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Isradipine, a calcium channel blocker, attenuates the ischemia-induced release of dopamine but not glutamate in rats
ELSEVIER
Neuroscience Letters 188 (1995) 151-154
ri[llill:[ IS[IS
Isradipine, a calcium channel blocker, attenuates the ischemia-induced release of dopamine but not glutamate in rats Hiroshi Nakane ,i,b,*, Hiroaki O o b o s h i a, Setsuro Ibayashi a, Hiroshi Y a o a, Seizo Sadoshima a, Masatoshi F u j i s h i m a a aSecond Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan bCenter for Emotional and Behavioral Disorders, Hizen National Mental Hospital, Saga, Japan Received 12 December 1994; revised version received 17 February 1995; accepted 20 February 1995
Abstract
This study was designed to investigate the role of the L-type voltage sensitive calcium channel blocker, isradipine, in the ischemiainduced release of neurotransmitters. Male spontaneously hypertensive rats were subjected to cerebral ischemia for 60 rain by bilateral carotid artery occlusion, and recirculated for 120 min. Isradipine (0.25 mg/kg n = 6) or vehicle (n = 6) was administered subcutaneously at 20 min before ischemia. In the striatum, cerebral blood flow was determined by the hydrogen clearance method and concentrations of extracellular dopamine and glutamate were measured by in vivo brain dialysis technique. Extracellular dopamine in the vehicle-treated group increased by 180-fold from the basal level, and glutamate by 24-fold during cerebral ischemia, lsradipine significantly attenuated the ischemic release of dopamine to 33-34% (P < 0.05) of the vehicle group, while it did not affect glutamate release. It is suggested that the release mechanism of dopamine and glutamate during cerebral ischemia may be different, especially in the dependence on the L-type calcium channels.
Keywords: Cerebral ischemia; Isradipine; Dopamine; Glutamate; Voltage sensitive calcium channel
Excessive release of neurotransmitters is known to play an important role in the development of ischemic cell damage. The ischemia-induced massive release of dopamine [5], glutamate [3] causes neuronal death probably due to an excitotoxic process. The major pathway of the excitotoxicity is believed to be the entry of extracellular calcium into ischemic neuronal cells. Isradipine, an L-type voltage sensitive calcium channel (VSCC) blocker, is one of the most powerful vasodilators, and is expected to increase cerebral blood flow (CBF) in the ischemic brain. In addition, isradipine seems to possess neuroprotective effects against cerebral ischemia by inhibiting the entry of excessive amount of calcium into neurons [1,10]. There are reports that neuroprotective effects of several drugs against cerebral ischemia are caused by attenuation of the release of excitatory amino acids from presynaptic neurons [13]. Recently, we showed partial inhibition of ischemia-induced release of dopamine by an * Corresponding author, Tel.: +81 92 6411151; Fax: +81 92 6322551.
infusion of isradipine into the striatum through a microdialysis probe [16]. Isradipine effectively attenuates the ischemic damage by local or systemic administration [1,10]. We speculate that such a favorable,, action of isradipine is partly attributable to the attenuation of an excessive release of these excitatory neurotransmitters. The aim of this study is to examine the role of L-type VSCC and its blocker, isradipine, in the release of dopamine and glutamate during cerebral ischemia. Seventeen male spontaneously hypertensive rats (SHRs), aged 7-9 months and weighing 350-450 g, were used. Just before each experiment, isradipine was dissolved in the vehicle (ethanol/polyethylene glycol 400, 1:1) and 0.25 mg/kg of solution was made in a dark room. The rats were anesthetized with amobarbital (100 mg/kg i.p.), and allowed to breathe spontaneously. Cerebral ischemia was developed by bilateral carotid artery occlusion as described previously [4]. Body temperature was maintained at 37°C using a heat lamp. Femoral arteries were cannulated for anaerobic sampling of blood and continuous recording of arterial blood pres-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 4 1 7 - R
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14. Nakane et al. / Neuroscience Letters 188 (1995) 151-154
sure. Twenty minutes after drug injection, both carotid arteries were tightly ligated for 60 rain and then reopened for 120 min to allow recirculation. Extracellular dopamine and glutamate, 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 [15,16,21,22]. A dialysis probe, 5 0 0 # m in outer diameter with a 3-ram dialysis membrane and a Teflon-coated platinum electrode attached with thermometer; 200 ktm in diameter with a l-ram uncoated portion at its tip, for CBF and brain temperature determination, were placed stereotaxically in the right striatum; 0.5 mm anterior and 3.0 mm lateral to the bregma and 4.5 mm in depth from the surface of the brain according to the atlas of Paxinos and Watson [17]. The striatum was perfused with Ringer solution through the dialysis probe at a rate of 4.0ktl/min and the perfusates were collected every 20 rain into a plastic tube. After determination of two baselines CBF and three concentrations of dopamine and glutamate during 60 min of resting period, isradipine was administered subcutaneously. Control rats (n = 6) received vehicle. CBF was measured at 20 rain after drug injection, 20 and 60 rain of ischemia, and 10 and 120 min of recirculation. Arterial blood was sampled during the control period, at 20 min after drug infusion, 60 min of ischemia, and 120 rain of recirculation. The position of the dialysis probe and CBF electrode was macroscopically determined after the experiment. The HPLC systems for dopamine and glutamate were described previously [15,22]. In each group, the concentrations of dopamine and glutamate in the last three samples during the resting period were referred to the basal values. The statistical differences in physiological parameters, mean arterial blood pressure, and CBF within or among groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test. The differences in the concentrations of dopamine and glutamate among the groups were also analyzed by one-way A N O V A followed by Scheffe's Ftest. All the values are presented as mean + SEM.
There were no significant differences in blood gases and pH between the groups during the experiment~ All rats showed respiratory alkalosis (pH 7.48 _+0.03, pCO 2 28.2 + 3.7, pO2 97.3 + 2.0 in the vehicle-treated group and 7.49 _+0.03, 26.5 + 3.8, 101.0 -+ 3.8 in the isradipinetreated group) during ischemia, but blood gases and pH returned to the control level after 120 rain of recirculation. Brain temperatures in the striatum at 60 min of ischemia were 34.5+_0.4°C in the vehicle group, 34.3 _+0.1 °C in the isradipine group. The changes in mean arterial blood pressure and CBF to the striatum are given in Table 1. Mean arterial blood pressure markedly fell after isradipine administration; from 185 _+5 to 122 + 6 m m H g (P < 0.01 versus at rest). These hypotensive changes lasted until the end of the experiment. Resting CBF were actually same between the groups; 48.5 _+6.5 ml/100 g per min in the vehicle group, 43.3 + 4.3 in the isradipine group. After administration of isradipine, CBF to the striatum significantly increased to 130% (P < 0.05) of the basal levels. During ischemia, CBF in each rat markedly decreased to less than 10% of the basal level. Although CBF in the vehicle group remained 68% of the basal level at 120 rain of recirculation, isradipine groups obtained considerable recovery of CBF during recirculation and revealed significantly higher CBF (169% at 120 min, P < 0.05) than the vehicle group. The averaged basal concentrations of dopamine and glutamate were 10.5 + 3.6 pg/20 rain and 3.9 -+ 1.6~M/ 20 min, in the vehicle group, 7.8 + 3.2 and 3.8 + 0.3, in the isradipine group. These variables were not significantly different between the groups. Administration of isradipine did not alter the concentrations of dopamine and glutamate under the resting condition. After induction of ischemia, concentrations of extracellular dopamine markedly increased within 20 rain by 180-fold of the basal value in the vehicle group, by l l0-fold in the isradipine group, without significant difference between the groups. Thereafter, concentrations of dopamine gradually decreased in all groups. In rats treated with isradipine, however, the level of dopamine was significantly smaller than the vehicle, resulting in 33% (P < 0.05) of the vehicle at 40 rain and 34% (P < 0.05) at 60 min. The elevated
Table 1 Time course of mean arterial blood pressure (MABP) and striatal cerebral blood flow (CBF) Rest
Vehicle (n = 6) MABP (mmHg) CBF (% of basal value) lsradipine, 0.25 mg/kg (n = 6) MABP (mmHg) CBF(% ofbasal value)
Drug
Recirculation
Ischemia 20 min
60 rain
10 min
190 _+4 100
193 _+4 88.5_+8.3
204 _+8 16.3+-4.3
185 -+7 5.0+2.0
171 _+6 112.0-+33.2
185 -+5 100
122 -+6** 130.4_+ 10.7"
99 -+9** 19.2+6.2
94 -+9** 8.4_+4.3
94 -+6** 147.1 _+38.6
120 rain 156 _+13 68.7+11.1 104 -+8* 168.9_+21.2"*
Values are mean _+SEM. MABP, mean arterial blood pressure. Resting CBF values are present in the text. **P < 0.01, *P < 0.05 versus vehicle.
153
H. Nakane et al. / Neuroscience Letters 188 (1995) 151-154
DOPAMINE
GLUTAMATE
14 -~
2000
~
.~
1500
.~
10
~
6
~
4
1000
I t
t'
-60-40-20
0
0 o...Q.,..,o~-.o.....o 20 40
Drug Ischemia
20 40 60 80
100 120
(rain)
Reeirculation
vehicle
isradipine 0.25mg/kg
: .~;~.
0 !-q, - q , 0
~ O....
-60-40-20
. . . . . . . . 0
20 40
0
Drug Ischemia
20 40 60 80
100120 (ram)
Recirculation
Fig. l. Changes in the concentration of extracellular dopamine and glutamate. At 40 and 60 rain of ischemia, significant reduction of elevated dopamine was determined by isradipine. There was no difference in the concentration of glutamate among the groups at 60 rain of ischemia. Values are mean _+SEM. **P < 0.01, *P < 0.05 versus vehicle. concentration of dopamine returned to the pre-ischemic levels at 40 rain of recirculation in both groups. Concentrations of glutamate, in contrast to dopamine, progressively increased and reached a peak at 60 min of ischemia; 24-fold of basal value in the vehicle group, 35-fold in the isradipine group. Elevated glutamate rapidly returned to the pre-ischemic level at 40 rain of recirculation. There were no significant difference between isradipineand vehicle-treated group (Fig. 1). Isradipine, a dihydropyridine-derivative, potently blocks the L-type VSCC [9]. Neuroprotective effects of this agent against cerebral ischemia are present in some reports, e.g. prevention of brain edema in Wistar rats [15] and attenuation of metabolic derangement in SHR [10]. There are two main mechanisms proposed for these neuroprotective effects; an improvement of CBF during ischemia by dilatation of cerebral arteries and an inhibition of the excessive influx of calcium through VSCCs in the neuron. Excessive neurotransmitters from presynaptic ternfinals play a role in the development of ischemic neuronal injury. Several investigators have revealed the critical role of calcium entry through N-type VSCCs in the release of dopamine by K + or electrical stimulation in the striatal slices [8]. In addition, an N-type VSCC blocker, soconotoxin, inhibits the release of dopamine induced by administration of nicotine or electrical stimuli [7]. We have also shown significant reductions in the concentration of extracellular dopamine by ~o-conotoxin both in the physiological state and during cerebral ischemia in vivo [15]. We speculate that N-type calcium channels are also essential for the ischemia-induced release of neurotransmitters. The involvement of L-type VSCCs is, however, controversial in neurotransmitter release. Nimodipine, diltiazem and verapamil, widely used L-type VSCC blockers,
did not alter the release of dopamine by nicotine, electrical stimulation or ischemia [2]. Meanwhile, Bay K 8644, a dihydropyridine agonist, potentiated the release of dopamine [20] and isradipine prevented the release of ischemia-induced dopamine [16]. In the present study, subcutaneous administration of isradipine significantly reduced the concentration of extracellular dopamine by 34-33% in the striatum during cerebral ischemia. Thus, we assume that calcium entry through L-type VSCCs is also involved in the massive release of dopamine during cerebral ischemia. Isradipine did not attenuate the release of glutamate. Glutamate increased rather gradually during cerebral ischemia although dopamine reached to the peak at 20 min of ischemia. Thus, we assume the mechanism of the release of glutamate may be different from dopamine. Several reports showed that the release of glutamate during ischemia is independent on calcium influx [6,11,12]. Rapid decline in ATP during anoxia is one of the factors for the inhibition of the calcium-dependent release [12]. Katayama et al. [11] revealed that glutamate was released by a calcium-dependent manner in the early phase of ischemia, and then, by a mainly calcium-independent mechanism. The other mechanism of the efflux of glutamate may be the impaired uptake in glial cell [191. Furthermore, glutamate exists in high amount in the cytoplasmic pool [18]. On the other hand, it is thought that the concentration of free dopamine is low in the cytosol, where it may be metabolized by enzymes and then taken up into the storage vesicles [18]. From these points of view, we assume that glutamate is released from vesicular and cytoplasmic pool and that dopamine is released mainly from vesicular pool during cerebral ischemia. This study was partly supported by a Research Grant for Cardiovascular Disease (2A-2) from the Ministry of
154
H. Nakane et al. /Neuroscience Letters 188 (1995) 151-154
H e a l t h a n d W e l f a r e o f J a p a n a n d t h e Social I n s u r a n c e A g e n c y C o n t r a c t F u n d c o m m i s s i o n e d to the J a p a n H e a l t h S c i e n c e s F o u n d a t i o n . I s r a d i p i n e w a s k i n d l y s u p p l i e d by S a n d o z Co. Ltd. (Basal, S w i t z e r l a n d ) . [1] Abe, K., Kogure, K. and Watanabe, T., Prevention of ischemic and postischemic brain edema by a novel calcium antagonist (PN200-110), J. Cereb. Blood Flow Metab., 8 (1988) 436-439. [2] Bentuf-Ferrer, D., Decombe, R., Saiag, B., Allain, H. and Van den Driessche, J., L-Type voltage-dependent calcium channels do not modulate aminergic neurotransmitter release induced by transient global cerebral ischaemia: an in vivo microdialysis study in rat, Exp. Brain Res., 93 (1993) 288-292. [3] Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. [4] Fujishima, M., Sugi, T., Morotomi, Y. and Omae, T., Effects of bilateral carotid artery ligation on brain lactate and pyruvate concentrations in normotensive and spontaneously hypertensive rats, Stroke, 6 (1975) 62-66. [5] Globus, M.Y.-T., Busto, R., Die~ch; W.D., Martinez, E., Valdrs, I. and Ginsberg, M.D,, Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric acid studied by intracerebral microdialysis, J. Neurochem., 51 (1988) 1455-1464. [6] Globus, M.Y.-T., Busto, R., Martinez, E., Vald6s, I. and Ginsberg, M.D., The effect of (s)-Emopamil, a novel calcium channel blocker, on the extracellular release of striatal glutamate and dopamine in a rat model of transient global ischemia. In Neurotransmission and Cerebrovascular Function I, Elsevier, Amsterdam, 1989, pp. 429-432. [7] Harsing, Jr., L.G., Sershen, H., Vizi, S.E. and Lajtha, A., N-Type calcium channels are involved in the dopamine releasing effect of nicotine, Neurochem. Res., 17 (1992) 729-734. [8] Herdon, H. and Naborski, S.R., Investigation of the roles of dihydropyridine and ~o-conotoxin-sensitive calcium channels in mediating depolarization-evoked endogenous dopamine release from striatal slices, Naunyn-Schmiedeberg's Arch. Pharmacol., 340 (1989) 36-40. [9] Hof, R.P., Scholtysik, G., Loutzenhiser, R., Vuorela, H.J. and Naumann, P., PN 200-110, a new calcium antagonist: electrophysiological, inotropic, and chronotropic effects on guinea pig myocardial tissue and effects on concentration and calcium uptake of rabbit aorta, J. Cardiovasc. Pharmacol., 6 (1984) 399-406.
[10] lbayashi, S., Sadoshima, S. and Fujishima, M., Calcium antagonist isradipine reduces metabolic derangement in acute cerebral ischemia in spontaneously hypertensive rats, J. Cereb. Blood Flow Metab., 11 (Suppl. 2) (1991) $717. [11] Katayama, Y., Kawamata, T., Tamura, T., Hovda, D.A., Becker, D.P. and Tsubokawa, T., Calcium-dependent glutamate release concomitant with massive potassium flux during cerebral ischemia in vivo, Brain Res., 558, 1991, 136-140. [12] Kauppinen, R.A., McMahon, H. and Nicholls, D.G., Ca 2+dependent and Ca2+-independent glutamate release, energy status and cytosolic free Ca2+-concentration in isolated nerve terminals following in vitro hypoglycemia and anoxia, Neuroscience, 27 (1988) 175-182. [13] Leach, M.J., Swan, J.H., Eisenthal, D., Dopson, M. and Nobbs, M., BW619C89, a glutamate release inhibitor, protects against focal cerebral ischemic damage, Stroke, 24 (1993) 1063-1067. [14] Nicholls, D.G. and Sihra, T.S., Synaptosomes possess an exocytotic pool of glutamate, Nature, 321 (1984) 772-773. [15] Ooboshi, H., Sadoshima, S., Yao, H., Nakahara, T., Uchimura, H. and Fujishima, M., Inhibition of ischemia-induced dopamine release by og-conotoxin, a calcium channel blocker, in the striatum of spontaneously hypertensive rats: in vivo brain dialysis study, J. Neurochem., 58 (1992) 298-303. [16] Ooboshi, H., Sadoshima, S., Ibayashi, S., Yao, H., Uchimura, H. and Fujishima, M., Isradipine attenuates the ischemia-induced release of dopamine in the striatum of the rat, Eur. J. Pharmacol., 233 (1993) 165-168. [17] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Tokyo, 1986. [18] Siegel, G., Agranoff, B., Albers, R.W. and Molinoff, P. (Eds.), Basic Neurochemistry, 4th edn., Raven Press, New York, 1989, pp. 236. [19] Szatkowski, M., Barbour, B. and Attwell, D., Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake, Nature, 348 (1990) 443-446. [20] Woodward, J.J. and Leslie, S.W., Bay K 8644 stimulation of calcium entry and endogenous dopamine release in rat striatal synaptosomes antagonized by nimodipine, Brain Res., 370 (1986) 397-400. [21] Yao, H., Sadoshima, S., lshitsuka, T., Nagao, T., Fujishima, M., Tsutsumi, T. and Uchimura, H., Massive striatal dopamine release in acute cerebral ischemia in rats, Experientia, 44 (1988) 506508. [22] Yao, H., Ooboshi, H., Ibayashi, S., Uchimura, H. and Fujishima, M., Cerebral blood flow and ischemia-induced neurotransmitter release in the striatum of aged spontaneously hypertensive rats, Stroke, 24 (1993) 577-580.
Neuroscience Letters 188 (1995) 151-154
ri[llill:[ IS[IS
Isradipine, a calcium channel blocker, attenuates the ischemia-induced release of dopamine but not glutamate in rats Hiroshi Nakane ,i,b,*, Hiroaki O o b o s h i a, Setsuro Ibayashi a, Hiroshi Y a o a, Seizo Sadoshima a, Masatoshi F u j i s h i m a a aSecond Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan bCenter for Emotional and Behavioral Disorders, Hizen National Mental Hospital, Saga, Japan Received 12 December 1994; revised version received 17 February 1995; accepted 20 February 1995
Abstract
This study was designed to investigate the role of the L-type voltage sensitive calcium channel blocker, isradipine, in the ischemiainduced release of neurotransmitters. Male spontaneously hypertensive rats were subjected to cerebral ischemia for 60 rain by bilateral carotid artery occlusion, and recirculated for 120 min. Isradipine (0.25 mg/kg n = 6) or vehicle (n = 6) was administered subcutaneously at 20 min before ischemia. In the striatum, cerebral blood flow was determined by the hydrogen clearance method and concentrations of extracellular dopamine and glutamate were measured by in vivo brain dialysis technique. Extracellular dopamine in the vehicle-treated group increased by 180-fold from the basal level, and glutamate by 24-fold during cerebral ischemia, lsradipine significantly attenuated the ischemic release of dopamine to 33-34% (P < 0.05) of the vehicle group, while it did not affect glutamate release. It is suggested that the release mechanism of dopamine and glutamate during cerebral ischemia may be different, especially in the dependence on the L-type calcium channels.
Keywords: Cerebral ischemia; Isradipine; Dopamine; Glutamate; Voltage sensitive calcium channel
Excessive release of neurotransmitters is known to play an important role in the development of ischemic cell damage. The ischemia-induced massive release of dopamine [5], glutamate [3] causes neuronal death probably due to an excitotoxic process. The major pathway of the excitotoxicity is believed to be the entry of extracellular calcium into ischemic neuronal cells. Isradipine, an L-type voltage sensitive calcium channel (VSCC) blocker, is one of the most powerful vasodilators, and is expected to increase cerebral blood flow (CBF) in the ischemic brain. In addition, isradipine seems to possess neuroprotective effects against cerebral ischemia by inhibiting the entry of excessive amount of calcium into neurons [1,10]. There are reports that neuroprotective effects of several drugs against cerebral ischemia are caused by attenuation of the release of excitatory amino acids from presynaptic neurons [13]. Recently, we showed partial inhibition of ischemia-induced release of dopamine by an * Corresponding author, Tel.: +81 92 6411151; Fax: +81 92 6322551.
infusion of isradipine into the striatum through a microdialysis probe [16]. Isradipine effectively attenuates the ischemic damage by local or systemic administration [1,10]. We speculate that such a favorable,, action of isradipine is partly attributable to the attenuation of an excessive release of these excitatory neurotransmitters. The aim of this study is to examine the role of L-type VSCC and its blocker, isradipine, in the release of dopamine and glutamate during cerebral ischemia. Seventeen male spontaneously hypertensive rats (SHRs), aged 7-9 months and weighing 350-450 g, were used. Just before each experiment, isradipine was dissolved in the vehicle (ethanol/polyethylene glycol 400, 1:1) and 0.25 mg/kg of solution was made in a dark room. The rats were anesthetized with amobarbital (100 mg/kg i.p.), and allowed to breathe spontaneously. Cerebral ischemia was developed by bilateral carotid artery occlusion as described previously [4]. Body temperature was maintained at 37°C using a heat lamp. Femoral arteries were cannulated for anaerobic sampling of blood and continuous recording of arterial blood pres-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 4 1 7 - R
152
14. Nakane et al. / Neuroscience Letters 188 (1995) 151-154
sure. Twenty minutes after drug injection, both carotid arteries were tightly ligated for 60 rain and then reopened for 120 min to allow recirculation. Extracellular dopamine and glutamate, 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 [15,16,21,22]. A dialysis probe, 5 0 0 # m in outer diameter with a 3-ram dialysis membrane and a Teflon-coated platinum electrode attached with thermometer; 200 ktm in diameter with a l-ram uncoated portion at its tip, for CBF and brain temperature determination, were placed stereotaxically in the right striatum; 0.5 mm anterior and 3.0 mm lateral to the bregma and 4.5 mm in depth from the surface of the brain according to the atlas of Paxinos and Watson [17]. The striatum was perfused with Ringer solution through the dialysis probe at a rate of 4.0ktl/min and the perfusates were collected every 20 rain into a plastic tube. After determination of two baselines CBF and three concentrations of dopamine and glutamate during 60 min of resting period, isradipine was administered subcutaneously. Control rats (n = 6) received vehicle. CBF was measured at 20 rain after drug injection, 20 and 60 rain of ischemia, and 10 and 120 min of recirculation. Arterial blood was sampled during the control period, at 20 min after drug infusion, 60 min of ischemia, and 120 rain of recirculation. The position of the dialysis probe and CBF electrode was macroscopically determined after the experiment. The HPLC systems for dopamine and glutamate were described previously [15,22]. In each group, the concentrations of dopamine and glutamate in the last three samples during the resting period were referred to the basal values. The statistical differences in physiological parameters, mean arterial blood pressure, and CBF within or among groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test. The differences in the concentrations of dopamine and glutamate among the groups were also analyzed by one-way A N O V A followed by Scheffe's Ftest. All the values are presented as mean + SEM.
There were no significant differences in blood gases and pH between the groups during the experiment~ All rats showed respiratory alkalosis (pH 7.48 _+0.03, pCO 2 28.2 + 3.7, pO2 97.3 + 2.0 in the vehicle-treated group and 7.49 _+0.03, 26.5 + 3.8, 101.0 -+ 3.8 in the isradipinetreated group) during ischemia, but blood gases and pH returned to the control level after 120 rain of recirculation. Brain temperatures in the striatum at 60 min of ischemia were 34.5+_0.4°C in the vehicle group, 34.3 _+0.1 °C in the isradipine group. The changes in mean arterial blood pressure and CBF to the striatum are given in Table 1. Mean arterial blood pressure markedly fell after isradipine administration; from 185 _+5 to 122 + 6 m m H g (P < 0.01 versus at rest). These hypotensive changes lasted until the end of the experiment. Resting CBF were actually same between the groups; 48.5 _+6.5 ml/100 g per min in the vehicle group, 43.3 + 4.3 in the isradipine group. After administration of isradipine, CBF to the striatum significantly increased to 130% (P < 0.05) of the basal levels. During ischemia, CBF in each rat markedly decreased to less than 10% of the basal level. Although CBF in the vehicle group remained 68% of the basal level at 120 rain of recirculation, isradipine groups obtained considerable recovery of CBF during recirculation and revealed significantly higher CBF (169% at 120 min, P < 0.05) than the vehicle group. The averaged basal concentrations of dopamine and glutamate were 10.5 + 3.6 pg/20 rain and 3.9 -+ 1.6~M/ 20 min, in the vehicle group, 7.8 + 3.2 and 3.8 + 0.3, in the isradipine group. These variables were not significantly different between the groups. Administration of isradipine did not alter the concentrations of dopamine and glutamate under the resting condition. After induction of ischemia, concentrations of extracellular dopamine markedly increased within 20 rain by 180-fold of the basal value in the vehicle group, by l l0-fold in the isradipine group, without significant difference between the groups. Thereafter, concentrations of dopamine gradually decreased in all groups. In rats treated with isradipine, however, the level of dopamine was significantly smaller than the vehicle, resulting in 33% (P < 0.05) of the vehicle at 40 rain and 34% (P < 0.05) at 60 min. The elevated
Table 1 Time course of mean arterial blood pressure (MABP) and striatal cerebral blood flow (CBF) Rest
Vehicle (n = 6) MABP (mmHg) CBF (% of basal value) lsradipine, 0.25 mg/kg (n = 6) MABP (mmHg) CBF(% ofbasal value)
Drug
Recirculation
Ischemia 20 min
60 rain
10 min
190 _+4 100
193 _+4 88.5_+8.3
204 _+8 16.3+-4.3
185 -+7 5.0+2.0
171 _+6 112.0-+33.2
185 -+5 100
122 -+6** 130.4_+ 10.7"
99 -+9** 19.2+6.2
94 -+9** 8.4_+4.3
94 -+6** 147.1 _+38.6
120 rain 156 _+13 68.7+11.1 104 -+8* 168.9_+21.2"*
Values are mean _+SEM. MABP, mean arterial blood pressure. Resting CBF values are present in the text. **P < 0.01, *P < 0.05 versus vehicle.
153
H. Nakane et al. / Neuroscience Letters 188 (1995) 151-154
DOPAMINE
GLUTAMATE
14 -~
2000
~
.~
1500
.~
10
~
6
~
4
1000
I t
t'
-60-40-20
0
0 o...Q.,..,o~-.o.....o 20 40
Drug Ischemia
20 40 60 80
100 120
(rain)
Reeirculation
vehicle
isradipine 0.25mg/kg
: .~;~.
0 !-q, - q , 0
~ O....
-60-40-20
. . . . . . . . 0
20 40
0
Drug Ischemia
20 40 60 80
100120 (ram)
Recirculation
Fig. l. Changes in the concentration of extracellular dopamine and glutamate. At 40 and 60 rain of ischemia, significant reduction of elevated dopamine was determined by isradipine. There was no difference in the concentration of glutamate among the groups at 60 rain of ischemia. Values are mean _+SEM. **P < 0.01, *P < 0.05 versus vehicle. concentration of dopamine returned to the pre-ischemic levels at 40 rain of recirculation in both groups. Concentrations of glutamate, in contrast to dopamine, progressively increased and reached a peak at 60 min of ischemia; 24-fold of basal value in the vehicle group, 35-fold in the isradipine group. Elevated glutamate rapidly returned to the pre-ischemic level at 40 rain of recirculation. There were no significant difference between isradipineand vehicle-treated group (Fig. 1). Isradipine, a dihydropyridine-derivative, potently blocks the L-type VSCC [9]. Neuroprotective effects of this agent against cerebral ischemia are present in some reports, e.g. prevention of brain edema in Wistar rats [15] and attenuation of metabolic derangement in SHR [10]. There are two main mechanisms proposed for these neuroprotective effects; an improvement of CBF during ischemia by dilatation of cerebral arteries and an inhibition of the excessive influx of calcium through VSCCs in the neuron. Excessive neurotransmitters from presynaptic ternfinals play a role in the development of ischemic neuronal injury. Several investigators have revealed the critical role of calcium entry through N-type VSCCs in the release of dopamine by K + or electrical stimulation in the striatal slices [8]. In addition, an N-type VSCC blocker, soconotoxin, inhibits the release of dopamine induced by administration of nicotine or electrical stimuli [7]. We have also shown significant reductions in the concentration of extracellular dopamine by ~o-conotoxin both in the physiological state and during cerebral ischemia in vivo [15]. We speculate that N-type calcium channels are also essential for the ischemia-induced release of neurotransmitters. The involvement of L-type VSCCs is, however, controversial in neurotransmitter release. Nimodipine, diltiazem and verapamil, widely used L-type VSCC blockers,
did not alter the release of dopamine by nicotine, electrical stimulation or ischemia [2]. Meanwhile, Bay K 8644, a dihydropyridine agonist, potentiated the release of dopamine [20] and isradipine prevented the release of ischemia-induced dopamine [16]. In the present study, subcutaneous administration of isradipine significantly reduced the concentration of extracellular dopamine by 34-33% in the striatum during cerebral ischemia. Thus, we assume that calcium entry through L-type VSCCs is also involved in the massive release of dopamine during cerebral ischemia. Isradipine did not attenuate the release of glutamate. Glutamate increased rather gradually during cerebral ischemia although dopamine reached to the peak at 20 min of ischemia. Thus, we assume the mechanism of the release of glutamate may be different from dopamine. Several reports showed that the release of glutamate during ischemia is independent on calcium influx [6,11,12]. Rapid decline in ATP during anoxia is one of the factors for the inhibition of the calcium-dependent release [12]. Katayama et al. [11] revealed that glutamate was released by a calcium-dependent manner in the early phase of ischemia, and then, by a mainly calcium-independent mechanism. The other mechanism of the efflux of glutamate may be the impaired uptake in glial cell [191. Furthermore, glutamate exists in high amount in the cytoplasmic pool [18]. On the other hand, it is thought that the concentration of free dopamine is low in the cytosol, where it may be metabolized by enzymes and then taken up into the storage vesicles [18]. From these points of view, we assume that glutamate is released from vesicular and cytoplasmic pool and that dopamine is released mainly from vesicular pool during cerebral ischemia. This study was partly supported by a Research Grant for Cardiovascular Disease (2A-2) from the Ministry of
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H. Nakane et al. /Neuroscience Letters 188 (1995) 151-154
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