ELSEVIER
Neuroscience Letters 191 (1995) 99-102
CCK, receptor activation protects CA1 neurons from ischemia-induced dysfunction in stroke-prone spontaneously hypertensive rats hippocampal slices Mitsuru Yasui, Kazuo Kawasaki* Division ofPharmacology,
Developmental Research Laboratories, Shionogi Received 16 February 1995;
& Co., Ltd., 3-I-l Futaba-cho, Toyonaka,
Osaku 5 6 1 , J a p a n
revised version received 10 April 1995; accepted 10 April 1995
Abstract
We examined the effect of cholecystokinin octapeptide sulfated type (CCK-8s) on dysfunction of CA1 pyramidal neurons induced by in vitro ischemic insult in hippocampal slices of stroke-prone spontaneously hypertensive rats (SHRSP). CCK-8S shortened the time required for partial recovery from block of a population spike produced by ischetnia. Furthermore. CCK-8S reduced ischemic insultinduced accumulation of K+ in extracellular space. Suppression of the K+ conductance by the CCKn receptor activation is suggested to contribute to neuroprotection by CCK-8s. Cholecystokinin octapeptide (CCK-8); Ischemic spontaneously hypertensive rats (SHRSP) Keywords:
Cholecystokinin octapeptide sulfated form (CCK-8s) is one of the brain-gut peptides and is distributed in both the periphery and central nervous system (CNS) [25], being particularly highly concentrated in the hippocampus [4]. This peptide has pleiotropic actions in the brain [14,17], Recently, Eigyo et al. [8] have shown that a potent analogue of CCK-8S, ceruletide, whose pharmacological and binding profiles are similar to CCK-8S [9,10,29], prevents hyperactivity, amnesia and selective neuronal cell death in the hippocampal CA1 region induced by bilateral occlusion of the gerbil common carotid arteries. CCK-8S and ceruletide also reduce glutamateinduced cell death in cultured neurons [2,18]. However, the neuroprotective effect of CCK-8S in these in vitro studies was observed only in cortical neurons. Moreover, peripherally applied CCK-8S lowers body and brain temperature [lo]; it should be noted that hypothermia by itself can prevent neuronal damage induced by ischemia [6]. Thus, it remains uncertain whether CCK-8S indeed rescues hippocampal neurons from ischemia-induced dysfunction.
* Corresponding author, Tel.: +81 6 331 8081; Fax:
Q 1995 Elsevier 0304-3940/95/$09.50 SSDI 0304-3940(95)11570-J
Science
+81 6 332 6385.
Ireland
Ltd. All
damage;
CA1
pyramidal
neuron; Hippocampal slice; Stroke-prone
Stroke-prone spontaneously hypertensive rats (SHRSP) have been regarded as an animal model of ischemic stroke in humans [ 10,151. Occlusion of bilateral common carotid arteries of SHRSP causes severe neuronal cell death in the hippocampus, compared with that in normotensive strain, Wistar-Kyoto rats [9]. Recently, we demonstrated that hippocampal CA1 pyramidal neurons of SHRSP are natively vulnerable to in vitro ischemic insult [28]. This vulnerability is suggested to be due, at least in part, to some abnormality in K+ channels of SHRSP hippocampal neurons. In addition, L-type Ca2+ channel blockers at submicromolar concentrations were found to show neuroprotective effects in the SHRSP hippocampal slices. These results prompted us to examine how CCK-8S affects dysfunction of the CA1 pyramidal neurons induced by ischemic insult in SHRSP hippocampal slice preparations. Transverse hippocampal slices (450 pm thick) were prepared from male SHRSP (78-88 days of age) bred in Aburahi Lab., Shionogi & Co., Ltd. The slice was perfused with artificial cerebrospinal fluid (aCSF) at 3435°C in a submersion recording chamber (flow rate 5 ml/min). The aCSF saturated with 95% O2 and 5% CO2 contained (mM): NaCl, 124; KCl, 3.5; NaH2P04, 1.24;
rights reserved
100
M. Yasui, K. Kawasaki i Neuroscience Letters 191 (1995) 99-102
MgSO,, 1.2; CaC12, 2.4; NaHCOs, 26; glucose, 10. Hypoxic and hypoglycemic (referred to below as ischemic) insult for 6 min was created by perfusion of aCSF containing low levels of glucose (2.5 mM), saturated with 95% N2 and 5% COZ. The evoked field potentials and the ground-referenced extracellular DC level were extracellularly recorded from the CA1 pyramidal cell layer with a glass micropipette of 2.5pm tip diameter filled with 2 M NaCl. Stimuli was applied to the Schaffer collateral/cornmissural fibers with a tungsten monopolar electrode at 0.1 Hz. Evoked potentials were digitized, and the population spike (PS) amplitude was calculated according to the method of Aitken [ 11. The stimulus strength was adjusted to evoke the PS with half-maximal amplitude throughout the experiment. The mean amplitude of 90 PSs recorded 15 min prior to the ischemic insult was taken as a control (100%). The extracellular K+ concentration ([K+],) was simultaneously measured with doublebarreled microelectrodes made of valinomycin (Sigma, St. Louis, MO). The field potential was recorded via the reference barrel of the microelectrode placed at a neighboring region. The preparation and calibration of the microelectrode were performed as described by Somjen
WI.
CCK-8S (Peptide Ltd.) was dissolved in distilled water. L364,718 was a gift from Merk & Co., Ltd. L365,216 was synthesized at Shionogi Research Laboratories and its quality was confirmed by binding and pharmacological studies. These two ligands were dissolved in dimethyl sulfoxide (DMSO) and diluted with aCSF (the final concentration of DMSO being 0.01%).
A
--mv 2
a,
The drug-free aCSF contained 0.01% of DMSO. Bach drug was applied for 36 min, starting 15 min before the ischemic insult. As described previously [27], the PS rapidly vanished during the ischemic insult (Fig. lAbi), but the multiple PSs were evoked transiently a few minutes after the disappearance of PS (Fig. lA,Bci). This transient event was termed the hyperexcitable period. The transient hyperexcitability was immediately followed by a sudden large negative shift of the DC level, spreading depression (S’D)like depolarization [22] (Fig. lAdi) and the PS disappeared again (Fig. lA,Bdi,e,). CCK-8S at a concentration of 10d7 M markedly reduced the generation of SD-like depolarization (Fig. lCd& and shortened the time required for partial recovery of the PS (Fig. 1C,De2), indicating protection of ischemia-induced CA1 neuronal dysfunction by CCK-SS. The effects of CCK-8s (10-8-10-6 M) on the alterations induced by ischemia are summarized in Table 1. First, the maximal negative shift of DC level was reduced. Second, the median of the time required for 25% recovery of the PS (T+,), an index of recovery from neuronal dysfunction, was shortened by CCK-8s. These reductions of maximal negative shift of DC level and ?“i, by CCK-8S were observed at doses larger than 1O-7 M. The CCK receptor can be classified pharmacologically into CCK, and CCK, subtypes 17,201. Coneurrent application of a selective CCKu receptor antagonist, L365,260 (10m6 M), significantly attenuated the protective effects of CCK-8s (10s7 M), whereas a selective CCKA receptor a n t a g o n i s t , L364,718 (10e6M) failed to affect the
I3
b, ci 4
1
1
1
0
-1 -2
-1 -2
111111111 I I 0 5
Ischemic Insult
I 10
I 15
I 20
Time after Ischemic Insult (min) Fig. I. Changes of electrical responses induced by ischemic insult, and their protection by CCK-8S in the SHRSP hippocampa! slice. (A) Time course of the changes in the PS amplitude (. ..a) and DC level (-) recorded in CA1 pyramidal cell layer. **mm’ shows the period of ischemic insult (6 mm). (B) Each wave-form (al - el) is recorded at the time indicated by the arrow in (A). Dot shows stimulus artifact. (CD) The same as in (A) and (B), respectively, except for the bath application of CCK-8S (10F7 M).
M. Yami, K . K a w a s a k i
I Neuroscience L e t t e r s 191(1995) 99-102
Table 1 The effects of CCK-8S and its antagonists on the alterations induced by ischemic insult in SHRSP hippocampal slices
Without drug CCK-8S 1O-8 M 1O-7 M 10-6M CCK-8S 1O-7 M + L365,260 1O-6 M CCK-8S 1O-7 M + L364,718 10-O M
Maximal negative shift of DC level (mV
T1/4 (midb
-1.9 f 0.3 -2.3 + 0.1 -0.9 f 0.4* -0.7 -r- 0.7* -2.2 + 0.5#
3 8 (9-60<) 46 (18-60~) 7+ (5-18) 11+ (4-19) 51$ (l&60<)
-1 .o + 0.4*
9+ (S-21)
a Mean + SEM. b Median (interquartial range). * P c 0.05 versus without, #I’ < 0.05 versus with CCK-8S 10m7 M (Duncan’s multiple range test). +P<0.05 versus without, *P
CCK-8s action (Table 1). Apparently, the neuroprotective effect of CCK-8S is mediated by the CCKn receptor. How might CCK-8S protect CA1 neurons from ischemia-induced dysfunction? To address this question, we examined the changes in [K+], levels produced by ischemic insult in the presence and absence of CCK-8s. An ischemic insult for 6 min caused marked elevation of [K+], (more than 15 n&I) [26] and resulted in severe dysfunction of synaptic responses. This hampered repeat of the ischemic insult in the same preparation. Thus, a shortterm ischemic insult (3 min) was repeatedly applied. As shown in Fig. 2, CCK-8S (low7 M) reduced the increase in [K+], to 61 it 8% (n = 4) of control. The present study demonstrates that CCK-8s can prevent ischemia-induced dysfunction of the CA1 pyramidal neurons of SHRSP hippocampal slices via activation of CCKn receptors. This protective action might be achieved by reducing the accumulation of [K+],. K+ accumulates in the extracellular space during ischemic insult in hippocampal slices [13]. Extracellular accumulation of K+ re-
sults in neuronal depolarization. Excess K+ accumulation is suggested to lead to SD-like depolarization, which is presumably responsible for the ensuing neuronal damage [22]. CCK-8s was found to attenuate the accumulation of [K+], (Fig. 2), thereby inhibiting the generation of SDlike depolarization (Table 1). We previously showed that hippocampal neurons in SHRSP are more sensitive to [K+], in the perfusate and more excitable than those in Wistar-Kyoto rats 1281. CCK-8s is reported to inhibit some voltage-dependent K+ currents in cultured hippocampal neurons [5]. This K+ channel-blocking action of CCK-8S might be related to reduced K+ accumulation in SHRSP hippocampal slices. Recently, CCKn receptor is shown to be a G-proteincoupled metabotropic receptor by cloning study [27]. In addition to closing some K+ channels [5], the activation of CCKa receptors result in stimulation of phosphoinositide hydrolysis and the rise in intracellular Ca2+ level [26]. This is reminiscent of metabotropic glutamate receptors, activation of which increases intracellular Ca2+ levels [ 191 and inhibits K+ currents [3,24]. Moreover, a metabotropic glutamate receptor agonist, (lS,3R)-ACPD, also protects CA1 neurons from ischemic insult [21]. It is tempting to speculate that a reduction of [K+], or an increase in intracellular Ca2+ or both may be critical for protection from ischemia-induced neuronal dysfunction. We thank Dr. Hiroshi Miyazaki for detailed instruction in preparing K+-sensitive electrodes. We are grateful to Dr. Motoi Kuno for critically reading the manuscript. 111 Aitken, P.J., Kainetic acid penicillin: differential effects on excita121
[31
[41 __
5.5 -
1st
- 2nd _.____ 3rd
5-
2 4.5 s 0 4 g 3.5 ’ /-. q-e-
151
161 . ..m....*....... lschemic Insult
3/
I
0
I
I
I
t
Time after Ischemic Insult (min)
I
I
1
5
Fig. 2. Suppression of ischemia-induced [K+lo rise by CCK-IS. Ischemic insult was applied three times with or without CCK-IS (10e7 M) at an interval of 40 mm. The 1st ischemic insult was applied without CCK-8s. The 2nd insult was applied during the application of CCK-IS. The 3rd insult was applied 60 mm after withdrawal of CCK8s.
101
[71
181
tory and inhibitory interactions in the CA1 region of the hippocampal slice, Brain Res., 325 (1985) 261-269. Akaike, A., Tamura Y., Sato, Y., Gzaki, K., Matsuoka, R., Miura, S. and Yoshinaga T., Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity, Brain Res., 557 (1991) 303-307. Baskys, A., Bernstein, N.K., Barolet, A.W. and Carlen, P.L., NMDA and quisqualate reduce a Ca-dependent K+ current by a protein k&se-mediated mechanism, Neurosci. Lett. 112 (1990) 76-81. Beinfeld, M.C. and Palkoviz, M., Distribution of cholecystokinin (CCK) in the hypothalamus and limbic system of the rat. Neuropeptide, 2 (1981) 123-129. Buckett, K.J. and Saint, D.A., Cholecystokinin modulates voltage dependent K’ currents in cultured rat hippocampal neurons, Neurosci. Len., 107 (1989) 162-166. Busto R., Dietrich, W.D., Globus, M.Y.-T., Valdes, I., Scheinberg, P. and Ginsberg, M.D., Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cerebral Blood Flow Metab., 7 (1987) 729-738. Chang, R.S.L., Lotti, V.J., Chen, T.B. and Kunkel, K.A. (1986) Characterization of the binding of [3H]-(+)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors, Mol. Pharmacol., 30, 212-217. Eigyo, M., Katsuura, G., Shintaku, H., Shinohara S., Katoh, A., Shiomi, T. and Matsushita, A., Systemic administration of a cholecystokinin analogue, cernletide, protects against ischemiainduced neurodegeneration in gerbils, Eur. J. Pharmacol., 214 (1992) 149-158.
102
A4. Yasui, K.
Kawasaki I Neuroscience Letters 191 (1995) 99402
[9] Erspamer, V., Bertaccini, G., De Caro, G., Endean, R. and Impicciatore, M., Pharmacological action of caerulein, Experientia, 23 (1967) 702-703. [IO] Fujimoto, M., Igano, K.. Watanabe, K., Irie, l., Inouye, K. and Okabayashi, T., Effects of caerulein-related peptides on cholecystokinin receptor bindings in brain and pancreas, Biochem. Pharmacol., 34 (1985) 1103-l 107. [11] Gemba, T., Matsunaga, K. and Ueda, M., Changes in extracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats, Neurosci. Lett., 135 (1992) 184-188. [12] Hanada, H., Stroke-prone and arteriolipidosis-prone SHR (YAMORI) as models for stroke and cerebrovascular atherogenesis in man. In spontaneous hypertension, DHEW Publ. No. (NIH), 394 (1977) 77-1179. [13] Hansen, A.J., Hounsgaard, J. and Jahnsen, H., Anoxia increase potassium conductance in the hippocampal nerve cells, Acta. Physiol. Stand., 11.5 (1982) 301-310. [14] Hughes, J., Boden, P., Costall, B., Domeney, A., Kelly, E., Horwell, D.C., Hunter, J.C., Pinnock, R.D. and Woodruff, G.N., Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity, Proc. Natl. Acad. Sci. USA, 87 (1990) 6728-6732. [15] Kakihana, M., Shino, A. and Nagaoka, A., Cardiovascular responses to cerebral ischemia following bilateral carotid artery occlusion in SHRSP, SHRSR and WKY rats, Jpn. J. Pharmacol., 33 (1983) 17-26. [I61 Kapas, L., Obal, Jr., F., Alfoldi, P., Rubiesek, G., Penke, B. and Obal, F., Effects of nocturnal intraperitoneal administration of cholecystokinin in rats: simultaneous increase in sleep, increase in EEG slow-wave activity, reduction of motor activity, suppression of eating, and decrease in brain temperature, Brain Res., 438 (1988) 155-164. [17] Katsuura, G. and Itoh, S., Potentiation of beta-endorphin effects by proglumide in rats, Eur. J. Pharmacol., 107 (1985) 365-366. [18] Katsuura, G. Shinohara, S., Shintaku, H., Eigyo, M. and Matsushita, A., Protective effect of CCK-8 and ceruletide on glutamate-induced neuronal cell death in rat neuron cultures: possible involvement of CCK-B receptors, Neurosci. Lett., 132 (1991) 159-162.
[19] Mayer, ML. and Miller, R.J., Excitatory ammo acid receptors, second messengers and regulation of intracellular Ca2+ in mammalian neurons, Trends Pharmacol. Sci., I I (1991) 254-260. [20] Miceli, M.O. and Steiner, M., Novel locabzations of central- and peripheral-type cholecystokinin binding sites in Syrian hamster brain as determined by autoradiography, Eur. J. Pharmacol., 169 (1989) 215-224. [21] Opitz, T. and Reymann, K.G., (lS,3R)-ACPD protects synaptic transmission from hypoxia in hippocampal slices, Neuropharmacology, 32 (1993) 103-104. [22] Somjem, G.G., Aitken, P.G., Balestrino, M., Herreras, 0. and Kawasaki, K. Spreading depression-like depolarization and se!ective vulnerability of neurons: a brief review, Stroke, 21 (suppl. III) (1990) 179-183. 1231 Somjen, G.G., The why and how of measuring the activity of ions in extracellular fluid of spinal cord and cerebral cortex. In Zeuthen (Ed.), The Application of Ion-Selective Microelectrodes, Elsevier/North-Holland Biomedical Press, Amsterdam, 198 L, pp. 175-193. 1241 Stratton, K.R., Worley, P.F. and Baraban, J.M., Excitation of hippocampal neurons by stimulation of glutamate Qp receptors, Eur. J. Pharmacol., 173 (1989) 235-237. [2.5] Vanderhaeghen, J.J., Signeau, J.C. and Gepts, W., New peptide in the vertebrate CNS reacting with antigastrin antibodies, Nature, 257 (1975) 604-605. [26] Wakui, M., Kase, H. and Petersen, OH., Cytoplasmic Ca2’ signals evoked by activation 01 cholecystokinin receptors: Ca’+dependent current recording in internally perfused pancreatic acinarcelis, J. Membr. Biol., 124 (1991) 179-187. 1271 Wank, S.A., Pisegna, J.R. and de Weerth, A., Brain and gastrointestinal cholecystokinin receptor family: structure and functional expression, Proc. Nat1 Acad. Sci. USA, 89 (1992) 8691-8695. [28] Yasui, M. and Kawasaki, K., Vulnerability of CA1 neurons m SHRSP hippocampal slices to ischemia, and its protection by Ca2+ channel blockers, Brain Res., 642 (1994) 146-152. [29] Zelter, G., Analgesia and ptosis caused by caerulein and cholecystokinin octapeptide (CCK-8), Nemopharmacology, 19 (1980) 415-422.