Life Sciences, Vol. 41, pp. 605-610
`Printed in the U.S.A.
`
`Pergamon Journals
`
`3'-5' CYCLIC-GUANOSINE MONOPHOSPHATE INCREASE IN RAT BRAIN HIPPOCAMPUS AFTER
`GAMMA-HYDROXYBUTYRATE ADMINISTRATION. PREVENTION BY VALPROATE AND NALOXONE.
`
`Philippe Vayer, Serge Gobaille, Paul Mandel and Michel Maitre
`
`Centre de Neurochimie du CNRS and INSERM U44, 5 rue Blaise Pascal,
`67084 STRASBOURG Cedex.
`
`(Received in final form May 21, 1987)
`Summary
`
`increase
`(123%) of cyclic GMP
`(cGMP) was observed
`An
`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
`( 10 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.
`
`increase of adenosine 3'-5' eye lic-adenos ine-monophosphate (cAMP) or
`An
`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,
`GHB
`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 (10,11), 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). The dissected brain regions were kept in liquid
`0024-3205/87 $3.00 + .00
`Copyright (c) 1987 Pergamon Journal~s~L~t~d~·~----~--~~~------------,
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`

`
`606
`
`GHB Increases cGMP in Rat Brain Hippocampus
`
`Vol. 41, No. 5, 1987
`
`nitrogen, weighed frozen and homogenized
`in 10 vols 1M
`ice cold perchloric
`acid.
`Protein was
`removed by centrifugation at 20,000 g for 25 min.
`The
`supernatants were neutralized with 3M K2co, and cyclic nucleotide contents were
`determined with
`the
`cAMP kit and <:he' cGMP 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 10 min during 120 min after
`injection at time zero of 500 mg/kg GHB. No significant changes were found for
`cGMP and cAMP in the cerebellum or in the striatum.
`cerebellum (3.02 ±
`(cGMP:
`0.48 pmole/mg protein),
`(0.25 ± 0.06 pmole/mg protein);
`striatum
`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 110 min,
`the basal level of cGMP is restored (Fig. 1). 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 valproate on the cGMP increase induced by GHB in hippocampus
`
`The animals were injected either with valproate (400 mg/kg IP) or with GHB
`(400 mg/kg IP) or pretreated with valproate (400 mg/kg IP) 15 min before GHB
`injection (400 mg/kg IP).
`In all 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 ccntent, but GHB
`increases cGMP in hippocampus by about 140% compared to controls injected with
`saline. However, pretreatment with valproate completly abolishes the GHB effect
`on cGMP 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 (10 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).
`
`Discussion
`
`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
`
`

`
`Vol. 41, No. 5, 1987
`
`GHB Increases cGMP in Rat Brain Hippocampus
`
`607
`
`I
`I In
`hi
`
`:II
`
`I
`ri
`
`0.7
`
`0.6
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`0
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`c 0.5
`.....
`"' ...,
`'-c. 0.4
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`..-<
`0
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`u
`
`0.1
`
`0
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`time
`
`(min)
`
`FIG. 1
`
`cGMP levels
`rng/kg IP).
`S.E.M .• The
`the control
`
`in hippocampus as a function of time after GHB administration ( 500
`Each point represents the mean of 3 different determinations ±
`cGMP levels between 20 and 100 min are significantly different from
`with p < 0.05 (Student's t test).
`
`I I
`
`1:
`
`I
`
`I
`
`]
`
`I
`
`0.6
`
`c
`..... ., ...,
`
`0
`'-
`c. 0.4
`0>
`.....
`E
`.,
`
`Ill
`
`..-<
`0
`E
`.9
`a. 0.2
`::E
`<.!J
`u
`
`0
`
`0
`
`100
`
`200
`
`300
`
`400
`
`500
`
`600
`
`700
`
`BOO
`
`GHB
`
`(mg/kg)
`
`FIG. 2
`
`Effect of various GHB doses on cGMP 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).
`
`

`
`608
`
`GHB Increases cGMP in Rat Brain Hippocampus
`
`Vol. 41, No. 5, 1987
`
`0.7
`
`0.6
`
`'2 0.5
`
`... .. ....
`
`0
`t.
`c. 0. 4
`"' E
`..... .,
`~ 0.3
`
`~ .s
`a.. 0.2
`~
`u
`
`0.1
`
`0
`
`A
`
`B
`
`c
`
`D
`
`FIG. 3
`
`Effect of valproate on cGMP increase induced by GHB in hippocampus.
`Animals
`injected
`(A) with valproate (400 mg/kg IP) or
`(B) pretreated with
`valproate (400 mg/kg) 15 min 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.
`(l<) p < 0.05 vs saline control (Student's t test).
`
`0. 7
`
`0.6
`
`~ 0.5
`
`(.
`c. 0. 4
`
`" "" 0
`"' E ' ~
`~ 0.3
`0
`E .s
`~ 0.2
`'.::
`
`0.1
`
`0
`
`B
`
`c
`
`D
`
`FIG. 4
`
`Effect of naloxone on cGMP increase induced by GHB in hippocampus.
`Animals
`injected
`(A) with naloxone
`(10 mg/kg IP) or
`(B) pretreated with
`naloxone ( 10 mg/kg) 15 min before GHB injection (400 mg/kg) or (C) with sa] ine
`or (D) injected with GHB (400 mg/kg IP). Each value is the mean of 6 different
`determinations ± SEM.
`(*) p < 0.05 vs saline control (Student's t test).
`
`

`
`Vol. 41, No. 5, 1987
`
`GHB Increases cGMP in Rat Brain Hippocampus
`
`609
`
`cGMP in hippocarnpus 1 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 (10-11). Moreover,
`cGMP has been implicated in seizure genesis and/or
`propagation (1). 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 (1).
`
`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
`is thus reduced. Under these conditions,
`GABAergic synapse,
`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 depress ion is also
`inhibited by pretreatment with naloxone (30). In the present studies the amount
`of naloxone injected (10 mg/kg) is too low to envisage a possible action on
`GABAergic
`receptors
`(31).
`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
`
`

`
`610
`
`GHB Increases cGMP in Rat Brain Hippoca11pus
`
`Vol. 41, No. 5, 1987
`
`enkephalins and
`(19),
`those of GHB
`to
`the opiates are similar
`effects of
`endorphins may be involved in the neurophysiological and neuropharmacological
`effects of GHB, particularly in the hippocampus.
`
`References
`
`1.
`
`2.
`
`J.A. FERRENDELLI, C.A. BLANK and A. R. GROSS, Brain Res., 200:93-103
`( 1980).
`Nucleotides Research, pp. 453- 464,
`J.A. FERRENDELLI, Advances in
`s. , Raven Press, New York
`(1978)
`W.J. George and L.J. Ignarro
`3. R.H. ROTH and N.J. GIARMAN, Biochem. Pharmacal., 19:1087-1093 (1970).
`4.
`J.D. DOHERTY, S.E. HATTOX, O.C.
`SNEAD and R.~ ROTH,
`J. Pharmacal.
`Experimental Ther., 207:130-139 (1978).
`5. H. LABORIT,
`J.
`JOVANY,
`J. GERARD and P.
`cology, 2:490-497 (1961).
`(1965).
`6. W.D. WINTERS and C.E. SPOONER, Int. J. Neuropharmacol., 4:197-200
`7. M. GODSCHALK, M.R. DZOLJIC and I.L. BONTA, Eur. J. Pharmacal., 44,
`105-
`111 ( 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).
`10. R.K.S. WANG and R.D. TRAUB, J. Neurophysiol., 49:442-458 (1983).
`11. D.C. MciNTYRE and R.J. RACINE, Progress in Neurobiol., 27:1-12
`(1986).
`12. E.H. RUBIN and J.A. FERRENDELLJ, J. Neurochem., 29:43-5r-(1977).
`13. R.B. MAILMAN, R.A. MUELLER and G.R. BREESE, --Life Sci., 23, 623-628
`(1978).
`-
`14. C.C. MAO, A. GUIDOTTI and E. COSTA, Molec. Pharmacal., 10:736-745
`15. G.L. GESSA, F. CRABAI, L. VARGIU and P.F. SPANO, J. Neurochem.,
`381 ( 1968).
`16. R.H. LENOX, G.J. KANT and J.L. MEYERHOFF, Handbook of Neurochemistry, pp.
`77-102, A. Lajtha (ed.), Plenum Press, (1982).
`LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chern.,
`17. O.H.
`193:265-275 (1951).
`18. w.J. KINNIER, D.M. CHUANG, D.L. CHENEY and E. COSTA, Neuropharmacology,
`19:1119-1123 (1980)
`19. O:c. SNEAD and L.J. BEARDEN, Neurology, 30:832-838 (1980).
`20. G.C.
`PALMER, S.J.
`PALMER and J.L. LEGENDRE, 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. Pharmacal., 33:2587-2590. (1984).
`23. C.C.
`MAO,
`A.
`GUIDOTTI and E.
`COSTA, Naunyn-Schmiedeberg's Arch.
`Pharmacal., 289:369-378 (1975).
`24. R.E. STUDY, ~Pharmacal. Exp. Ther., 215:575-581 (1980).
`25.
`J.A. FERRENDELLI and D.A. KINSCHERF,-y: Pharmacal. Exp. Ther., 207:787-
`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,
`( 1980).
`SCHMITT, J.J. BOURGUIGNON, P. MANDEL and M. MAITRE, FEBS
`29. P. VAYER, M.
`Lett., 190:55-60 (1985).
`ITO, E. KAUFMAN, T. NELSON and L. SOKOLOFF, Brain Res.,
`30. G. CROSBY, M.
`275:194-197 (1983).
`31. R. DINGLEDINE, L.L. IVERSEN and E. BRENKER, Eur. J. Pharmacal., 47:19-27
`(1978).
`32. M.E. CORCORAN and J.A. WADA, Life Sci., 24:791-796 (1979).
`33. H. FRENK, L. PAUL and J. DIAZ, Neurosci.-xbs., 4:142 (1978).
`34. G.J. GWYNN and E. COSTA, Proc. Natl. Acad. Sci.-USA 79:690-694 (1982).
`
`FABIANI, Neuropsychopharma(cid:173)
`
`(1974).
`lj:377-
`
`19:47-52