Life Sciences, Vol. 41, pp. 605-610 Pergamon Journals Printed in the U.S.A. 3'-5' CYCLIC-GUANOSINE MONOPHOSPHATE INCREASE IN RAT BRAIN HIPPOCAMPUS AFTER GAMMA-HYDROXYBUTYRATE ADMINISTRATION. PREVENTION BY VAI2ROATE AND NALOXONE. Philippe Vayer, Serge Gobaille, Paul Mandel and Michel Maitre Centre de Neurochimie du CNRS and INSERM Uz~, 5 rue Blaise Pascal, 67084 STRASBOURG Cedex. (Received in final form May 21, 1987) Summary An increase (123~) of cyclic GMP (cGMP) was observed in the hippocampus of the rat killed by microwave irradiation 45 min after administration of 500 mg/kg y-hydroxybutyrate (GHB) IP. This increase is time and dose dependent. No modification in cyclic nucleotide content was observed in striatum and in cerebellum. As the role of GHB has been implicated in neurotransmission, the fact that this compound increases cyclic GMP accumulation in hippocampus in vivo may represent a mechanism by which the actions of GHB are mediated at the cellular level. Valproate (400 mg/kg) or naloxone (I0 mg/kg) pretreatment completely abolish the cGMP increase due to GHB. A GABAergic and/or opiate phenomenon may be involved in the mechanism of GHB induced increase of cGMP. An increase of adenosine 3'-5' cyclic-adenosine-monophosphate (cAMP) or of 3'-5' cyclic-guanosine-monophosphate (cGMP) or both have been observed after administration of several convulsant drugs and agents in experimental animals (1,2). Moreover, agents that lead to behavioral excitation tend to increase cGMP levels whereas those that depress motor activity decrease its levels (2). Interestingly, y-hydroxybutyrate (GHB) which occurs naturally in the brains of several mammalian species (3) including man (4), induces when administered to animals a state of behavioral sedation often called sleep or anaesthesia (5). In addition, GBB induces hypersynchronism in the electroencephalographic pattern in rat, rabbit and man (6,7,8). These effects have been described as epileptoid E.E.G. seizures which can be antagonized by anti-petit mal drugs. Besides these effects, GHB is a good candidate for a role in neurotransmission or neuromodulation (9). The cyclic nucleotides, involved in the cellular action of numerous neurotransmitters, can also mediate the neuroregulatory effects of GHB in mammalian brain. The aim of this paper is to investigate the effect of exogenous GHB on the level of cyclic nucleotides in three regions of the rat brain : hippocampus, which is considered as the burst generator for several acute epilepsy models (I0, ii), cerebellum, where cyclic nucleotides have been extensively studied (12,13,14), and striatum where GHB interacts with the dopaminergic system (8-15). Materials and Methods Male adult Wistar rats weighing about 300 g were used in all studies. The animals were injected IP with GHB (sodium salt) and/or with the other test substances (sodium valproate, or naloxone hydrochloride). The rats were sacrified after appropriate times by exposing the head to focused microwave irradiation (7.5 kW, 1.6 sec. exposure) which prevents post-mortem changes in cyclic nucleotide levels (16). l~e dissected brain regions were kept in liquid 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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`606 GHB Increases cGMP in Rat Brain Hippocampus Vol. 41, No. 5, 1987 nitrogen, weighed frozen and homogenized in I0 vols IM ice cold perchloric acid. Protein was removed by centrifugation at 20,000 g for 25 min. The supernatants were neutralized with 3M KoCO o and cyclic nucleotide contents were determined with the cAMP kit and ~h~ cGHP R.I.A. kit from Amersham (Radiochemical Center). Protein contents of the different pellets were measured by the Lowry method (17) after solubilization in 2N NaOH. Results Cyclic nucleotides as a function of time after GHB administration Cyclic nucleotide levels were measured every I0 min during 120 min after injection at time zero of 500 mg/kg GHB. No significant changes were found for cGHP and cAMP in the cerebellum or in the striatum. (cGMP: cerebellum (3.02 ± 0.48 pmole/mg protein), striatum (0.25 ± 0.06 pmole/mg protein)~ cAMP: cerebellum (6.29 ± 0.53 pmole/mg protein), striatum (3.14 ± 0.56 pmole/mg protein). In the hippocampus, the level of cGMP (0.28 ± 0.05 pmole/mg protein) increases with time. The rise of cGMP was first noted 20 min after injection of GHB, with a maximum at 30-50 min (0.63 ± 0.04 pmole/mg protein). After II0 min, the basal level of cGMP is restored (Fig. I). For cAMP, no significant changes were found (3.57 ± 0.96 pmole/mg protein). Effect of various concentrations of GHB on cGMP levels in hippocampus 200 mg/kg to 700 mg/kg GHB were administered IP to rats which were killed after 45 min by microwave irradiation, cGMP levels were determined in the dissected hippocampus. Fig. 2 shows that the maximum increase in cGMP occurs for 400 - 500 mg/kg. Higher doses induced less accumulation of cGMP. Effect of v alproate on the cGMP increase induced by GHB in hi~pocampus The animals were injected either with valproate (400 mg/kg IP) or with GHB (400 mg/kg IP) or pretreated with vaiproate (400 mg/kg IP) 15 min before GHB injection (400 mg/kg IP). In al] cases, 45 min after the last injection, the animals were killed as described above and cGMP was determined in the hippocampus. Fig. 3 shows that valproate does not modify cGMP content, but GHB increases cGHP in hippoeampus by about 140~ compared to controls injected with saline. However, pretreatment with valproate completly abolishes the GHB effect on cGHP levels (Fig. 3). Under these conditions, no modifications of cGMP levels are observed compared to controls injected with saline or with valproate alone. Effect of naloxone on the cGMP increase induced by GHB For these experiments, the same protocol as described for the experiment with valproate was adopted, but this latter compound was replaced by administration of naloxone (I0 mg/kg IP). As indicated by SNEAD et al. (19) naloxone completely blocked behavioral changes induced by administration of GHB. In particular, no catalepsy was observed in animals receiving both naloxone and GHB. Pretreatment with naloxone blocks the GHB effect on cGMP levels (Fig. 4). Discussi on This work demonstrates the increase of cGMP accumulation induced by GHB in rat brain hippocampus. The control values of cGMP are identical to those previously described for hippocampus of rats sacrificed by microwave irradiation (18). No changes were found in the other regions studied either for cGMP or for cAMP levels. GHB caused a time and dose dependent accumulation of
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`Vol. 41, No. 5, 1987 GHB Increases cGMP in Rat Brain Hippocampus 607 0.7 0.6 c 0.5 o L ~ 0.4 ~ 0.3 ,--t a. 0.2 ¢..i o.i 0 I I I I I I 0 20 .40 60 80 100 12o time (mln) 140 FIG. 1 cGMP levels in hippocampus as a function of time after GHB administration (500 mg/kg IP). Each point represents the mean of 3 different determinations ± S.E.M.. The cGMP levels between 20 and lOO min are significantly different from the control with p < 0.05 (Student's t test). c o Q. o O- L~ 0.6 0.,4 0.2 0 I I I 1 I I I 0 ~.00 200 300 400 500 600 700 GHB (mg/kg) BOO FIG. 2 Effect of various GHB doses on rOMP levels in hippocampus. The rats were killed 45 min after GHB injection IP. Each point represents the mean of 3 different determinations ± S.E.M. The cGMP levels between 400 and 600 mg/kg are significantly different from the control with p < 0.05 (Student's t test).
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`608 GHB Increases cGMP in Rat Brain Hippocampus Vol. 41, No. 5, 1987 0.7 O.B ~ 0.5 o ~-0.4 ~ 0.3 ,--i o. 0.2 lg u 0.t ////~ "///z ////~ ?'/.v'Z A D 7 rill1 y//z r/ill ////, F///~ z/l% F//// !'.44~ "///A • ~//J/Z B C FIG. 3 Effect of valproate on cGMP increase induced by OHB in hippocampus. Animals injected (A) with valproate (400 mg/kg IP) or (B) pretreated with valproate (400 mg/kg) 15 rain before GHB injection (400 mg/kg IP) or (C) with saline or (D) injected with GHB (400 mg/kg IP). Each value is the mean of 6 different determinations + S.E.M. (*~) p < 0.05 vs saline control (Student's t test). 0.7 0.6 "~ 0.5 ,= Q- 0.4 o cL 0.2 u 0.1 x A B C D FIO. 4 Effect of naloxone on cGMP increase induced by OttB in hippocampus. Animals injected (A) with naloxone (lO mg/kg IP) or (B) pretreated with naloxone (lO mg/kg) 15 rain before GHB injection (400 mg/kg) or (C) with saline or (D) injected with GHB (400 mg/kg IP). Each value is the mean of 6 different determinations + S~t. (~) p < 0.05 vs saline control (Student's t test).
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`Vol. 41, No. 5, 1987 GHB Increases cGMP in Rat Brain Hippocampus 609 cGMP in hippocampus~ this effect is maximum 30-50 minutes after injection for a dose of 400 mg/kg which induce a strong sedation with loss of righting reflex. Behavioral modification was accompanied by an EEG pattern similar to those occuring during petit mal epilepsy (8). These phenomena are consistent with the reported hypothesis of an involvement of hippocampus in several acute epilepsy models (I0-Ii). Moreover, cGMP has been implicated in seizure genesis and/or propagation (I). Some evidence indicates that the cGMP synthesis preceding the onset of epileptoid episodes most likely results from massive depolarization elicited by excitatory firing from cholinergic and glutamatergic neurons which are largely represented in hippocampus. (20). Consistently, a modification of brain acetylcholine after GHB administration has been reported (8). However, there is no apparent correlation between the onset and degree of behavioral change with the onset and magnitude of the rise in cGMP in hippocampus in response to GHB treatment. EEG paroxysms occur within 2 to 3 minutes of administration (19) and catalepsy with loss of righting reflex is strongly established a few minutes after IP administration of 700 mg/kg GHB. Nevertheless, the fact that the naturally occurring substance, GHB, increases cGMP accumulation in rat hippocampus in vivo, raises the possibility that this response may represent a mechanism by which the actions of GHB in brain are mediated at the neuronal level. In fact, several demonstrated properties of GHB in brain are in favor of a neurotransmitter or a neuromodulator role for this substance (9). In this respect, it should be noted that hippocampus is the richest human and rat brain region with regard to density of GHB high affinity binding sites (21-22). Thus an increase of the cGMP level might represent a transduction signal for GHB receptor stimulation. In contrast, cerebellum is practically devoid of GHB binding sites and striatum contains low densities of these receptors (21,22). The anticonvulsant drug valproate, which inhibits the epileptoid pattern of the EEG in animals injected with GHB, completely prevents cGMP accumulation in hippocampus induced by GHB administration. This result is not surprising in view of the fact that in general, many anticonvulsant drugs antagonize the increase of cGMP levels associated with experimental seizures (23). However, it is generally accepted that it is doubtful that the alteration of cGMP is a mechanism by which anticonvulsants exert their effects (24). Valproate is known to inhibit depolarization induced increases in cyclic nucleotide levels (24-25). These effects may be important in seizure control induced by GHB because elevated cyclic nucleotide levels have been implicated in the maintainance of sustained seizure discharge (I). Valproate is thought to act via a reinforcement of GABAergic transmission (26-27). However, this anticonvulsant drug also increases GHB levels possibly by inhibition of its catabolism (28,29). The GABA pool formed from GHB breakdown which might be involved in the negative feedback regulation of a GABAergic synapse, is thus reduced. Under these conditions, an increase in GABAergic brain activity which has been shown to decrease cGMP accumulation is conceivable (2). In contrast, GHB administration increases the GABA pool derived from GHB and thus exerts a feed back inhibition on certain GABA synapses, reducing inhibitory input in brain, leading to cGMP accumulation and epileptic phenomena. However, it is evident that further studies are required to support this hypothesis. Naloxone also antagonizes the GHB induced increase in cGMP in hippocampus. This opiate antagonist has been shown to block the petit mal epilepsy model provided by GHB treated animals (19). Cerebral metabolic depression is also inhibited by pretreatment with naloxone (JO). In the present studies the amount of naloxone injected (I0 mg/kg) is too low to envisage a possible action on GABAergic receptors (Jl). In addition, naloxone has no effects on pentylenetetrazol induced seizures, amygdaloid kindling or human seizures (32- 33). Opioid receptor agonists elicit a dose-dependent and transient elevation of cGMP content in neuroblastoma cells (34). Thus, as EEG and behavioral
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`610 GHB Increases cGMP in Rat Brain Hippocal~pus Vol. 41, No. 5, 1987 effects of the opiates are similar to those of GHB (19), enkephalins and endorphins may be involved in the neurophysiological and neuropharmacological effects of GHB, particularly in the hippocampus. References I. J.h. FERRENDELLI, C.A. BLANK and A. R. GROSS, Brain Res., 200:93-103 (1980). 2. J.A. FERRENDELLI, Advances in Cyclic Nucleotides. Research, pp. 453- 464, W.J. George and L.J. Ignarro (eds.), Raven Press, New York (1978) 3. R.H. ROTH and N.J. OlARMAN, Biochem. Pharmacol., 19:1087-1093 (1970). 4. J.D. DOHERTY, S.E. HATTOX, O.C. SNEAD and R.H. ROTH, J. Pharmacol. Experimental Ther., 207:130-139 (1978). 5. H. LABORIT, J. JOUA"-'NY, J. GERARD and P. FABIANI, Neuropsychopharma- cology, 2:490-497 (1961). 6. W.D. WIN~ERS and C.E. SPOONER, Int. J. Neuropharmacol., -4:197-200 (1965). 7. M. GODSCHALK, M.R. DZOIJIC and I.L. BONTA, Eur. J. Pharmacol., 44, 105- III (1977). 8. O.C. SNEAD, Life Sci., 20:1935-1944 (1977). 9. M. MAITRE and P. MANDEL~'-C.R. Acad. Sci., Paris, 298:341-345 (1984). I0. R.K.S. WANG and R.D. TRAUB, J. Neurophysiol., 49:4"~-458 (1983). II. D.C. McINTYRE and R.J. RACINE, Progress in Neu~-obiol., 27:1-12 (1986). 12. E.H. RUBIN and J.A. FERRENDELLI, J. Neurochem., 2~9:43-5T'(1977). 13. R.B. MAILMAN, R.A. MUELLER and G.R. BREESE, Life Sci., 2.~3, 623-628 (1978). 14. C.C. MAO, A. GUIDOTTI and E. COSTA, Molec. Pharmacol., 10:736-745 (1974). 15. G.L. GESSA, F. CRABAI, L. VARGIU and P.F. SPANO, J. Neurochem., 15:377- 381 (1968). 16. R.H. LENOX, G.J. KANT and J.L. MEYERHOFF, Handbook of Neurochemistry, pp. 77-102, A. Lajtha (ed.), Plenum Press, (198~). 17. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem., 19.3:265-275 (1951). 18. W.J. KINNIER, D.M. CHUANG, D.L. CHENEY and E. COSTA, Neuropharmacology, 19: II 19-1123 (1980) 19. O-'~C. SNEAD and L.J. BEARDEN, Neurology, 30:832-838 (1980). 20. G.C. PALMI~, S.J. PALMER and J.L. LEGE-'NDRE, Exper. Neurol., 71:601- 614 (1981). 21. J. BENAVIDES, J.F. RUMIGNY, J.J. BOURGUIGNON, C. CASH, C.G. WERMUTH, P. MANDEL, G. VINCENDON and M. MAITRE, Life Sci., 30:953-961 (1982). 22. O.C. SNEAD and C.C. LIU, Biochem. Pharmacol., 3~.2587-2590. (1984). 23. C.C. MAO, A. GUIDOTTI and E. COSTA, Naunyn-Schmiedeberg's Arch. Pharmacol., 289:369-378 (1975). 24. R.E. STUDY, J. Pharmacol. Exp. Ther., 215:575-581 (1980). 25. J.A. FERRENDELLI and D.A. KINSCHERF, J. Pharmacol. Exp. Ther., 20___Z:787- 793 (1978). 26. K. GALE and M.J. IADAROLA, Science, 208:288-291 (1980). 27. S. SIMLER, L. CIESIELSKI, M. MAITRE, H. RANDRIANARISOA and P. MANDEL, Biochem. Pharmac., 22:1701-1708 (1973). 28. O.C. SNEAD, L.J. BEARDEN and V. PEGRAM, Neuropharmacology, 19:47-52 (1980). 29. P. VAYER, M. SCHMITT, J.J. BOURGUIGNON, P. MANDEL and M. MAITRE, FEBS Lett., 190:55-60 (1985). 30. G. CROSBY, M. ITO, E. KAUFMAN, T. NEI~ON and L. SOKOLOFF, Brain Res., 275:194-197 (1983). 31. R. DINGLEDINE, L.L. IVERSEN and E. BRENKER, Eur. J. Pharmacol., 47:19-27 (1978). 32. M.E. CORCORAN and J.A. WADA, Life Sci., 2.44: 791-796 (1979). 33. B. FRENK, L. PAUL and J. DIAZ, Neurosci. Abs., _4:142 (1978). 34. G.J. GWYNN and E. COSTA, Proc. Natl. Acad. Sci. USA 79:690-694 (1982).