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Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord
Neuroscience Letters, 83 (1987) 173-178 Elsevier Scientific Publishers Ireland Ltd.
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Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord R.E. Rodriguez*, R.G. Hill a n d J. H u g h e s Parke-Davis Research Unit, Addenbrooke Hospital Site, Cambridge ( U.K.)
(Received 16 June 1987; Revised version received 10 August 1987; Accepted 14 August 1987) Key words: Cholecystokinin; ?-Aminobutyric acid; In vivo release; Desensitization
The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (I nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK:8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10/zM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.
Cholecystokinin octapeptide (CCK(26-33)) is the most abundant peptide in the human central nervous system (CNS) and this octapeptide is present throughout the CNS of a number of mammalian species [1, 6]. It is found in high concentration in synaptosomes [7], from which it can be released in a calcium-dependent manner [9]. When applied to the hippocampus, CCK has an excitatory action [8], and similar effects have been noted in the medulla [18] and in the dorsal horn of the spinal cord [11, 19]. These observations are consistent with the proposal that CCK may act as a neurotransmitter substance within the CNS. It has recently been suggested t h a t s o m e o f the spinal c o n t e n t o f C C K , n a m e l y that f o u n d in p r i m a r y sensory neurones, m i g h t in fact be a n o t h e r peptide, calcitonin generelated p e p t i d e ( C G R P ) [12]. It is p r o b a b l e t h a t at least s o m e o f ' t h e C C K in spinal cord, p a r t i c u l a r l y t h a t c o n t a i n e d in n e u r o n e s intrinsic to the C N S , in genuine C C K , however, as p r e c u r s o r p e p t i d e for C C K p r o d u c t i o n has been l o c a t e d there [2]. T h e m o s t i m p o r t a n t issue f r o m the s t a n d p o i n t o f the p r e s e n t s t u d y is t h a t there is clear evidence for f u n c t i o n a l C C K r e c e p t o r s within the spinal c o r d [11]. *Present address: Dept. of Biochemistry, Faculty of Medicine, University of Salamanca, 37007 Salamanca, Spain. Correspondence: R.G. Hill, Parke-Davis Research Unit, Addenbrookes Hospital Site, Hills Road, Cambridge CBQ 2QB, U.K. 0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd,
174
It has been shown that CCK, when given either peripherally or centrally to animals, produces antinociception under a variety of experimental conditions [13]. As CCK is capable of producing antinociception but has predominantly excitatory actions on single neurons, it may be acting to excite inhibitory interneurones. Such a mechanism has been suggested to explain the antinociceptive action of substance P [18], and other peptides. ~-Aminobutyric acid (GABA) is believed to act as transmitter of both pre- and postsynaptic inhibition at the level of the spinal cord [15, 16, 18] and in higher centres [10]. An interaction of CCK with the GABA release mechanism may help to explain the antinociceptive actions of CCK. We have accordingly examined the effects of CCK on the releases of GABA from spinal cord neurones 'in vivo'. Experiments were performed in male Wistar rats weighing 500-700 g under urethane (1.25 mg/kg) anaesthesia. The animals were artificially ventilated through a tracheal cannula. The heart rate and blood pressure were continuously monitored, and the central temperature was kept constant by means of an homeothermic blanket system. For detailed methods see legend to Fig. l. The baseline release of [3H]GABA showed an exponential fall during the course of the experiment. Both CCK-8 and high potassium-medium released [3H]GABA without changing the rate of washout of [14C]suerose, which was included as a space marker (Fig. l). The rate of onset of the CCK-8 evoked response was similar to that of the response evoked by perfusion with high potassium. It is likely that the release of [3H]GABA is due to a direct spinal effect since the evoked release of [3H]GABA occurred soon after beginning the CCK-8 perfusion. Removal of Ca 2+ from the perfusate accompanied by raising the concentration of Mg 2+ to 4 mM did not affect the baseline release of [3H]GABA but markedly diminished the CCK-8-induced (10 /tM) release of [3H]GABA, and the effect produced by 50 mM K + was completely abolished (not shown). The lowest concentration of CCK-8 in the perfusate found to produce an effect on [3H]GABA release was 1 nM. Increasing concentrations of CCK-8 in the perfusate thereafter produced a progressive loss of response (Fig. 3A). This phenomenon was not a result of exhaustion of [3H]GABA in the tissue, since [3H]GABA was released from the same animals when high potassium (50 mM) artificial cerebrospinal fluid (ACSF) was subsequently superfused. Desensitization of electrophysiological responses to CCK has been shown in cultures of spinal neurones [17], in hippocampal CA1 neurones [7] and in ventromedial hypothalamic neurones [14], although these findings are not universal [3]. We do not have an explanation for the desensitization to the transmitter releasing action of CCK in our experiments. This effect lasted some three or more hours and such prolonged desensitization is unusual for classical postsynaptic receptor desensitization. The phenomenon may therefore involve other mechanisms although, as mentioned above, probably not transmitter depletion. It should be noted that release of radiolabel and not of endogenous GABA is being studied. The addition of substance P (SP; 10 #M) to the superfusate had no effect on the [3H]GABA release when applied twice in 4 animals (Fig. 2). In the same animals,
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Fig. 1. Release of [3H]GABA from superfused rat spinal cord in response to (A) CCK administration (10 /+M) in the perfusate, (B) high potassium (50 mM) in the perfusate. To perfuse the spinal space, an exposure of the cisterna magna was made by a midline incision and blunt dissection, followed by incision of the dura and arachnoid membranes. A piece of 10 cm of polyethylene tubing (0.28 mm i.d., 0.61 mm o.d., Portex) was advanced down the subarachnoid space to the lumbar area [20]. The tip of this tubing served as an inflow cannula and ACSF was pumped in at a rate of 110 pl/min with a peristaltic pump (Gilson). The ACSF was withdrawn with another polyethylene tube positioned at the opening of the cisterna magna at a rate of 100 pl/min using a second peristaltic pump. Before entering the animal's body, the perfusion fluid was warmed (37 + 5°C) and was continuously oxygenated (95% Oz-5% CO2). Normal ACSF medium consisted of (in mM): NaCI 134, KC1 5, KH2PO4 1.25, MgSO4 2, CaCI2 2, NaHCO3 16, glucose 10. To study the effect of high potassium on [3H]GABA release, the concentration of potassium chloride was raised to produce a final concentration of 50 mM K + while a corresponding amount of NaCI was removed from the ACSF solution to minimize undesirable osmotic effects. Experiments were conducted by first flushing the subarachnoid space with ACSF for 1 h to allow stabilization of the preparation, then incubating the cord with 6.6/zM [3H]GABA (2.2 x 106 dpm) and 3.6 x 10-4 M [14C]sucrose (4.4 x 106 dpm) for a further 1 h. The rats were pretreated with 40 mg/kg amino-oxyacetic acid, 4 h before the incubation with [3H]GABA to prevent metabolism of the amino acid. After the incubation period, the cord was again perfused with ACSF for 50 min, then appropriate test substances were added to the perfusate and 5-rain fractions (500/zl) were collected. The radioactive content of the superfusate was determined by liquid scintillation spectrometry after adding 6 ml of CP-scintillation fluid (Beckman) and 50/ll formic acid to suppress chemiluminescence. For the statistical analysis of data Student's t-test was used. The figure shows the change in dpm × 104 after introducing CCK-8 (10/zM) in the perfusing medium for 0.5 h (A) (open bar) and after perfusing with high potassium (50/zM) for 0.5 h (B) (hatched bar). The baseline 15 rain prior to the administration of drugs is shown to indicate the basal levels of [3H] release. The filled circles represent the release of [t4C]sucrose. The results shown are the mean+S+E,M, from 3 animals. Both the CCK-8 and the K +-induced effect were statistically significant as assessed by Student's t-test (P < 0.05). The peak values were compared with the mean of the release value obtained immediately before and immediately after superfusion with drugs.
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Time (rains) Fig. 2. Failure to release [3HJGABA (open circles) from superfused rat spinal cord in response to SP addition to the superfusate. SP (10/~M) (vertically striped bars) was administered twice at 30-rain intervals for a 30-rain period. The subsequent addition of CCK-8 (10/~M) to the perfusate (diagonally striped bar), 0.5 h after SP produced [3H]GABA release. Neither SP nor CCK-8 produced an effect on the release of [J4C]sucrose (closed circles). The data represent the mean__+S.E.M. from 4 rats. The CCK-8 peak was statistically significant when compared with controls ( P < 0.05).
superfusion with CCK-8 (10 /tM) after SP superfusion released an amount of [3H]GABA comparable to that observed when CCK-8 was administered first (compare with Fig. 1). This finding emphasises that although CCK-8 and SP are both excitory peptides found in the dorsal horn of the spinal cord [19], the mechanisms by which they act are discrete and different. Proglumide, the putative CCK receptor antagonist [5], when given in a dose of 150 mg/kg i.p., totally blocked the CCK-8 evoked released of [3H]GABA (Fig. 3). This finding is in agreement with studies in which proglumide was found to block the GABA release evoked by CCK-8 from tissue slices of rat cerebral cortex [5], but should be interpreted with caution, since in our hands proglumide also blocked the high potassium induced release of [3H]GABA. It is possible, although unlikely, that the high potassium stimulus is evoking release of endogenous CCK as has been described by Yaksh et al. [20], and that this CCK is evoking GABA release. This would then account for the sensitivity of both the action of CCK and that of potassium to proglumide. If this was to be the case, it would be difficult to explain our observation that high potassium retains its ability to release [3H]GABA when the action of exogenous CCK has been desensitized. There is also by no means general agreement that proglumide is an effective CCK antagonist in the CNS and it should be noted that it does not compete for binding with CCK in radioligand studies [4]. In our own electrophysiological studies in the spinal cord and hippocampus of the rat 'in vitro' we have failed to demonstrate any
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Fig. 3. Release of [~H]GABA (open circles) from the superfused spinal cord of the rat before and after treatment with proglumide. A: 1 nM (closed bar) CCK-8, 10 p M CCK-8 (open bar) and 50 mM K + (hatched bar) were superfused at 30-min intervals for 30-min periods. The results obtained are consistent with those obtained previously (Figs. 1 and 2). B: proglumide (150 mg/kg i.p.) was injected and half an hour afterwards, 1 nM CCK-8 was added to the superfusate and the same experimental protocol described in A was followed. Proglumide itself had no effect on the basal release of either [3H]GABA (open circles) or [14C]sucrose (closed circles). The release evoked by either CCK-8 or high potassium was completely blocked. Data shown represents mean + S.E.M. for 3 different rats. The 1 nM CCK-8 peak and the high potassium peak in A are both statistically different (P < 0.05) when compared with controls.
antagonism of the actions of CCK with proglumide (Hill et al., unpublished observations). Proglumide therefore cannot be assumed to be a selective CCK-8 antagonist in the spinal cord.
178 It is p o s s i b l e t h a t [ 3 H ] G A B A a l t h o u g h t a k e n u p a n d r e l e a s e d b y t h e s p i n a l c o r d is n o t b e h a v i n g in e x a c t l y t h e s a m e m a n n e r as e n d o g e n o u s
GABA.
m e n t s a r e t h e r e f o r e n e e d e d to i n v e s t i g a t e t h e a c t i o n s o f C C K
Further experi-
upon the release of
endogenous GABA. It is c l e a r , h o w e v e r , t h a t t h e r e s u l t s o b t a i n e d in t h i s s t u d y a r e c o n s i s t e n t w i t h t h e suggestion that the antinociceptive actions of intrathecally injected CCK are indirect and via the release of an inhibitory neurotransmitter substance. I Beinfeld, M.C., Cholecystokinin in the central nervous system: a mini review, Neuropeptides, 3 (1983) 411 427. 2 Beinfeld, M.C., Cholecystokinin (CCK) gene-related peptides; distribution and characterization of immunoreactive pro-CCK and an amino-terminal pro-CCK fragment in rat brain, Brain Res., 344 (1985) 351 355. 3 Brooks, P.A. and Kelly, J.S., Cholecystokinin as a potent excitant of neurons of the dentate gyrus of rats, Ann. N. Y. Acad. Sci., 448 (1985) 361 374. 4 Clark, C.R., Daum, P. and Hughes, J., Lack of competition between two reputed peripheral cholecystokinin receptor antagonists for central cholecystokinin binding sites, Ann. N.Y. Acad. Sci., 448 (1985) 581-582. 5 Chiodo, L.A. and Bunney, B.S., Proglumide: selective antagonism of excitatory effects ofcholecystokinin in central nervous system, Science, 219 (1983) 1449-1451. 6 Dockray, G., Immunohistochemical evidence of cholecystokinin-like peptides in brain, Nature (London), 264 (1976) 568 570. 7 Dodd, P.R., Edwardson, J.A. and Dockray, G.J., The depolarization-induced release of cholecystokinin C-terminal octapeptides (CCK-8) from rat synaptosomes and brain slices, Regul. Peptides, 1 (1980) t7 29. 8 Dodd, J. and Kelly, J.S., The actions of cholecystokinin and related peptides on pyramidal neurons of the mammalian hippocampus, Brain Res., 205 (1981) 337-350. 9 Emson, P.C., Lee, C.M. and Rehfeld, J.F., Cholecystokinin octapeptides: vesicular localization and calcium dependent release from rat brain in vitro, Life Sci., 26 (1980) 2157-2163. 10 Iversen, L.L., Mitchell, J.F. and Srinivasan, V., The release of y-aminobutyric acid during inhibition in the cat visual cortex, J. Physiol. (London), 212 (1971) 519--534. 11 Jeftinija, S., Miletic, V. and Randic, M., Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro, Brain Res., 213 (1981) 231 236. 12 Ju, G., H6kfelt, T., Fischer, J.A., Frey, P., Rehfeld, J.F. and Dockray, G.J., Does cholecystokinin-like immunoreactivity in rat primary sensory neurones represent calcitonin gene-related peptide?, Neurosci. Left., 68 (1986) 305 310. 13 Jurna, J. and Zetler, G., Antinociceptive effect of centrally administered caerulein and cholecystokinin octapeptide (CCK-8), Eur. J. Pharmacol., 73 (1981) 323-331. 14 Kow, L.M. and Pfaff, D.W., CCK-8 stimulation of ventromedial hypothalamic neurons in vitro: a feeding relevant event?, Peptides, 7 (I 986) 473-479. 15 Krnjevic, K., Pre- and post-synaptic inhibition. In P. Mandel and F.V. De Feudis (Eds.), GABA Biochemistry and CNS Functions. Plenum, New York, 1979, pp. 27~286. 16 Levy, R.A., Presynaptic control of input to the central nervous system, Can. J. Pharmacol., 58 (1980) 751 766. 17 Rogawski, M.A., Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons, Peptides, 3 (1982) 545-551. 18 Salt, T.E. and Hill, R.G., The effects of C-terminal fragments ofcholecystokinin on the firing of single neurons in the caudal trigeminal nucleus of the rat, Neuropeptides, 2 (1982) 301 306. 19 Salt, T.E. and Hill, R.G., Neurotransmitter candidates of somatosensory primary afferent fibres, Neuroscience, 10 (1983) 1083 1103. 20 Yaksh, T.L., Abay, E.O. and Go, V.L.W., Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord, Brain Res., 242 (1982) 279 290.
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Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord R.E. Rodriguez*, R.G. Hill a n d J. H u g h e s Parke-Davis Research Unit, Addenbrooke Hospital Site, Cambridge ( U.K.)
(Received 16 June 1987; Revised version received 10 August 1987; Accepted 14 August 1987) Key words: Cholecystokinin; ?-Aminobutyric acid; In vivo release; Desensitization
The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (I nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK:8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10/zM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.
Cholecystokinin octapeptide (CCK(26-33)) is the most abundant peptide in the human central nervous system (CNS) and this octapeptide is present throughout the CNS of a number of mammalian species [1, 6]. It is found in high concentration in synaptosomes [7], from which it can be released in a calcium-dependent manner [9]. When applied to the hippocampus, CCK has an excitatory action [8], and similar effects have been noted in the medulla [18] and in the dorsal horn of the spinal cord [11, 19]. These observations are consistent with the proposal that CCK may act as a neurotransmitter substance within the CNS. It has recently been suggested t h a t s o m e o f the spinal c o n t e n t o f C C K , n a m e l y that f o u n d in p r i m a r y sensory neurones, m i g h t in fact be a n o t h e r peptide, calcitonin generelated p e p t i d e ( C G R P ) [12]. It is p r o b a b l e t h a t at least s o m e o f ' t h e C C K in spinal cord, p a r t i c u l a r l y t h a t c o n t a i n e d in n e u r o n e s intrinsic to the C N S , in genuine C C K , however, as p r e c u r s o r p e p t i d e for C C K p r o d u c t i o n has been l o c a t e d there [2]. T h e m o s t i m p o r t a n t issue f r o m the s t a n d p o i n t o f the p r e s e n t s t u d y is t h a t there is clear evidence for f u n c t i o n a l C C K r e c e p t o r s within the spinal c o r d [11]. *Present address: Dept. of Biochemistry, Faculty of Medicine, University of Salamanca, 37007 Salamanca, Spain. Correspondence: R.G. Hill, Parke-Davis Research Unit, Addenbrookes Hospital Site, Hills Road, Cambridge CBQ 2QB, U.K. 0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd,
174
It has been shown that CCK, when given either peripherally or centrally to animals, produces antinociception under a variety of experimental conditions [13]. As CCK is capable of producing antinociception but has predominantly excitatory actions on single neurons, it may be acting to excite inhibitory interneurones. Such a mechanism has been suggested to explain the antinociceptive action of substance P [18], and other peptides. ~-Aminobutyric acid (GABA) is believed to act as transmitter of both pre- and postsynaptic inhibition at the level of the spinal cord [15, 16, 18] and in higher centres [10]. An interaction of CCK with the GABA release mechanism may help to explain the antinociceptive actions of CCK. We have accordingly examined the effects of CCK on the releases of GABA from spinal cord neurones 'in vivo'. Experiments were performed in male Wistar rats weighing 500-700 g under urethane (1.25 mg/kg) anaesthesia. The animals were artificially ventilated through a tracheal cannula. The heart rate and blood pressure were continuously monitored, and the central temperature was kept constant by means of an homeothermic blanket system. For detailed methods see legend to Fig. l. The baseline release of [3H]GABA showed an exponential fall during the course of the experiment. Both CCK-8 and high potassium-medium released [3H]GABA without changing the rate of washout of [14C]suerose, which was included as a space marker (Fig. l). The rate of onset of the CCK-8 evoked response was similar to that of the response evoked by perfusion with high potassium. It is likely that the release of [3H]GABA is due to a direct spinal effect since the evoked release of [3H]GABA occurred soon after beginning the CCK-8 perfusion. Removal of Ca 2+ from the perfusate accompanied by raising the concentration of Mg 2+ to 4 mM did not affect the baseline release of [3H]GABA but markedly diminished the CCK-8-induced (10 /tM) release of [3H]GABA, and the effect produced by 50 mM K + was completely abolished (not shown). The lowest concentration of CCK-8 in the perfusate found to produce an effect on [3H]GABA release was 1 nM. Increasing concentrations of CCK-8 in the perfusate thereafter produced a progressive loss of response (Fig. 3A). This phenomenon was not a result of exhaustion of [3H]GABA in the tissue, since [3H]GABA was released from the same animals when high potassium (50 mM) artificial cerebrospinal fluid (ACSF) was subsequently superfused. Desensitization of electrophysiological responses to CCK has been shown in cultures of spinal neurones [17], in hippocampal CA1 neurones [7] and in ventromedial hypothalamic neurones [14], although these findings are not universal [3]. We do not have an explanation for the desensitization to the transmitter releasing action of CCK in our experiments. This effect lasted some three or more hours and such prolonged desensitization is unusual for classical postsynaptic receptor desensitization. The phenomenon may therefore involve other mechanisms although, as mentioned above, probably not transmitter depletion. It should be noted that release of radiolabel and not of endogenous GABA is being studied. The addition of substance P (SP; 10 #M) to the superfusate had no effect on the [3H]GABA release when applied twice in 4 animals (Fig. 2). In the same animals,
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Fig. 1. Release of [3H]GABA from superfused rat spinal cord in response to (A) CCK administration (10 /+M) in the perfusate, (B) high potassium (50 mM) in the perfusate. To perfuse the spinal space, an exposure of the cisterna magna was made by a midline incision and blunt dissection, followed by incision of the dura and arachnoid membranes. A piece of 10 cm of polyethylene tubing (0.28 mm i.d., 0.61 mm o.d., Portex) was advanced down the subarachnoid space to the lumbar area [20]. The tip of this tubing served as an inflow cannula and ACSF was pumped in at a rate of 110 pl/min with a peristaltic pump (Gilson). The ACSF was withdrawn with another polyethylene tube positioned at the opening of the cisterna magna at a rate of 100 pl/min using a second peristaltic pump. Before entering the animal's body, the perfusion fluid was warmed (37 + 5°C) and was continuously oxygenated (95% Oz-5% CO2). Normal ACSF medium consisted of (in mM): NaCI 134, KC1 5, KH2PO4 1.25, MgSO4 2, CaCI2 2, NaHCO3 16, glucose 10. To study the effect of high potassium on [3H]GABA release, the concentration of potassium chloride was raised to produce a final concentration of 50 mM K + while a corresponding amount of NaCI was removed from the ACSF solution to minimize undesirable osmotic effects. Experiments were conducted by first flushing the subarachnoid space with ACSF for 1 h to allow stabilization of the preparation, then incubating the cord with 6.6/zM [3H]GABA (2.2 x 106 dpm) and 3.6 x 10-4 M [14C]sucrose (4.4 x 106 dpm) for a further 1 h. The rats were pretreated with 40 mg/kg amino-oxyacetic acid, 4 h before the incubation with [3H]GABA to prevent metabolism of the amino acid. After the incubation period, the cord was again perfused with ACSF for 50 min, then appropriate test substances were added to the perfusate and 5-rain fractions (500/zl) were collected. The radioactive content of the superfusate was determined by liquid scintillation spectrometry after adding 6 ml of CP-scintillation fluid (Beckman) and 50/ll formic acid to suppress chemiluminescence. For the statistical analysis of data Student's t-test was used. The figure shows the change in dpm × 104 after introducing CCK-8 (10/zM) in the perfusing medium for 0.5 h (A) (open bar) and after perfusing with high potassium (50/zM) for 0.5 h (B) (hatched bar). The baseline 15 rain prior to the administration of drugs is shown to indicate the basal levels of [3H] release. The filled circles represent the release of [t4C]sucrose. The results shown are the mean+S+E,M, from 3 animals. Both the CCK-8 and the K +-induced effect were statistically significant as assessed by Student's t-test (P < 0.05). The peak values were compared with the mean of the release value obtained immediately before and immediately after superfusion with drugs.
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Time (rains) Fig. 2. Failure to release [3HJGABA (open circles) from superfused rat spinal cord in response to SP addition to the superfusate. SP (10/~M) (vertically striped bars) was administered twice at 30-rain intervals for a 30-rain period. The subsequent addition of CCK-8 (10/~M) to the perfusate (diagonally striped bar), 0.5 h after SP produced [3H]GABA release. Neither SP nor CCK-8 produced an effect on the release of [J4C]sucrose (closed circles). The data represent the mean__+S.E.M. from 4 rats. The CCK-8 peak was statistically significant when compared with controls ( P < 0.05).
superfusion with CCK-8 (10 /tM) after SP superfusion released an amount of [3H]GABA comparable to that observed when CCK-8 was administered first (compare with Fig. 1). This finding emphasises that although CCK-8 and SP are both excitory peptides found in the dorsal horn of the spinal cord [19], the mechanisms by which they act are discrete and different. Proglumide, the putative CCK receptor antagonist [5], when given in a dose of 150 mg/kg i.p., totally blocked the CCK-8 evoked released of [3H]GABA (Fig. 3). This finding is in agreement with studies in which proglumide was found to block the GABA release evoked by CCK-8 from tissue slices of rat cerebral cortex [5], but should be interpreted with caution, since in our hands proglumide also blocked the high potassium induced release of [3H]GABA. It is possible, although unlikely, that the high potassium stimulus is evoking release of endogenous CCK as has been described by Yaksh et al. [20], and that this CCK is evoking GABA release. This would then account for the sensitivity of both the action of CCK and that of potassium to proglumide. If this was to be the case, it would be difficult to explain our observation that high potassium retains its ability to release [3H]GABA when the action of exogenous CCK has been desensitized. There is also by no means general agreement that proglumide is an effective CCK antagonist in the CNS and it should be noted that it does not compete for binding with CCK in radioligand studies [4]. In our own electrophysiological studies in the spinal cord and hippocampus of the rat 'in vitro' we have failed to demonstrate any
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Fig. 3. Release of [~H]GABA (open circles) from the superfused spinal cord of the rat before and after treatment with proglumide. A: 1 nM (closed bar) CCK-8, 10 p M CCK-8 (open bar) and 50 mM K + (hatched bar) were superfused at 30-min intervals for 30-min periods. The results obtained are consistent with those obtained previously (Figs. 1 and 2). B: proglumide (150 mg/kg i.p.) was injected and half an hour afterwards, 1 nM CCK-8 was added to the superfusate and the same experimental protocol described in A was followed. Proglumide itself had no effect on the basal release of either [3H]GABA (open circles) or [14C]sucrose (closed circles). The release evoked by either CCK-8 or high potassium was completely blocked. Data shown represents mean + S.E.M. for 3 different rats. The 1 nM CCK-8 peak and the high potassium peak in A are both statistically different (P < 0.05) when compared with controls.
antagonism of the actions of CCK with proglumide (Hill et al., unpublished observations). Proglumide therefore cannot be assumed to be a selective CCK-8 antagonist in the spinal cord.
178 It is p o s s i b l e t h a t [ 3 H ] G A B A a l t h o u g h t a k e n u p a n d r e l e a s e d b y t h e s p i n a l c o r d is n o t b e h a v i n g in e x a c t l y t h e s a m e m a n n e r as e n d o g e n o u s
GABA.
m e n t s a r e t h e r e f o r e n e e d e d to i n v e s t i g a t e t h e a c t i o n s o f C C K
Further experi-
upon the release of
endogenous GABA. It is c l e a r , h o w e v e r , t h a t t h e r e s u l t s o b t a i n e d in t h i s s t u d y a r e c o n s i s t e n t w i t h t h e suggestion that the antinociceptive actions of intrathecally injected CCK are indirect and via the release of an inhibitory neurotransmitter substance. I Beinfeld, M.C., Cholecystokinin in the central nervous system: a mini review, Neuropeptides, 3 (1983) 411 427. 2 Beinfeld, M.C., Cholecystokinin (CCK) gene-related peptides; distribution and characterization of immunoreactive pro-CCK and an amino-terminal pro-CCK fragment in rat brain, Brain Res., 344 (1985) 351 355. 3 Brooks, P.A. and Kelly, J.S., Cholecystokinin as a potent excitant of neurons of the dentate gyrus of rats, Ann. N. Y. Acad. Sci., 448 (1985) 361 374. 4 Clark, C.R., Daum, P. and Hughes, J., Lack of competition between two reputed peripheral cholecystokinin receptor antagonists for central cholecystokinin binding sites, Ann. N.Y. Acad. Sci., 448 (1985) 581-582. 5 Chiodo, L.A. and Bunney, B.S., Proglumide: selective antagonism of excitatory effects ofcholecystokinin in central nervous system, Science, 219 (1983) 1449-1451. 6 Dockray, G., Immunohistochemical evidence of cholecystokinin-like peptides in brain, Nature (London), 264 (1976) 568 570. 7 Dodd, P.R., Edwardson, J.A. and Dockray, G.J., The depolarization-induced release of cholecystokinin C-terminal octapeptides (CCK-8) from rat synaptosomes and brain slices, Regul. Peptides, 1 (1980) t7 29. 8 Dodd, J. and Kelly, J.S., The actions of cholecystokinin and related peptides on pyramidal neurons of the mammalian hippocampus, Brain Res., 205 (1981) 337-350. 9 Emson, P.C., Lee, C.M. and Rehfeld, J.F., Cholecystokinin octapeptides: vesicular localization and calcium dependent release from rat brain in vitro, Life Sci., 26 (1980) 2157-2163. 10 Iversen, L.L., Mitchell, J.F. and Srinivasan, V., The release of y-aminobutyric acid during inhibition in the cat visual cortex, J. Physiol. (London), 212 (1971) 519--534. 11 Jeftinija, S., Miletic, V. and Randic, M., Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro, Brain Res., 213 (1981) 231 236. 12 Ju, G., H6kfelt, T., Fischer, J.A., Frey, P., Rehfeld, J.F. and Dockray, G.J., Does cholecystokinin-like immunoreactivity in rat primary sensory neurones represent calcitonin gene-related peptide?, Neurosci. Left., 68 (1986) 305 310. 13 Jurna, J. and Zetler, G., Antinociceptive effect of centrally administered caerulein and cholecystokinin octapeptide (CCK-8), Eur. J. Pharmacol., 73 (1981) 323-331. 14 Kow, L.M. and Pfaff, D.W., CCK-8 stimulation of ventromedial hypothalamic neurons in vitro: a feeding relevant event?, Peptides, 7 (I 986) 473-479. 15 Krnjevic, K., Pre- and post-synaptic inhibition. In P. Mandel and F.V. De Feudis (Eds.), GABA Biochemistry and CNS Functions. Plenum, New York, 1979, pp. 27~286. 16 Levy, R.A., Presynaptic control of input to the central nervous system, Can. J. Pharmacol., 58 (1980) 751 766. 17 Rogawski, M.A., Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons, Peptides, 3 (1982) 545-551. 18 Salt, T.E. and Hill, R.G., The effects of C-terminal fragments ofcholecystokinin on the firing of single neurons in the caudal trigeminal nucleus of the rat, Neuropeptides, 2 (1982) 301 306. 19 Salt, T.E. and Hill, R.G., Neurotransmitter candidates of somatosensory primary afferent fibres, Neuroscience, 10 (1983) 1083 1103. 20 Yaksh, T.L., Abay, E.O. and Go, V.L.W., Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord, Brain Res., 242 (1982) 279 290.