehicle-treated group Ž n s 7.. After determining two baseline CBF values and four determinations of amino acid concentrations during a 40 min of resting period, the administration of H7, 5 = 10y4 M or HA1004, 5 = 10y4 M, was started through the dialysis probe. H7 ŽSigma, St. Louis. and HA1004 ŽSigma, St. Louis. were
Fig. 1. Changes in the concentration of extracellular glutamate. H7 attenuated the glutamate release in the striatum during ischemia. HA1004 did not affect the ischemia-induced glutamate release. The values are the mean " S.D. ) p - 0.05 vs. vehicle.
H. Nakane et al.r Brain Research 782 (1998) 290–296
293
for perfusion into each group of rats. In the control rats, dialysis probes were perfused with vehicle. Twenty min after starting drug infusion, both carotid arteries were tightly ligated for 20 min and then reopened for 60 min to allow for recirculation. The cerebral blood flow was measured at 20 min after drug perfusion, 20 min of ischemia, and 10 and 60 min of recirculation. Arterial blood was sampled during the control period, at 20 min after drug infusion, 20 min of ischemia, and 60 min of recirculation. The position of the dialysis probe and CBF electrode was then macroscopically determined after the experiment. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986. 2.6. Statistics
Fig. 2. Changes in the concentration of extracellular aspartate. H7 attenuated the aspartate release in the striatum during ischemia. HA1004 did not affect the ischemia-induced aspartate release. The values are the mean" S.D. ) p- 0.05 vs. vehicle.
dissolved in Ringer solution Ž147 mM Naq, 2.3 mM Ca2q, 4 mM Kq, 155.5 mM Cly, pH 6.8.. 5 = 10y4 M solutions of H7 ŽpH 6.2. or HA1004 ŽpH 6.4. solution were made
In each group, the concentrations of the amino acids in the last fourth sample during the resting period were referred as the basal values. The statistical differences in physiological parameters, mean arterial blood pressure, and CBF among groups were analyzed by a one-way analysis of variance ŽANOVA. followed by Dunnett’s t-test. The differences in the concentrations of amino acids among the groups were analyzed by a two-way repeated ANOVA regarding such factors as the group and the time. To assess further differences among the groups, we used a one-way ANOVA followed by Scheffe’s multiple comparison. All the values were presented as the mean " S.D.
Fig. 3. Changes in the concentration of extracellular taurine. H7 and HA1004 attenuated the taurine release in the striatum during ischemia. However, the degree of reduction by HA1004 was smaller than that by H7. The values are the mean" S.D. ) ) p - 0.01 and ) p - 0.05 vs. vehicle.
294
H. Nakane et al.r Brain Research 782 (1998) 290–296
3. Results 3.1. Physiological parameters during cerebral ischemia Table 1 shows the physiological parameters in the three groups of rats. There were no significant differences in paCO 2 , pH, hematocrit among the three groups during the experiment. The arterial oxygen tension in the HA1004 group was slightly but significantly higher than in the vehicle group. However, this change was within the physiological range. The rats showed hyperventilation and consequent respiratory alkalosis during ischemia, as is usually seen in our model w16x. The changes in the mean arterial blood pressure, striatal CBF and brain temperature are given in Table 2. The mean arterial blood pressure slightly increased during ischemia in all groups but no significant differences were observed among the three groups throughout the experiment. The resting CBF values were not significantly different among the groups; 61.5 " 27.6 mlr100 grmin in the control group, 44.2 " 12.6 in the H7 group and 56.8 " 20.5 in the HA1004 group and were unchanged after 20 min of H7 and HA1004 perfusion. During ischemia, the CBF in each rat markedly decreased Ž p - 0.01. to less than 10% of the resting value. Although the control group showed postischemic hyperemia immediately after reopening the carotid arteries, followed by hypoperfusion, the H7 and HA1004 groups showed neither postischemic hyperemia nor hypoperfusion. The H7 treated group thus demonstrated a considerable recovery of CBF during recirculation and revealed a significantly higher CBF value Ž114% of resting value at 60 min, p - 0.05. than did the control group Ž54% of resting value.. 3.2. Effects of H7 and HA1004 on the extracellular concentration of amino acids The basal levels of glutamate, aspartate, taurine of each group were 0.74 " 0.63 m M, 0.23 " 0.15 m M, and 1.59 " 0.67 m M in the vehicle group, 1.03 " 1.15 m M, 0.22 " 0.16 m M and 1.52 " 0.49 m M in the H7-treated group, 0.74 " 0.20 m M, 0.28 " 0.11 m M and 1.56 " 0.49 m M in HA1004-treated group, respectively and there were no significant differences among the three groups. The basal level of GABA could not be detected in this study. H7 and HA1004 did not affect the extracellular concentrations of amino acids during the resting period. The concentrations of excitatory amino acids, glutamate and aspartate, increased and reached to a peak at 20 min of ischemia; 21-fold and 19-fold of the basal value in the vehicle group, respectively and then rapidly returned to the preischemic level at 20 min of recirculation. H7 significantly inhibited the increases in glutamate and aspartate; 3-fold Ž p - 0.05 vs. vehicle. and 3-fold Ž p - 0.05 vs. vehicle. of basal value, respectively ŽFigs. 1 and 2.. HA1004 did not show any significant inhibition of the release of glutamate and aspartate during ischemia. The ischemia-induced release of GABA, an inhibitory amino acid, was also attenuated by
H7 Ž p - 0.05 vs. vehicle. but not HA1004 Žvehicle; 2.46 " 1.26 m M, H7; 0.62 " 0.75 m M, HA1004; 1.58 " 1.06 m M.. H7 strongly inhibited the release of taurine; 16-fold in the vehicle group, 4-fold Ž p - 0.01 vs. vehicle. and the inhibition lasted throughout recirculation. HA1004 also showed an inhibitory effect on the ischemia-induced release of taurine; 7-fold Ž p - 0.05 vs. vehicle. but the effect was smaller than that of H7 ŽFig. 3.. 4. Discussion The present study suggests that the activation of PKC may be one of the major mechanisms by which ischemia causes a massive release of amino acids. A large amount of PKC appears to be present in the brain w25x, especially at the presynaptic nerve terminals w18x, and thus plays an important role in neuronal transmission w7,44x, neurotrophic activity w30x, and neuronal plasticity w34x. This is the first study which shows the effects of PKC on the release of amino acids under ischemic conditions by using in vivo brain dialysis methods. A major concern in the studies using PKC inhibitor is the specificity of PKC inhibition. Staurosporine, which has an antifungal activity, is one of the potent PKC inhibitors ŽIC50 is 2.7 nM. w42x. However, it also inhibits cyclic AMP ŽcAMP.- and cyclic GMP ŽcGMP.-dependent kinases. H7 is known to inhibit PKC by interfering with the interaction of ATP and protein substrates within the catalytic domain. The Ki values of H7 against PKC, cAMP- and cGMP-dependent protein kinase are 6.0, 3.0 and 5.8 m M, respectively w23x. In the concentration range used in the present study, this agent also inhibits cAMP and cGMP dependent protein kinases. We thus used another derivative of isoquinoline sulfonamide, HA1004, which has a much less inhibitory effect on PKC Žthe Ki values against PKC, cAMP- and cGMPdependent protein kinase are 40, 2.3 and 1.3 m M, respectively. w23x. In this study, HA1004 also inhibited the release of amino acids during ischemia, but the effect was much smaller than that of H7. Taken together, the effect of H7 in the present study may be specific for PKC. 4.1. Role of PKC in amino acid release Several reports have shown that activated PKC enhances the release of neurotransmitters under nonischemic conditions w2,10,17,28,29,33x. At least four different mechanisms, Ž1. calcium influx from voltage sensitive calcium channels ŽVSCCs., in particular, N- and P-types of VSCCs w8,24x, Ž2. modulation by metabotropic glutamate ŽmGlu. receptors w19,22x, Ž3. phosphorylation of synapsin I by Ca2q-calmodulin-dependent protein ŽCaM. kinase II w20x, Ž4. depolymerization and repolymerization of actin assembly w4x, are considered to play a role in neurotransmitter release under nonischemic condition. Dekker et al. w6x showed that phosphorylation of B-50, which is an important presynaptic PKC substrate, was closely correlated with neurotransmitter release in rat hippocampal slices. Some
H. Nakane et al.r Brain Research 782 (1998) 290–296
reports also revealed that PKC regulated the ion channels, e.g., VSCCs w12,40,43x and calcium-activated potassium channels w1x. Thus, PKC may be involved in several steps of neurotransmitter release under nonischemic conditions. In the present study, H7, an inhibitor of PKC, markedly attenuated the ischemia-induced increase of the extracellular glutamate, aspartate, GABA, and taurine. Lu et al. w27x showed that glutamate accumulation under ischemic condition was inhibited by H7 in vitro. Phillis and O’Regan w38x have recently shown that PKC contributes to ischemiaevoked glutamate and aspartate release in vivo. The findings of their studies are also similar to ours. Our study is the first report on the involvement of PKC in ischemia-induced amino acids release by using an in vivo brain dialysis model. We showed that ischemia-induced release of GABA and taurine were also inhibited by H7. Especially, taurine was more strongly inhibited by H7 and HA1004 than the other amino acids. The reason for this difference, however, is still unknown, because the release mechanism of taurine has not been clarified. We previously reported that the ischemia-induced release of taurine was reduced in aged rats w36x. The release mechanism of excitatory and inhibitory amino acids therefore appears to be different. Under ischemic conditions, two different mechanisms are considered about increase of extracellular glutamate concentrations. In the early phase of ischemia, glutamate could be released from the neurotransmitter pool by a calcium dependent process w9x. In the late phase, the extracellular concentration of glutamate could be elevated by reversal of the glutamate transporter because of depletion of ATP supplies w32,41x. The role of PKC in the ischemia-induced neurotransmitter release is not clear. However, the present study and previous studies w27,38x support the conclusion that PKC also plays a role under ischemic conditions. Phillis and O’regan w38x showed that destabilization and deterioration of the plasma membrane would be important in ischemia-induced release of excitatory amino acids. In this process, hydrolysis of membrane phospholipids, e.g., by phospholipase A 2 and C, occurred and PKC was involved in the cascade of events. In the present study, the release of both excitatory and inhibitory amino acids were attenuated by the PKC inhibitor. This result suggests that not only excitatory amino acids but also inhibitory amino acids could diffuse into the extracellular space from neurons or glia cells by the deterioration of the membrane during ischemia. We postulate that PKC may be involved in neurotransmitter release under ischemic conditions by modulating the stabilization of the membrane.
5. Conclusion In summary, H7 strongly inhibited the ischemia-induced release of amino acids. These results thus strongly
295
suggest the conclusion that PKC may play an important role in neurotransmitter release during ischemia. Acknowledgements This study was partly supported by the Social Insurance Agency Contract Fund commissioned by the Japan Health Sciences Foundation. References w1x J. Barban, S. Snyder, B. Alger, PKC regulates ionic conductance in hippocampal pyramidal neurons: electrophysiological effects of phorbol esters, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 2538–2542. w2x A.P. Barrie, D.G. Nicholls, J. Sanchez-Prieto, T.S. Sihra, An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes, J. Neurochem. 57 Ž1991. 1398–1404. w3x H. Benveniste, J. Drejer, A. Shousboe, N.H. Diemer, Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem. 43 Ž1984. 1369–1374. w4x B.W. Bernstein, J.R. Bamburg, Cycling of actin assembly in synaptosomes and neurotransmitter release, Neuron 3 Ž1989. 257– 265. w5x D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system ŽReview., Neuron 1 Ž1988. 623–634. w6x L. Dekker, P.D. Graan, D. Versteeg, A. Oestreicher, W. Gispen, Phosphorylation of B-50 ŽGAP43. is correlated with neurotransmitter release in rat hippocampal slices, J. Neurochem. 52 Ž1989. 24–30. w7x S.A. DeRiemer, J.A. Strong, K.A. Albert, P. Greengard, L.K. Kaczmarek, Enhancement of calcium current in Aplysia neurons by phorbol ester and protein kinase C, Nature 313 Ž1985. 313–316. w8x D.J. Dooley, A. Lupp, G. Hertting, Inhibition of central neurotransmitter release by v-conotoxin GIVA, a peptide modulator of the N-type voltage-sensitive calcium channel, Naunyn–Schmideberg’s Arch. Pharmacol. 336 Ž1987. 467–470. w9x J. Drejer, H. Benveniste, N.H. Diemer, A. Schousboe, Cellular origin of ischemia-induced glutamate release from brain tissue in vivo and in vitro, J. Neurochem. 45 Ž1985. 145–151. w10x M.L. Eboli, M.T. Ciotti, D. Mercanti, P. Calissano, Differential involvement of protein kinase C in transmitter release and response to excitatory amino acids in cultured cerebellar neurons, Neurochem. Res. 18 Ž1993. 133–138. w11x V. Felipo, M.-D. Minana, S. Grisolıa, ˜ ´ Inhibitiors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures, Brain Res. 604 Ž1993. 192–196. w12x F. Fournier, P. Charnet, E. Bourinet, C. Vilbert, F. Matifat, G. Charpentier, P. Navarre, G. Brule, D. Marlot, Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebral mRNA, FEBS 317 Ž1993. 118–124. w13x M. Fujishima, T. Sugi, M. Morotomi, T. Omae, Effects of bilateral carotid artery ligation on brain lactate and pyruvate concentrations in normotensive and spontaneously hypertensive rats, Stroke 6 Ž1975. 62–66. w14x M. Fujishima, T. Omae, Carotid back pressure following bilateral carotid occlusion in normotensive and spontaneously hypertensive rats, Experientia 32 Ž1976. 1021–1022. w15x M. Fujishima, J. Ogata, T. Sugi, T. Omae, Mortality and cerebral metabolism after bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats, J. Neurol. Neurosurg. Psychiat. 39 Ž1976. 212–217. w16x M. Fujishima, Y. Nakatomi, K. Tamaki, J. Ogata, T. Omae, Cerebral
296
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
H. Nakane et al.r Brain Research 782 (1998) 290–296 ischemia induced by bilateral carotid occlusion in spontaneously hypertensive rats, J. Neurol. Sci. 33 Ž1977. 1–11. V.C. Gandhi, D.J. Jones, Protein kinase C modulates the release of w 3 Hx5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels, Neuropharmacology 31 Ž1992. 1101–1109. P. Girard, G. Massei, J. Wood, J. Kuo, Polyclonal antibodies to phospholipidrCa2q-independent protein kinase and immunocytochemical localization of the enzyme in rat brain, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 3030–3034. S.R. Glaum, R.J. Miller, The metabotropic glutamate receptors: acute regulation of synaptic transmission by metabotropic glutamate receptors, in: P.J. Conn, J. Patel ŽEds.., Humana Press, Clifton, NJ, 1995, pp. 142–172. P. Greengard, F. Valtorta, A.J. Czernik, F. Benfenati, Synaptic vesicle phosphoproteins and regulation of synaptic function, Science 259 Ž1993. 780–784. H. Hara, H. Onodera, M. Yoshidomi, Y. Matsuda, K. Kogure, Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab. 10 Ž1990. 646–653. I. Herrero, M.T. Miras-Portugal, J. Sanchez-Prieto, Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation, Nature 360 Ž1992. 163–166. H. Hidaka, M. Inagaki, S. Kawamoto, Y. Sasaki, Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C, Biochemistry 23 Ž1984. 5036– 5041. M. Kimura, Y. Yamanishi, T. Hanada, T. Kagaya, M. Kuwada, T. Watanabe, K. Katayama, Y. Nishizawa, Involvement of P-type calcium channels in high potassium-eliceted release of neurotransmitters from rat brain slices, Neuroscience 66 Ž1995. 609–615. J.F. Kuo, R.G.G. Andersson, B.C. Wise, L. Mackerlova, I. Salomonsson, N.L. Brackett, N. Katoh, M. Shoji, R.W. Wernn, Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine, Proc. Natl. Acad. Sci. U.S.A. 77 Ž1980. 7039–7043. P. Lindroth, K. Mopper, High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolum fluorescence derivatization with o-phthalaldehyde, Anal. Chem. 51 Ž1979. 1667–1674. Y.M. Lu, B.F. Lu, F.O. Zhao, Y.L. Yan, X.P. Ho, Accumulation of glutamate is regulated by calcium and protein kinase C in rat hippocampal slices exposed to ischemic states, Hippocampus 3 Ž1993. 221–227. R.C. Malenka, D.V. Madison, R.A. Nicoll, Potentiation of synaptic transmission in the hippocampus by phorbol esters, Nature 321 Ž1986. 175–177. R.C. Malenka, G.S. Ayoub, R.A. Nicoll, Phorbol esters enhance transmitter release in rat hippocampal slices, Brain Res. 403 Ž1987. 198–203.
w30x H.P.M. Montz, G.E. Davis, S.D. Skaper, M. Manthorpe, S. Varon, Tumor-promoting phorbol diester mimics two distinct neurotrophic factors, Dev. Brain Res. 23 Ž1985. 150–154. w31x H. Nakane, H. Ooboshi, S. Ibayashi, H. Yao, S. Sadoshima, M. Fujishima, Isradipine, a calcium channel blocker, attenuates the ischemia-induced release of dopamine but not glutamate in rats, Neurosci. Lett. 188 Ž1995. 151–154. w32x D. Nicholls, D. Attwell, The release and uptake of excitatory amino acids, Trends Pharmac. Sci. 11 Ž1990. 462–468. w33x R.A. Nichols, J.W. Haycock, J.K.T. Wang, P. Greengard, Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes, J. Neurochem. 48 Ž1987. 615–621. w34x Y. Nishizuka, Studies and perspectives of protein kinase C, Science 233 Ž1986. 305–312. w35x J. Ogata, M. Fujishima, Y. Morotomi, T. Omae, Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study, Stroke 7 Ž1976. 54–60. w36x H. Ooboshi, S. Sadoshima, H. Yao, S. Ibayashi, T. Matsumoto, H. Uchimura, M. Fujishima, Ischemia-induced release of amino acids in the hippocampus of aged hypertensive rats, J. Cereb. Blood Flow Metab. 15 Ž1995. 227–234. w37x G. Paxinos, C. Watson, The rat brain in stereotaxic coodinates, 2nd edn., Academic Press, Tokyo, 1986. w38x J.W. Phillis, M.H. O’Regan, Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex, Brain Res. 730 Ž1996. 150–164. w39x S. Satoh, I. Ikegaki, Y. Suzuki, T. Asano, M. Shibuya, H. Hidaka, Neuroprotective properties of a protein kinase inhibitor against ischaemia-induced neuronal damage in rats and gerbils, Br. J. Pharmacol. 118 Ž1996. 1592–1596. w40x K.J. Swartz, A. Merritt, B.P. Bean, D.M. Lovinger, Protein kinase C modulates glutamate receptor inhibition of Ca2q channels and synaptic transmission, Nature 361 Ž1993. 165–168. w41x M. Szatkowski, D. Attwell, Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms, Trends Neurol. Sci. 17 Ž1994. 359–365. w42x T. Tamaoki, H. Nomoto, I. Takahashi, Y. Kato, M. Morimoto, F. Tomita, Staurosporine, a potent inhibitor of phospholipidrCa2q dependent protein kinase, Biochem. Biophys. Res. Commun. 135 Ž1986. 397–402. w43x D.M. Terrian, D.K. Ways, Persistent enhancement of sustained calcium-dependent glutamate release by phorbol esters: role of calmodulin-independent serinerthreonine phosphorylation and actin disassembly, J. Neurochem. 64 Ž1995. 181–190. w44x L.A. Witters, C.A. Vater, G.E. Lienhard, Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C, Nature 315 Ž1985. 777–778. w45x H. Yao, H. Ooboshi, S. Ibayashi, H. Uchimura, M. Fujishima, Cerebral blood flow and ischemia-induced neurotransmitter release in the striatum of aged spontaneously hypertensive rats, Stroke 24 Ž1993. 577–580.
Research report
Protein kinase C modulates ischemia-induced amino acids release in the striatum of hypertensive rats Hiroshi Nakane a
a,b,)
, Hiroshi Yao a,b , Setsuro Ibayashi a , Takanari Kitazono a , Hiroaki Ooboshi a , Hideyuki Uchimura b , Masatoshi Fujishima a
Second Department of Internal Medicine, Faculty of Medicine, Kyushu UniÕersity, Fukuoka, Japan b Center for Emotional and BehaÕioral Disorders, Hizen National Mental Hospital, Saga, Japan Accepted 21 October 1997
Abstract The role of protein kinase C ŽPKC. in mediating the ischemia-induced release of amino acids in the striatum was studied using an in vivo brain dialysis technique in the striatum of spontaneously hypertensive rats ŽSHRs.. Using HPLC combined with fluorescence detection methods, we investigated the concentrations of amino acids in the dialysates produced by 20 min of transient forebrain ischemia. We studied the effects of an inhibitor of PKC, 1-Ž5-isoquinolinesulfonyl.-2-methylpiperazine dihydrochloride ŽH7. and another isoquinoline analog ŽHA1004. with less inhibitory effect on the C kinase in ischemia-induced amino acids release. Bilateral carotid artery occlusion caused a marked reduction in the striatal blood flow by 91 " 6%. The extent of the cerebral blood flow ŽCBF. reduction were essentially the same among H7-, HA1004-, and the vehicle-treated groups. Forebrain ischemia produced a marked increase in glutamate Ž21-fold of the basal concentration., aspartate Ž19-fold. and taurine Ž16-fold.. Pretreatment with H7 markedly attenuated the ischemia-induced release of these three amino acids to 3, 3 and 4-fold of the basal values, respectively. Increase of g-aminobutyric acid ŽGABA. was also attenuated by H7 Žvehicle; 2.46 " 1.26 m M, H7; 0.62 " 0.75 mM.. HA1004 did not affect the release of glutamate, aspartate or GABA during ischemia. The ischemia-induced release of taurine was significantly inhibited by HA1004 but the effect was much smaller than that of H7. These results thus indicate that PKC plays a major role in the ischemia-induced release of amino acids in the striatum of SHR. q 1998 Elsevier Science B.V. Keywords: Cerebral ischemia; Protein kinase C; Glutamate; Aspartate; Taurine; GABA
1. Introduction A massive release of glutamate occurs during cerebral ischemia w3x, which produces an accumulation of intracellular Ca2q, and thereby causes neuronal cell death w5x. It was recently reported that staurosporine, a potent PKC inhibitor w21x and fasudil ŽHA1077., a novel protein kinase inhibitor w39x were effective in protecting hippocampal CA1 neurons against ischemic damage and thus significantly improved the neurological function in both rats and gerbils. Other PKC inhibitors, 1-Ž5- isoquinolinesulfonyl.-2-methylpiperazine dihydrochloride ŽH7. and calphostin C, were also reported to prevent glutamate toxicity in primary neuronal cultures w11x. The protective mechanism for the PKC inhibitor is not clear, but one
possibility is PKC may be involved in the ischemia-induced massive release of excitatory amino acids from presynaptic nerve terminals, glial cells, and postsynaptic elements. Although several reports have shown that activated PKC enhances the release of neurotransmitters under nonischemic conditions w2,10,17,28,29,33x, there are a few reports about the involvement of PKC on ischemia-induced neurotransmitter release w27,38x. To clarify the involvement of PKC in ischemic amino acids release, we evaluated the effects of two inhibitors of PKC, H7 and an isoquinoline sulfonamid, HA1004, on the amino acids release in transient forebrain ischemia in rats. 2. Materials and methods 2.1. Animals
)
Corresponding author. Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan. Telex: q81-92-632-2551; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 3 1 - 0
Male spontaneously hypertensive rats ŽSHRs., aged 7–9 months and weighing 350–450 g, were used in the present
H. Nakane et al.r Brain Research 782 (1998) 290–296
study. As hypertension is one of the most important risk factors of cerebral infarction, we have used SHRs to examine the pathophysiology of cerebral ischemia. The rats were maintained in the Kyushu University Animal Center and fed a standard chow diet and tap water ad libitum. 2.2. Brain ischemia The rats were anesthetized with amobarbital Ž100 mgrkg, i.p.., and allowed to breathe spontaneously. Forebrain ischemia was produced by bilateral carotid artery occlusion according to the original method w13x as described previously w31,36x. In our previous studies, bilateral carotid artery occlusion caused an extremely high mortality w15x, a marked increase in anaerobic glycolytic metabolites of the brain w13x, and diffuse-extensive cerebral infarcts in SHR w35x. Although the configuration of the circle of Willis is essentially identical in both normotensive rat and SHR w35x, SHR shows a marked fall of carotid back pressure to below the critical perfusion pressure of cerebral autoregulation w14x. Thus this hemodynamic change is thought to be a major causative factor of diffuse and extensive cerebral infarction following bilateral carotid artery occlusion. Their body temperature was maintained at 378C using a heat pad. The femoral arteries were cannulated with PE 50 tubing for the anaerobic sampling of blood and the continuous recording of the arterial blood pressure with an AP 601 G electromanometer ŽNihon Koden, Tokyo, Japan.. Arterial blood gases and pH were determined with an IL meter model 1304 ŽInstrumentation Laboratories, Lexington, KY, USA.. Both common carotid arteries were exposed through a ventral midline incision in
291
the neck, carefully separated from the vagosympathetic trunks, and loosely encircled with sutures for the later ligation. 2.3. In ÕiÕo brain dialysis and CBF measurement Extracellular amino acids Žglutamate, aspartate, GABA, taurine., and regional CBF in the striatum were simultaneously determined using an in vivo brain dialysis technique and a hydrogen clearance method, respectively, as described previously w45x. The rats were fixed in a head holder, and a small burr hole was made in the skull. A dialysis probe, measuring 500 m m in outer diameter Ž3-mm dialysis membrane; molecular cut off approximately 20,000 Da; Carnegie Medicine, Stockholm, Sweden. and a Teflon-coated platinum CBF electrode attached to a thermometer, 200 m m in diameter, were placed stereotaxically into the right striatum Ž0.5 mm anterior and 3.0 mm lateral to the bregma and 4.5 mm in depth from the brain surface. according to the atlas of Paxinos and Watson w37x. The distance between the microdialysis membrane and the hydrogen clearance electrode was about 1 mm. The striatum was perfused with Ringer solution through a dialysis probe at a rate of 3.0 m lrmin with a syringe pump Žmodel EP-60, Eicom, Kyoto, Japan. and the perfusates were collected every 10 min into a plastic tube. 2.4. Biochemical assay The concentrations of amino acids were determined using HPLC combined with fluorescence detection after precolum derivatization with 4 mM o-phthalaldehyde w26x. Each 24 m l of perfusate and 8 m l of o-phthalaldehyde
Table 1 Physiological parameters Ž1. Rest
Drug
pH Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
7.42 " 0.01 7.42 " 0.03 7.43 " 0.02
7.42 " 0.02 7.42 " 0.02 7.44 " 0.02
7.52 " 0.06 7.57 " 0.06 7.53 " 0.08
7.42 " 0.03 7.43 " 0.03 7.44 " 0.02
pa CO2 Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
38.2 " 1.0 37.7 " 2.3 38.6 " 0.8
40.5 " 1.8 38.6 " 2.6 37.5 " 3.1
31.2 " 5.2 25.6 " 5.0 27.0 " 10.2
39.6 " 3.0 38.4 " 1.5 34.4 " 5.9
pa O2 Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
87.3 " 2.8 89.5 " 4.6 90.5 " 2.9
85.6 " 4.6 89.0 " 3.3 93.5 " 3.1a
98.3 " 6.4 102.7 " 5.5 94.3 " 12.0
83.8 " 5.5 89.6 " 4.5 85.8 " 21.5
Ht (%) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
41 " 5 43 " 3 42 " 3
43 " 3 42 " 5 43 " 1
44 " 3 43 " 4 45 " 3
42 " 1 44 " 4 46 " 2
Values are mean " S.D. ) p - 0.05 vs. Vehicle.
Ischemia
Recirculation
H. Nakane et al.r Brain Research 782 (1998) 290–296
292 Table 2 Physiological parameters Ž2. Rest
Drug
Ischemia
Recirculation 10 min
60 min
MABP (mmHg) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
194 " 14 193 " 26 184 " 22
191 " 13 187 " 15 184 " 18
208 " 35 211 " 25 198 " 29
174 " 24 168 " 11 160 " 14
180 " 24 181 " 11 151 " 9
CBF (% of rest) Vehicle 100 H7 5 = 10y4 M HA1004 5 = 10y4 M
100 100 100
119 " 19 137 " 27 101 " 19
9.1 " 5.5 7.0 " 3.2 7.0 " 6.2
168 " 74 119 " 80 95 " 34
54 " 17 114 " 23 79 " 46
Brain temparature (8C) Vehicle H7 5 = 10y4 M HA1004 5 = 10y4 M
35.7 " 0.2 35.7 " 0.2 35.8 " 0.3
35.8 " 0.2 35.8 " 0.2 36.0 " 0.3
34.3 " 0.6 34.7 " 0.4 34.8 " 0.8
36.4 " 0.3 35.7 " 0.4 ) 36.2 " 0.5
35.8 " 0.5 35.8 " 0.3 35.6 " 0.4
Values are mean " S.D. ) p - 0.05 vs. Vehicle.
solution was reacted for 2 min, and then 30 m l of the mixed solution were injected into the chromatography using an autoinjector Žmodel 231-401, Gilson Medical Electronics, Villiers le Bel, France.. The chromatographic system consisted of an LC-6A pump ŽShimadzu, Kyoto, Japan. at a flow rate of 1.0 mlrmin, a reverse-phase column ŽEicompac MA-5ODS, 4.6 = 150 mm, Eicom, Kyoto, Japan. and a fluorescent detector ŽRF-535, Shimadzu, Tokyo, Japan.. The mobile phase was 0.1 M sodium phosphate ŽpH 6.0. containing 30% Žvol.rvol.. methanol.
2.5. Experimental protocol We divided the rats into three groups, consisting of the H7-treated group Ž n s 6., the HA1004-treated group Ž n s 4., and the vehicle-treated group Ž n s 7.. After determining two baseline CBF values and four determinations of amino acid concentrations during a 40 min of resting period, the administration of H7, 5 = 10y4 M or HA1004, 5 = 10y4 M, was started through the dialysis probe. H7 ŽSigma, St. Louis. and HA1004 ŽSigma, St. Louis. were
Fig. 1. Changes in the concentration of extracellular glutamate. H7 attenuated the glutamate release in the striatum during ischemia. HA1004 did not affect the ischemia-induced glutamate release. The values are the mean " S.D. ) p - 0.05 vs. vehicle.
H. Nakane et al.r Brain Research 782 (1998) 290–296
293
for perfusion into each group of rats. In the control rats, dialysis probes were perfused with vehicle. Twenty min after starting drug infusion, both carotid arteries were tightly ligated for 20 min and then reopened for 60 min to allow for recirculation. The cerebral blood flow was measured at 20 min after drug perfusion, 20 min of ischemia, and 10 and 60 min of recirculation. Arterial blood was sampled during the control period, at 20 min after drug infusion, 20 min of ischemia, and 60 min of recirculation. The position of the dialysis probe and CBF electrode was then macroscopically determined after the experiment. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986. 2.6. Statistics
Fig. 2. Changes in the concentration of extracellular aspartate. H7 attenuated the aspartate release in the striatum during ischemia. HA1004 did not affect the ischemia-induced aspartate release. The values are the mean" S.D. ) p- 0.05 vs. vehicle.
dissolved in Ringer solution Ž147 mM Naq, 2.3 mM Ca2q, 4 mM Kq, 155.5 mM Cly, pH 6.8.. 5 = 10y4 M solutions of H7 ŽpH 6.2. or HA1004 ŽpH 6.4. solution were made
In each group, the concentrations of the amino acids in the last fourth sample during the resting period were referred as the basal values. The statistical differences in physiological parameters, mean arterial blood pressure, and CBF among groups were analyzed by a one-way analysis of variance ŽANOVA. followed by Dunnett’s t-test. The differences in the concentrations of amino acids among the groups were analyzed by a two-way repeated ANOVA regarding such factors as the group and the time. To assess further differences among the groups, we used a one-way ANOVA followed by Scheffe’s multiple comparison. All the values were presented as the mean " S.D.
Fig. 3. Changes in the concentration of extracellular taurine. H7 and HA1004 attenuated the taurine release in the striatum during ischemia. However, the degree of reduction by HA1004 was smaller than that by H7. The values are the mean" S.D. ) ) p - 0.01 and ) p - 0.05 vs. vehicle.
294
H. Nakane et al.r Brain Research 782 (1998) 290–296
3. Results 3.1. Physiological parameters during cerebral ischemia Table 1 shows the physiological parameters in the three groups of rats. There were no significant differences in paCO 2 , pH, hematocrit among the three groups during the experiment. The arterial oxygen tension in the HA1004 group was slightly but significantly higher than in the vehicle group. However, this change was within the physiological range. The rats showed hyperventilation and consequent respiratory alkalosis during ischemia, as is usually seen in our model w16x. The changes in the mean arterial blood pressure, striatal CBF and brain temperature are given in Table 2. The mean arterial blood pressure slightly increased during ischemia in all groups but no significant differences were observed among the three groups throughout the experiment. The resting CBF values were not significantly different among the groups; 61.5 " 27.6 mlr100 grmin in the control group, 44.2 " 12.6 in the H7 group and 56.8 " 20.5 in the HA1004 group and were unchanged after 20 min of H7 and HA1004 perfusion. During ischemia, the CBF in each rat markedly decreased Ž p - 0.01. to less than 10% of the resting value. Although the control group showed postischemic hyperemia immediately after reopening the carotid arteries, followed by hypoperfusion, the H7 and HA1004 groups showed neither postischemic hyperemia nor hypoperfusion. The H7 treated group thus demonstrated a considerable recovery of CBF during recirculation and revealed a significantly higher CBF value Ž114% of resting value at 60 min, p - 0.05. than did the control group Ž54% of resting value.. 3.2. Effects of H7 and HA1004 on the extracellular concentration of amino acids The basal levels of glutamate, aspartate, taurine of each group were 0.74 " 0.63 m M, 0.23 " 0.15 m M, and 1.59 " 0.67 m M in the vehicle group, 1.03 " 1.15 m M, 0.22 " 0.16 m M and 1.52 " 0.49 m M in the H7-treated group, 0.74 " 0.20 m M, 0.28 " 0.11 m M and 1.56 " 0.49 m M in HA1004-treated group, respectively and there were no significant differences among the three groups. The basal level of GABA could not be detected in this study. H7 and HA1004 did not affect the extracellular concentrations of amino acids during the resting period. The concentrations of excitatory amino acids, glutamate and aspartate, increased and reached to a peak at 20 min of ischemia; 21-fold and 19-fold of the basal value in the vehicle group, respectively and then rapidly returned to the preischemic level at 20 min of recirculation. H7 significantly inhibited the increases in glutamate and aspartate; 3-fold Ž p - 0.05 vs. vehicle. and 3-fold Ž p - 0.05 vs. vehicle. of basal value, respectively ŽFigs. 1 and 2.. HA1004 did not show any significant inhibition of the release of glutamate and aspartate during ischemia. The ischemia-induced release of GABA, an inhibitory amino acid, was also attenuated by
H7 Ž p - 0.05 vs. vehicle. but not HA1004 Žvehicle; 2.46 " 1.26 m M, H7; 0.62 " 0.75 m M, HA1004; 1.58 " 1.06 m M.. H7 strongly inhibited the release of taurine; 16-fold in the vehicle group, 4-fold Ž p - 0.01 vs. vehicle. and the inhibition lasted throughout recirculation. HA1004 also showed an inhibitory effect on the ischemia-induced release of taurine; 7-fold Ž p - 0.05 vs. vehicle. but the effect was smaller than that of H7 ŽFig. 3.. 4. Discussion The present study suggests that the activation of PKC may be one of the major mechanisms by which ischemia causes a massive release of amino acids. A large amount of PKC appears to be present in the brain w25x, especially at the presynaptic nerve terminals w18x, and thus plays an important role in neuronal transmission w7,44x, neurotrophic activity w30x, and neuronal plasticity w34x. This is the first study which shows the effects of PKC on the release of amino acids under ischemic conditions by using in vivo brain dialysis methods. A major concern in the studies using PKC inhibitor is the specificity of PKC inhibition. Staurosporine, which has an antifungal activity, is one of the potent PKC inhibitors ŽIC50 is 2.7 nM. w42x. However, it also inhibits cyclic AMP ŽcAMP.- and cyclic GMP ŽcGMP.-dependent kinases. H7 is known to inhibit PKC by interfering with the interaction of ATP and protein substrates within the catalytic domain. The Ki values of H7 against PKC, cAMP- and cGMP-dependent protein kinase are 6.0, 3.0 and 5.8 m M, respectively w23x. In the concentration range used in the present study, this agent also inhibits cAMP and cGMP dependent protein kinases. We thus used another derivative of isoquinoline sulfonamide, HA1004, which has a much less inhibitory effect on PKC Žthe Ki values against PKC, cAMP- and cGMPdependent protein kinase are 40, 2.3 and 1.3 m M, respectively. w23x. In this study, HA1004 also inhibited the release of amino acids during ischemia, but the effect was much smaller than that of H7. Taken together, the effect of H7 in the present study may be specific for PKC. 4.1. Role of PKC in amino acid release Several reports have shown that activated PKC enhances the release of neurotransmitters under nonischemic conditions w2,10,17,28,29,33x. At least four different mechanisms, Ž1. calcium influx from voltage sensitive calcium channels ŽVSCCs., in particular, N- and P-types of VSCCs w8,24x, Ž2. modulation by metabotropic glutamate ŽmGlu. receptors w19,22x, Ž3. phosphorylation of synapsin I by Ca2q-calmodulin-dependent protein ŽCaM. kinase II w20x, Ž4. depolymerization and repolymerization of actin assembly w4x, are considered to play a role in neurotransmitter release under nonischemic condition. Dekker et al. w6x showed that phosphorylation of B-50, which is an important presynaptic PKC substrate, was closely correlated with neurotransmitter release in rat hippocampal slices. Some
H. Nakane et al.r Brain Research 782 (1998) 290–296
reports also revealed that PKC regulated the ion channels, e.g., VSCCs w12,40,43x and calcium-activated potassium channels w1x. Thus, PKC may be involved in several steps of neurotransmitter release under nonischemic conditions. In the present study, H7, an inhibitor of PKC, markedly attenuated the ischemia-induced increase of the extracellular glutamate, aspartate, GABA, and taurine. Lu et al. w27x showed that glutamate accumulation under ischemic condition was inhibited by H7 in vitro. Phillis and O’Regan w38x have recently shown that PKC contributes to ischemiaevoked glutamate and aspartate release in vivo. The findings of their studies are also similar to ours. Our study is the first report on the involvement of PKC in ischemia-induced amino acids release by using an in vivo brain dialysis model. We showed that ischemia-induced release of GABA and taurine were also inhibited by H7. Especially, taurine was more strongly inhibited by H7 and HA1004 than the other amino acids. The reason for this difference, however, is still unknown, because the release mechanism of taurine has not been clarified. We previously reported that the ischemia-induced release of taurine was reduced in aged rats w36x. The release mechanism of excitatory and inhibitory amino acids therefore appears to be different. Under ischemic conditions, two different mechanisms are considered about increase of extracellular glutamate concentrations. In the early phase of ischemia, glutamate could be released from the neurotransmitter pool by a calcium dependent process w9x. In the late phase, the extracellular concentration of glutamate could be elevated by reversal of the glutamate transporter because of depletion of ATP supplies w32,41x. The role of PKC in the ischemia-induced neurotransmitter release is not clear. However, the present study and previous studies w27,38x support the conclusion that PKC also plays a role under ischemic conditions. Phillis and O’regan w38x showed that destabilization and deterioration of the plasma membrane would be important in ischemia-induced release of excitatory amino acids. In this process, hydrolysis of membrane phospholipids, e.g., by phospholipase A 2 and C, occurred and PKC was involved in the cascade of events. In the present study, the release of both excitatory and inhibitory amino acids were attenuated by the PKC inhibitor. This result suggests that not only excitatory amino acids but also inhibitory amino acids could diffuse into the extracellular space from neurons or glia cells by the deterioration of the membrane during ischemia. We postulate that PKC may be involved in neurotransmitter release under ischemic conditions by modulating the stabilization of the membrane.
5. Conclusion In summary, H7 strongly inhibited the ischemia-induced release of amino acids. These results thus strongly
295
suggest the conclusion that PKC may play an important role in neurotransmitter release during ischemia. Acknowledgements This study was partly supported by the Social Insurance Agency Contract Fund commissioned by the Japan Health Sciences Foundation. References w1x J. Barban, S. Snyder, B. Alger, PKC regulates ionic conductance in hippocampal pyramidal neurons: electrophysiological effects of phorbol esters, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 2538–2542. w2x A.P. Barrie, D.G. Nicholls, J. Sanchez-Prieto, T.S. Sihra, An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes, J. Neurochem. 57 Ž1991. 1398–1404. w3x H. Benveniste, J. Drejer, A. Shousboe, N.H. Diemer, Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem. 43 Ž1984. 1369–1374. w4x B.W. Bernstein, J.R. Bamburg, Cycling of actin assembly in synaptosomes and neurotransmitter release, Neuron 3 Ž1989. 257– 265. w5x D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system ŽReview., Neuron 1 Ž1988. 623–634. w6x L. Dekker, P.D. Graan, D. Versteeg, A. Oestreicher, W. Gispen, Phosphorylation of B-50 ŽGAP43. is correlated with neurotransmitter release in rat hippocampal slices, J. Neurochem. 52 Ž1989. 24–30. w7x S.A. DeRiemer, J.A. Strong, K.A. Albert, P. Greengard, L.K. Kaczmarek, Enhancement of calcium current in Aplysia neurons by phorbol ester and protein kinase C, Nature 313 Ž1985. 313–316. w8x D.J. Dooley, A. Lupp, G. Hertting, Inhibition of central neurotransmitter release by v-conotoxin GIVA, a peptide modulator of the N-type voltage-sensitive calcium channel, Naunyn–Schmideberg’s Arch. Pharmacol. 336 Ž1987. 467–470. w9x J. Drejer, H. Benveniste, N.H. Diemer, A. Schousboe, Cellular origin of ischemia-induced glutamate release from brain tissue in vivo and in vitro, J. Neurochem. 45 Ž1985. 145–151. w10x M.L. Eboli, M.T. Ciotti, D. Mercanti, P. Calissano, Differential involvement of protein kinase C in transmitter release and response to excitatory amino acids in cultured cerebellar neurons, Neurochem. Res. 18 Ž1993. 133–138. w11x V. Felipo, M.-D. Minana, S. Grisolıa, ˜ ´ Inhibitiors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures, Brain Res. 604 Ž1993. 192–196. w12x F. Fournier, P. Charnet, E. Bourinet, C. Vilbert, F. Matifat, G. Charpentier, P. Navarre, G. Brule, D. Marlot, Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebral mRNA, FEBS 317 Ž1993. 118–124. w13x M. Fujishima, T. Sugi, M. Morotomi, T. Omae, Effects of bilateral carotid artery ligation on brain lactate and pyruvate concentrations in normotensive and spontaneously hypertensive rats, Stroke 6 Ž1975. 62–66. w14x M. Fujishima, T. Omae, Carotid back pressure following bilateral carotid occlusion in normotensive and spontaneously hypertensive rats, Experientia 32 Ž1976. 1021–1022. w15x M. Fujishima, J. Ogata, T. Sugi, T. Omae, Mortality and cerebral metabolism after bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats, J. Neurol. Neurosurg. Psychiat. 39 Ž1976. 212–217. w16x M. Fujishima, Y. Nakatomi, K. Tamaki, J. Ogata, T. Omae, Cerebral
296
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
H. Nakane et al.r Brain Research 782 (1998) 290–296 ischemia induced by bilateral carotid occlusion in spontaneously hypertensive rats, J. Neurol. Sci. 33 Ž1977. 1–11. V.C. Gandhi, D.J. Jones, Protein kinase C modulates the release of w 3 Hx5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels, Neuropharmacology 31 Ž1992. 1101–1109. P. Girard, G. Massei, J. Wood, J. Kuo, Polyclonal antibodies to phospholipidrCa2q-independent protein kinase and immunocytochemical localization of the enzyme in rat brain, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 3030–3034. S.R. Glaum, R.J. Miller, The metabotropic glutamate receptors: acute regulation of synaptic transmission by metabotropic glutamate receptors, in: P.J. Conn, J. Patel ŽEds.., Humana Press, Clifton, NJ, 1995, pp. 142–172. P. Greengard, F. Valtorta, A.J. Czernik, F. Benfenati, Synaptic vesicle phosphoproteins and regulation of synaptic function, Science 259 Ž1993. 780–784. H. Hara, H. Onodera, M. Yoshidomi, Y. Matsuda, K. Kogure, Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab. 10 Ž1990. 646–653. I. Herrero, M.T. Miras-Portugal, J. Sanchez-Prieto, Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation, Nature 360 Ž1992. 163–166. H. Hidaka, M. Inagaki, S. Kawamoto, Y. Sasaki, Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C, Biochemistry 23 Ž1984. 5036– 5041. M. Kimura, Y. Yamanishi, T. Hanada, T. Kagaya, M. Kuwada, T. Watanabe, K. Katayama, Y. Nishizawa, Involvement of P-type calcium channels in high potassium-eliceted release of neurotransmitters from rat brain slices, Neuroscience 66 Ž1995. 609–615. J.F. Kuo, R.G.G. Andersson, B.C. Wise, L. Mackerlova, I. Salomonsson, N.L. Brackett, N. Katoh, M. Shoji, R.W. Wernn, Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine, Proc. Natl. Acad. Sci. U.S.A. 77 Ž1980. 7039–7043. P. Lindroth, K. Mopper, High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolum fluorescence derivatization with o-phthalaldehyde, Anal. Chem. 51 Ž1979. 1667–1674. Y.M. Lu, B.F. Lu, F.O. Zhao, Y.L. Yan, X.P. Ho, Accumulation of glutamate is regulated by calcium and protein kinase C in rat hippocampal slices exposed to ischemic states, Hippocampus 3 Ž1993. 221–227. R.C. Malenka, D.V. Madison, R.A. Nicoll, Potentiation of synaptic transmission in the hippocampus by phorbol esters, Nature 321 Ž1986. 175–177. R.C. Malenka, G.S. Ayoub, R.A. Nicoll, Phorbol esters enhance transmitter release in rat hippocampal slices, Brain Res. 403 Ž1987. 198–203.
w30x H.P.M. Montz, G.E. Davis, S.D. Skaper, M. Manthorpe, S. Varon, Tumor-promoting phorbol diester mimics two distinct neurotrophic factors, Dev. Brain Res. 23 Ž1985. 150–154. w31x H. Nakane, H. Ooboshi, S. Ibayashi, H. Yao, S. Sadoshima, M. Fujishima, Isradipine, a calcium channel blocker, attenuates the ischemia-induced release of dopamine but not glutamate in rats, Neurosci. Lett. 188 Ž1995. 151–154. w32x D. Nicholls, D. Attwell, The release and uptake of excitatory amino acids, Trends Pharmac. Sci. 11 Ž1990. 462–468. w33x R.A. Nichols, J.W. Haycock, J.K.T. Wang, P. Greengard, Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes, J. Neurochem. 48 Ž1987. 615–621. w34x Y. Nishizuka, Studies and perspectives of protein kinase C, Science 233 Ž1986. 305–312. w35x J. Ogata, M. Fujishima, Y. Morotomi, T. Omae, Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study, Stroke 7 Ž1976. 54–60. w36x H. Ooboshi, S. Sadoshima, H. Yao, S. Ibayashi, T. Matsumoto, H. Uchimura, M. Fujishima, Ischemia-induced release of amino acids in the hippocampus of aged hypertensive rats, J. Cereb. Blood Flow Metab. 15 Ž1995. 227–234. w37x G. Paxinos, C. Watson, The rat brain in stereotaxic coodinates, 2nd edn., Academic Press, Tokyo, 1986. w38x J.W. Phillis, M.H. O’Regan, Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex, Brain Res. 730 Ž1996. 150–164. w39x S. Satoh, I. Ikegaki, Y. Suzuki, T. Asano, M. Shibuya, H. Hidaka, Neuroprotective properties of a protein kinase inhibitor against ischaemia-induced neuronal damage in rats and gerbils, Br. J. Pharmacol. 118 Ž1996. 1592–1596. w40x K.J. Swartz, A. Merritt, B.P. Bean, D.M. Lovinger, Protein kinase C modulates glutamate receptor inhibition of Ca2q channels and synaptic transmission, Nature 361 Ž1993. 165–168. w41x M. Szatkowski, D. Attwell, Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms, Trends Neurol. Sci. 17 Ž1994. 359–365. w42x T. Tamaoki, H. Nomoto, I. Takahashi, Y. Kato, M. Morimoto, F. Tomita, Staurosporine, a potent inhibitor of phospholipidrCa2q dependent protein kinase, Biochem. Biophys. Res. Commun. 135 Ž1986. 397–402. w43x D.M. Terrian, D.K. Ways, Persistent enhancement of sustained calcium-dependent glutamate release by phorbol esters: role of calmodulin-independent serinerthreonine phosphorylation and actin disassembly, J. Neurochem. 64 Ž1995. 181–190. w44x L.A. Witters, C.A. Vater, G.E. Lienhard, Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C, Nature 315 Ž1985. 777–778. w45x H. Yao, H. Ooboshi, S. Ibayashi, H. Uchimura, M. Fujishima, Cerebral blood flow and ischemia-induced neurotransmitter release in the striatum of aged spontaneously hypertensive rats, Stroke 24 Ž1993. 577–580.