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Mechanisms of inhibition of LHRH release by alcohol and cannabinoids
I.S. Parhar (Ed.) Progress in Brain Research, Vol. 141 0 2002 Elsevier Science B.V. All rights reserved
CHAPTER
13
Mechanisms of inhibition of LHRH release by alcohol and cannabinoids Valeria Rettori ‘$*, Alejandro Lomniczi ‘, Claudia Mohn ‘, Camila Scorticati ‘, Paula Vissio ‘, Mercedes Lasaga*, Ana Franchi ‘, Samuel M. McCann 3 I Centro
de Estudios Farmacoldgicos y Botdnicos CONICET, Serrano 669, 1414, Buenos Aires, Argentina 2 Centro de Investigaciones en Reproduccidn, Fat. Medicina, UBA, Buenos Aires, Argentina 3 Pennington Biomedical Research Center; LSU, Baton Rouge, LA, USA
Introduction This paper will review our research and that of our associates on the effects of alcohol and cannabinoids on reproduction. Today, alcohol is classified as a psychotropic drug, like delta-9 tetrahydrocannabinol (THC), the active ingredient of marihuana. A large number of neurotransmitters not only the classical ones, but also a host of neuropeptides that can act as neutransmitters or neuromodulators, exist in the central nervous system (CNS) and there is abundant evidence that alcohol and cannabinoids can affect a number of them. Therefore, mechanisms of action of these drugs in the CNS are very complex. Addiction to alcohol or marihuana produces numerous deleterious effects in the organism. Among these alterations is the suppression of reproduction in humans, monkeys and small rodents by inhibition of the release of luteinizing hormone (LH). This inhibition of LH secretion is caused mainly by hypothalamic action to inhibit the release of luteinizing hormone-releasing hormone (LHRH), in vivo and in vitro. In conscious, ovariectomized rats, intragastric administration of alcohol (3 g/kg), a dose that
* Correspondence to: V. Rettori, CEFYBO-CONICET, Serrano 669, 1414, Buenos Aires, Argentina. Fax: +54114963-4473; Tel: +541 l-4855-7204; E-mail: [email protected]
causes mild intoxication, has been shown to produce a marked decrease in plasma LH concentrations on comparison with the unaffected LH values of diluent administered animals (Dees et al., 1985). There was a highly significant decrease in the area under the secretion curve of LH, with a reduction of LH pulses. On the other hand, following a challenge with exogenous LHRH, the response of LH was the same as in controls, indicating that pituitary responsiveness was the same for alcohol and saline groups. In contrast to LH, alcohol did not significantly alter pulsatile FSH secretion, indicating that alcohol selectively inhibited pulsatile release of LHRH but not the putative FSHRF. Furthermore, the secretion of LH by pituitaries incubated in vitro in the presence of different concentrations of alcohol (50-100 mM) was the same as without alcohol. Similar results were obtained when THC was injected into the third cerebral ventricle. A single dose of THC (2 ~1 of lop6 M) significantly decreased serum LH levels as compared to values in vehicle injected rats (Wenger et al., 1987). Also, there was no effect on plasma FSH levels as seen with alcohol. Furthermore, the response to a challenge dose of LHRH on LH secretion by cultured dispersed pituitary cells was the same in the presence of THC or vehicle. Therefore, we investigated the effect of both drugs on LHRH release from medial basal hypothalami (MBH) incubated in an in vitro system.
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Effect of alcohol on LHRH release To understand the inhibitory pathways involved in the inhibition of LHRH release by alcohol, it is necessary to put them into the context of our previous work on the NOergic control of LHRH release. Our previous work indicated that the release of LHRH is controlled by nitric oxide (NO) (Rettori et al., 1993; Canteros et al., 1996). NO is formed by conversion of arginine to citrulline and NO in equimolar concentrations by the action of nitric oxide synthase (NOS). There are three isoforms of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutive, and need the presence of Ca2+ to form NO, the inducible NOS (iNOS) is Ca2+ independent and is induced mostly by endotoxins as well by cytokines (Moncada et al., 1991). In the present work we will be referring to constitutive NOS, mainly to nNOS. nNOS has been demonstrated by immunocytochemical methods in neurons in some areas of the CNS including some regions of hypothalamus, as the median eminencearcuate region. Previous research has indicated that NO stimulates the release of LHRH both in vivo and in vitro. On the basis of in vitro experiments using incubations of MBH in a static incubation system, it has been determined that norepinephrine (NE) activates constitutive NOS in this region. The NO released from these neurons diffuses to LHRH terminals, where it induces the release of LHRH. It has been shown that NO not only activates guanylate cyclase followed by increased cyclic guanosine monophosphate (cGMP) release but also activates cyclooxygenase (COX) that increases release of eicosanoids (Rettori et al., 1992). Prostaglandin E2 (PGE2) by activating adenylate cyclase (Ojeda et al., 1979) with consequent increase in cyclic adenosine monophosphate (CAMP) evokes exocytosis of LHRH granules by activation of protein kinase A. The LHRH released diffuses into the hypophyseal portal vessels that deliver it to the anterior pituitary gland where it acts on gonadotropes to release LH. Support for this theoretical pathway stems from the ability of inhibitors of NOS, such as NG-monomethyl-L-arginine, to inhibit LHRH release, whereas releasers of NO, such as sodium nitroprusside (NP), induce LHRH release as well as that of PGE2 from MBH (Rettori et al., 1992). The release of LHRH is not only under the control of stimulatory neurotransmitters such as NE (Rettori
et al., 1993) and glutamic acid (Rettori et al., 1994) via NO, but is also under the control of inhibitory neurotransmiters such as gamma-amino butyric acid (GABA) (Masotto et al., 1989) and beta-endorphin (Lomniczi et al., 2000) both of which inhibit LHRH release. The inhibitory action of GABA on LHRH release could be prevented by hemoglobin (a scavenger of NO), indicating that NO has a stimulatory action on GABA release. It is possible that the increase in GABA release during LHRH secretion induced by NO could be a mechanism to terminate the pulses of LHRH (Seilicovich et al., 1995). Beta-endorphin also can inhibit LHRH release, probably by stimulating p-opiate receptors on NOergic neurons because we have shown that betaendorphin inhibits the activity of NOS in MBH, whereas naltrexone, a p-opiate receptor antagonist, increased the activity of NOS in this tissue. Betaendorphin also blocked the action of sodium nitroprussiate (NP) (a NO donor) on PGE2 release and consequently LHRH secretion (Faletti et al., 1999). Since alcohol has been shown to increase the release of GABA and of beta-endorphin we studied the interactions between GABA, beta-endorphin and alcohol. We confirmed that alcohol increases the release of GABA and beta-endorphin. Furthermore, beta-endorphin also stimulated GABA release, but GABA had no stimulatory action on beta-endorphin release and NP significantly inhibited the release of beta-endorphin from MBH in our in vitro experimental model. Furthermore, alcohol diminished significantly the N-methyl-D-aspartate (NMDA) stimulated NOS and this inhibition could be reversed by addition of naltrexone and bicuculline (a GABA-A receptor antagonist). However, bicuculline ( 10m4 M) could reverse the alcohol induced block of NMDAstimulated LHRH release only when the concentration of alcohol used was 50 mM. At a higher concentration of alcohol such as 100 mM, the inhibitory action of alcohol could be reversed by naltrexone (lop6 M) and not by bicuculline. These results suggest that the primary effect of alcohol is to stimulate beta-endorphin release which in turn stimulates GABA release. Both of these inhibitory neurotransmiters then act together to suppress LHRH release. Since beta-endorphin decreases NOS activity while GABA and alcohol are without effect on NOS activity, they are acting down stream from NOS that is,
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they block the NOergic activation of cyclooxigenase (COX), that is necessary for LHRH release. This conclusion was confirmed, since the addition of an effective concentration of 8-bromo cGMP (a stable analogue of cGMP) to correct for a possible blockade of guanylate cyclase did not reverse the action of GABA or alcohol (Lomniczi et al., 2000). Addition of arachidonate to provide more substrate for COX also failed to reverse this inhibition, suggesting that the primary action of both, GABA and alcohol was by inhibition of COX. This conclusion is supported also by the fact that addition of PGE2 reversed the alcohol block on LHRH release. Recent preliminary results show that alcohol not only inhibits the activity of COX as measured by radio-conversion assay of 14C-arachidonic acid to eicosanoids, such as PGE2 (Canteros et al., 1995) but also decreases COX content in MBH (unpublished observation from a collaborative work with V. Svivaslava and W.L. Dees). In Fig. 1 we present the diagrammatic representation of the postulated mechanism of action of alcohol to suppress NMDA-stimulated LHRH release that we described above.
Effect of the active cannabinoid, delta-9tetrahydrocannabinol (THC) on LHRH release All previous studies including ours in several species (Ayalon et al., 1977; Almirea et al., 1983; Wenger et al., 1987) indicate that the inhibitory effect of THC on the reproductive axis is exerted mainly at the hypothalamic level with the inhibition mainly of LH secretion by the pituitary and consequent alteration of reproductive function. Studies performed by our group (Rettori et al., 1990) using a static incubation system to incubate medial basal hypothalamic (MBH) explants in the presence of different concentrations of THC (IO-” to IO-’ M) showed that THC was without effect on basal release of LHRH. Since it was reported previously (Negro-Vilar et al., 1979), that catecholamines stimulate LHRH release, we used this approach to study the effect of THC on stimulated LHRH release and found as expected, that norepinephrine (NE) (5 x 10e5) as well as dopamine (5 x lop5 M) stimulated significantly the release of LHRH and that it was inhibited by the addition of THC (10s8 M). Since it is also established that PGEz is stimulated by NE and is also part of the
secretory pathway of LHRH release, we measured the release of PGE2 from MBH in the presence of THC and found that THC (lop7 M) lowered significantly the release of PGE:! from MBH as measured by RIA. The inhibition of PGE2 release could also be due to an inhibition of COX activity. Therefore, we performed radio-conversion studies using 14Carachidonic acid and measured the eicosanoids that are formed by the action of COX and found that addition of THC to MBH incubated with 14C-arachidonic acid had a dramatic inhibitory effect on COX activity, since all the eicosanoids measured such as 6-keto Ft,, PGFzc(,PGE2 and TxBz were highly significantly inhibited by THC (10e8 and lop7 M) as compared to values in controls (Rettori et al., 1990). Our findings are in agreement with the known fact that THC and other cannabinoids inhibit adenylate cyclase in a reversible, dose-dependent and stereoselective manner (Bidau-Russell et al., 1990). These facts taken together (inhibition of PGE2 and PGEZ-stimulated CAMP release) explain the inhibition of LHRH release with the consequent lowering of plasma LH levels. The effects of THC as well as of other cannabinoids were believed to be due to a non-specific interaction with the membrane lipids, since cannabinoids are highly lipophilic molecules. But since the discovery of cannabinoid receptor CBl in the brain (Devane et al., 1988), its abundance and anatomical localization, together with the behavioral effects of THC provide the molecular basis for the action of cannabinoids. Till now, two cannabinoid receptors: CBl localized mostly in the brain (Herkenham et al., 1990) and CB2 receptor localized mostly in peripheral tissues and immune cells (Galiegue et al., 1995) have been described. Since then, complimentary DNAs have been cloned, the expression of their genes, and their functional domains have been described. The structure of the CBl receptor exhibits the basic structure of a G-protein-coupled receptor with a molecular weight of 64 kDA (Ameri, 1999). The distribution of CBl has been well described in rat brain by Herkenham et al. (1991) who found that these binding sites are not homogenously distributed. Although they present high density binding by radioautographic method in some areas such as hippocampus, the hypothalamus present sparse binding that is slightly elevated in the ventromedial nu-
-------__ -.__ GABAn
!
LHRH-n ,‘,A -
PGE,
! Lipid
cGMP
1
t GC ’
AC
k
LHRH
Fig. 1. Diagrammatic representation of the postulated mechanism of action of alcohol (EtOH) to suppress NMDA-stimulated LHRH release. For explanation, see text. b-End, beta-endorphin; ~LR, p-opioid receptor; GABA-n, GABA neuron; NO-n, NO-ergic neuron; NE-n, noradrenergic neuron; IX,,, ~11 adrenergic receptor; NMDA-r, NMDA receptor; Glut-n, glutamergic neuron; LHRHn, LHRH neuronal terminal; lipids, membrane phospholipids; PLA2, phospholipase As; GC, guanylate cyclase; AC, adenylate cyclase. Solid arrow indicates stimulation, Dashed arrow indicates inhibition. (From Lomniczi et al., 2000, Proc. Natl. Acad. Sci. USA, 97: 2337-2342.)
cleus (Mailleux and Vanderhaeghen, 1992). In order to find out if the CBl receptors in the hypothalamus are localized on LHRH neurons and terminals,
we performed immunohistochemical studies using an antiserum raised in rabbits against CBl receptors (anti-rat CB 1, working dilution 1 : 200, kindly do-
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Fig. 2. Transversal sections (B) Detail of a neuron with
(20 pm) of rat hypothalamus CB 1 receptors.
immunostained
nated by Dr. K. Mackie, Dept. of Anesthesiology, Univ. of Washington, Seattle, WA, USA). We observed a scattered distribution in the hypothalamus (Fig. 2A and B). Although ir-CBl receptors were distributed in the hypothalamic area where LHRH neurons are found, using double immunohistochemistry techniques, we did not observed colocalization of CB 1 receptors with LHRH (data not shown). These studies suggest that THC most probably is acting on LHRH release by affecting neurotransmitters that are involved in the pathway of LHRH release (Murphy et al., 1998). There is evidence that cannabinoids can inhibit pre-synaptic release of glutamate in rat hippocampus (Shen et al., 1996).
Effect of endogenous cannabinoid, on LHRH release
anandamide,
The discovery of specific cannabinoid receptors mediating the effects of marihuana raised the possibility of the existence of endogenous ligands, similar
with
anti-CBl
receptor
(red).
(A) 3v: third
cerebral
ventricle.
to the endogenous ligands for opiate receptors, such as beta-endorphin. Endogenous substances that bind to cannabinoid receptors and mimic the action of THC isolated from nervous and peripheral tissues are amides and esters of eicosanoid-like fatty acids. The first such substance isolated from porcine brain by Devane et al., 1992 is N-arachidonylethanolamine, named ‘anandamide’ (‘ananda’ means ‘inner bliss’ in Sanskrit) and amide for chemical bonding. Anandamide possess all the properties of a cannabinoid agonist for CBl and CB2 receptors (Felder et al., 1993) and as THC causes inhibition of adenylate cyclase. Another endocannabinoid is 2arachidonylglycerol (Mechoulam et al., 1998). The pathway of anandamide formation is from the hydrolysis of Narachidonoyl-phophatidylethanolamine catalyzed by a phospholipase D-like enzyme (Di Marzo et al., 1994). This pathway suggests that anandamide is formed ‘on demand’ by stimulated cells and accounts for very low levels found in the brain. As with THC, anandamide was found to be able
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to lower plasma LH levels (Wenger et al., 1995). Therefore, we studied the effect of different concentrations of anandamide on LHRH release from MBH in the in vitro system described here. As seen with THC, anandamide (10e9 to 10e6 M) did not modify significantly the basal release of LHRH from MBH incubated in vitro. When LHRH release was stimulated with NMDA (20 mM) the addition of anandamide together with NMDA inhibited the increase in LHRH release that was evoked by NMDA. This inhibition could be completely reversed by addition of GABA-A antagonist (bicuculline, 10m5 M) but not by the opioid antagonist naltrexone (lop6 M). Therefore we studied the effect of anandamide on GABA release from MBH in vitro. The addition of anandamide ( lop9 M) stimulated highly significantly GABA release from MBH. Studies on the effect of anandamide on LH secretion from pituitaries incubated in vitro, showed that only the concentration of lop9 M induced a significant decrease of LH secretion and did not modify the stimulatory response to exogenous LHRH. All these results indicate that anandamide lowers plasma LH mainly by inhibiting the pulsatility of LHRH (since basal release was not modified) and that the pathway is by increased GABA release, a well known inhibitory neurotransmitter that inhibits LHRH release. In conclusion, all these studies indicate that alcohol and plant derived cannabinoid (THC) as well as endogenous compound (anandamide) have a deleterious effect on reproduction in adult male rats, mainly by lowering plasma LH levels exerting its action mainly at the hypothalamic level by inhibiting LHRH release. Also, they share a common pathway by stimulating inhibitory neurotransmitters such as GABA and consequently inhibiting cyclooxygenase with consequent decrease of PGE2 release and inhibition of adenylate cyclase with inhibition of CAMP that is necessary for the extrusion of LHRH from its terminals into hypophyseal portal vessels in order to reach the pituitary gland and release LH from gonadotropes. Acknowledgements This work was supported by Ministery of Public Health ‘Carrillo-Ofiativia’ grant 2001 and BID 802/OC-AR PICDCT No. 5-6117.
References Almirea, R.G., Smith, C.G. and Asch, R.H. (1983) The effects of marihuana and delta-9-tetrahydroanabinol on luteal function in the rhesus monkey. Fert. StetiZ., 39: 212-217. Ameri, A. (1999) The effects of cannabinoids on the brain. Prog. Neurobiol., 58: 315-348. Ayalon, D., Nir, L., Cordova, T., Bauminger, S., Puder, M., Naor, Z., Kashi, B., Zor, U., Harrell, A. and Lindner, H.R. (1977) Acute effect of delta-9-tetrahydrocannabinol on hypothalamopituitary-ovarian axis in the rat. Neuroendocrinology, 23: 3142. Bidau-Russell, M., Devane, W.A. and Howlett, A.C. (1990) Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain. J. Neurochem., 55: 2 l-26. Canteros, G., Rettori, V., Franchi, A., Genaro, A., Cebral, E., Faletti, A., Gimeno, M. and McCann, SM. (1995) Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide. Proc. Narl. Acad. Sci. USA, 92: 3416-3420. Canteros, G., Rettori, V., Genaro, A., Suburo, A., Gimeno, M. and McCann, S.M. (1996) Nitric Oxide synthase content of hypothalamic explants: Increased by norepinephrine and inactivated by NO and cGMP. Proc. Natl. Acad. Sci. USA, 93: 4246-4250. Dees, W.L., Rettori, V., Kozzlowski, G.P. and McCann, S.M. (1985) Ethanol and the pulsatile release of luteinizing hormone, follicle stimulating hormone and prolactin in ovariectomized rats. Alcohol, 2: 641-646. Devane, W.A., Dysarz, EA., Johnson, M.R., Melvin, L.S. and Howlett, A.C. (1988) Determination and characterization of a cannabinoid receptor in rat brain. Mol. Pharmacol., 34: 605613. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor, Science, 258: 1946-1949. Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.C. and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature, 372: 686-69 1. Faletti, A., Mastronardi, CA., Lomniczi, A., Seilicovich, A., Gimeno, M., McCann, S.M. and Rettori, V. (1999) Betaendorphin blocks luteinizing hormone-releasing hormone release by inhibiting the nitric oxidergic pathway controlling its release. Proc. Natl. Acad. Sci. USA, 96: 1722-1726. Felder, C.C., Briley, E.M., Axelrod, J., Simpson, J.T., Mackie, K. and Devane, W.A. (1993) Anandamide, an endogenous cannabinomimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction. Proc. Natl. Acad. Sci. USA, 90: 7656-7660. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carridre, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G. and Casellas, P. (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eu,: J. Biochem., 232: 54-61.
181 Herkenham, M., Lynn, A.B., Little, M.D., Johnson, M.R., Melvin, LX, de Costa, B.R. and Rice, K.C. (1990) Cannabinoid receptor localization in brain. Pmt. N&Z. Acad. Sci. USA, 87: 1932-1936. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, LX, de Costa, B.R. and Rice, K.L. (1991) Characterization and localization of cannabinoid receptors in the rat brain: a quantitative in vitro autoradiographic study. J. Neurosci., 11: 563-583. Lomniczi, A., Mastronardi, C.A., Faletti, A.G., Seilicovich, A., De Laurentiis, A., McCann, SM. and Rettori, V. (2000) Inhibitory pathways and the inhibition of luteinizing hormonereleasing hormone release by alcohol. Proc. Natl. Acad. Sci. USA, 97: 2337-2342. Mailleux, P. and Vanderhaeghen, J.J. (1992) Distribution of the neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry. Neuxwcience, 48: 655-688. Masotto, C., Wisnieski, G. and Negro-Vilar, A. (1989) Different gamma-aminobutyric acid receptor subtypes are involved in the regulation of opiate-dependent and independent luteinizing hormone-releasing hormone secretion. Endocrinology, 125: 548-553. Mechoulam, R., Fide, E. and Di Marzo, V. (1998) Endocannabinoids. Em J. Phamacol., 359: I-18. Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev., 43: 109-l 15. Murphy, L.L., Muhoz, R.M., Adrian, B.A. and Villanua, M.A. (1998) Function of cannabinoid receptors in the neuroendocrine regulation of hormone secretion. Neurobiol. Dis., 5: 432-446. Negro-Vilar, A., Ojeda, S.R. and McCann, S.M. (1979) Catecholaminergic modulation of luteinizing hormone-releasing hormone release by median eminence terminals in vitro. Endocrinology, 104: 1749-1757. Ojeda, S.R., Negro-Vilar, A. and McCann, SM. (1979) Release of prostaglandin E2 by hypothalamic tissue: evidence for their involvement in catecholamine-induced luteinizing hormonereleasing hormone release. Endocrinology, 104: 617-624.
Rettori, V., Aguila, M.C., Gimeno, M.F., Franchi, A.M. and McCann, S.M. (1990) In vitro effect of delta-9-tetrahydrocannabinol to stimulate somatostatin release and block that of luteinizing hormone-releasing hormone by suppression of the release of prostaglandin Ez. Proc. Natl. Acad. Sci. USA, 87: 10063-10066. Rettori, V., Gimeno, M., Lyson, K. and McCann, S.M. (1992) Nitric oxide mediates norepinephrine-induced prostaglandin Es release from the hypothalamus. Proc. Natl. Acad. Sci. USA, 89: 11543-l 1546. Rettori, V., Belova, N., Dees, W.L., Lyson, K., Dees, W.L., Gimeno, M. and McCann, S.M. (1993) Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc. Natl. Acad. Sci. USA, 90: 1013010134. Rettori, V., Kamat, A. and McCann, S.M. (1994) Nitric oxide mediates the stimulation of luteinizing-hormone releasing hormone release induced by glutamic acid in vitro. Brain Rex Bull., 33: 501-503. Seilicovich, A., Duvilanski, B.H., Pisera, D., Thea, S., Gimeno, M., Rettori, V. and McCann, S.M. (1995) Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid. Proc. Natl. Acad. Sci. CJSA,92: 3421-3424. Shen, M., Piser, T.M., Seybold, VS. and Thayer, S.A. (1996) Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures. J. Neurosci., 16: 4322-4334. Wenger, T., Rettori, V., Snyder, G.D., Dalterio, S. and McCann, S.M. (1987) Effects of delta-9-tetrahydrocannabinol on the hypothalamic-pituitary control of luteinizing hormone and follicle-stimulating-hormone secretion in adult male rats. Neuroendocrinology, 46: 488-493. Wenger, T., Toth, B.E. and Martin, B.R. (1995) Effects of anandamide (endogen cannabinoid) on anterior pituitary hormone secretion in adult ovariectomized rats. Life Sci., 56: 20572063.
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Mechanisms of inhibition of LHRH release by alcohol and cannabinoids Valeria Rettori ‘$*, Alejandro Lomniczi ‘, Claudia Mohn ‘, Camila Scorticati ‘, Paula Vissio ‘, Mercedes Lasaga*, Ana Franchi ‘, Samuel M. McCann 3 I Centro
de Estudios Farmacoldgicos y Botdnicos CONICET, Serrano 669, 1414, Buenos Aires, Argentina 2 Centro de Investigaciones en Reproduccidn, Fat. Medicina, UBA, Buenos Aires, Argentina 3 Pennington Biomedical Research Center; LSU, Baton Rouge, LA, USA
Introduction This paper will review our research and that of our associates on the effects of alcohol and cannabinoids on reproduction. Today, alcohol is classified as a psychotropic drug, like delta-9 tetrahydrocannabinol (THC), the active ingredient of marihuana. A large number of neurotransmitters not only the classical ones, but also a host of neuropeptides that can act as neutransmitters or neuromodulators, exist in the central nervous system (CNS) and there is abundant evidence that alcohol and cannabinoids can affect a number of them. Therefore, mechanisms of action of these drugs in the CNS are very complex. Addiction to alcohol or marihuana produces numerous deleterious effects in the organism. Among these alterations is the suppression of reproduction in humans, monkeys and small rodents by inhibition of the release of luteinizing hormone (LH). This inhibition of LH secretion is caused mainly by hypothalamic action to inhibit the release of luteinizing hormone-releasing hormone (LHRH), in vivo and in vitro. In conscious, ovariectomized rats, intragastric administration of alcohol (3 g/kg), a dose that
* Correspondence to: V. Rettori, CEFYBO-CONICET, Serrano 669, 1414, Buenos Aires, Argentina. Fax: +54114963-4473; Tel: +541 l-4855-7204; E-mail: [email protected]
causes mild intoxication, has been shown to produce a marked decrease in plasma LH concentrations on comparison with the unaffected LH values of diluent administered animals (Dees et al., 1985). There was a highly significant decrease in the area under the secretion curve of LH, with a reduction of LH pulses. On the other hand, following a challenge with exogenous LHRH, the response of LH was the same as in controls, indicating that pituitary responsiveness was the same for alcohol and saline groups. In contrast to LH, alcohol did not significantly alter pulsatile FSH secretion, indicating that alcohol selectively inhibited pulsatile release of LHRH but not the putative FSHRF. Furthermore, the secretion of LH by pituitaries incubated in vitro in the presence of different concentrations of alcohol (50-100 mM) was the same as without alcohol. Similar results were obtained when THC was injected into the third cerebral ventricle. A single dose of THC (2 ~1 of lop6 M) significantly decreased serum LH levels as compared to values in vehicle injected rats (Wenger et al., 1987). Also, there was no effect on plasma FSH levels as seen with alcohol. Furthermore, the response to a challenge dose of LHRH on LH secretion by cultured dispersed pituitary cells was the same in the presence of THC or vehicle. Therefore, we investigated the effect of both drugs on LHRH release from medial basal hypothalami (MBH) incubated in an in vitro system.
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Effect of alcohol on LHRH release To understand the inhibitory pathways involved in the inhibition of LHRH release by alcohol, it is necessary to put them into the context of our previous work on the NOergic control of LHRH release. Our previous work indicated that the release of LHRH is controlled by nitric oxide (NO) (Rettori et al., 1993; Canteros et al., 1996). NO is formed by conversion of arginine to citrulline and NO in equimolar concentrations by the action of nitric oxide synthase (NOS). There are three isoforms of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutive, and need the presence of Ca2+ to form NO, the inducible NOS (iNOS) is Ca2+ independent and is induced mostly by endotoxins as well by cytokines (Moncada et al., 1991). In the present work we will be referring to constitutive NOS, mainly to nNOS. nNOS has been demonstrated by immunocytochemical methods in neurons in some areas of the CNS including some regions of hypothalamus, as the median eminencearcuate region. Previous research has indicated that NO stimulates the release of LHRH both in vivo and in vitro. On the basis of in vitro experiments using incubations of MBH in a static incubation system, it has been determined that norepinephrine (NE) activates constitutive NOS in this region. The NO released from these neurons diffuses to LHRH terminals, where it induces the release of LHRH. It has been shown that NO not only activates guanylate cyclase followed by increased cyclic guanosine monophosphate (cGMP) release but also activates cyclooxygenase (COX) that increases release of eicosanoids (Rettori et al., 1992). Prostaglandin E2 (PGE2) by activating adenylate cyclase (Ojeda et al., 1979) with consequent increase in cyclic adenosine monophosphate (CAMP) evokes exocytosis of LHRH granules by activation of protein kinase A. The LHRH released diffuses into the hypophyseal portal vessels that deliver it to the anterior pituitary gland where it acts on gonadotropes to release LH. Support for this theoretical pathway stems from the ability of inhibitors of NOS, such as NG-monomethyl-L-arginine, to inhibit LHRH release, whereas releasers of NO, such as sodium nitroprusside (NP), induce LHRH release as well as that of PGE2 from MBH (Rettori et al., 1992). The release of LHRH is not only under the control of stimulatory neurotransmitters such as NE (Rettori
et al., 1993) and glutamic acid (Rettori et al., 1994) via NO, but is also under the control of inhibitory neurotransmiters such as gamma-amino butyric acid (GABA) (Masotto et al., 1989) and beta-endorphin (Lomniczi et al., 2000) both of which inhibit LHRH release. The inhibitory action of GABA on LHRH release could be prevented by hemoglobin (a scavenger of NO), indicating that NO has a stimulatory action on GABA release. It is possible that the increase in GABA release during LHRH secretion induced by NO could be a mechanism to terminate the pulses of LHRH (Seilicovich et al., 1995). Beta-endorphin also can inhibit LHRH release, probably by stimulating p-opiate receptors on NOergic neurons because we have shown that betaendorphin inhibits the activity of NOS in MBH, whereas naltrexone, a p-opiate receptor antagonist, increased the activity of NOS in this tissue. Betaendorphin also blocked the action of sodium nitroprussiate (NP) (a NO donor) on PGE2 release and consequently LHRH secretion (Faletti et al., 1999). Since alcohol has been shown to increase the release of GABA and of beta-endorphin we studied the interactions between GABA, beta-endorphin and alcohol. We confirmed that alcohol increases the release of GABA and beta-endorphin. Furthermore, beta-endorphin also stimulated GABA release, but GABA had no stimulatory action on beta-endorphin release and NP significantly inhibited the release of beta-endorphin from MBH in our in vitro experimental model. Furthermore, alcohol diminished significantly the N-methyl-D-aspartate (NMDA) stimulated NOS and this inhibition could be reversed by addition of naltrexone and bicuculline (a GABA-A receptor antagonist). However, bicuculline ( 10m4 M) could reverse the alcohol induced block of NMDAstimulated LHRH release only when the concentration of alcohol used was 50 mM. At a higher concentration of alcohol such as 100 mM, the inhibitory action of alcohol could be reversed by naltrexone (lop6 M) and not by bicuculline. These results suggest that the primary effect of alcohol is to stimulate beta-endorphin release which in turn stimulates GABA release. Both of these inhibitory neurotransmiters then act together to suppress LHRH release. Since beta-endorphin decreases NOS activity while GABA and alcohol are without effect on NOS activity, they are acting down stream from NOS that is,
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they block the NOergic activation of cyclooxigenase (COX), that is necessary for LHRH release. This conclusion was confirmed, since the addition of an effective concentration of 8-bromo cGMP (a stable analogue of cGMP) to correct for a possible blockade of guanylate cyclase did not reverse the action of GABA or alcohol (Lomniczi et al., 2000). Addition of arachidonate to provide more substrate for COX also failed to reverse this inhibition, suggesting that the primary action of both, GABA and alcohol was by inhibition of COX. This conclusion is supported also by the fact that addition of PGE2 reversed the alcohol block on LHRH release. Recent preliminary results show that alcohol not only inhibits the activity of COX as measured by radio-conversion assay of 14C-arachidonic acid to eicosanoids, such as PGE2 (Canteros et al., 1995) but also decreases COX content in MBH (unpublished observation from a collaborative work with V. Svivaslava and W.L. Dees). In Fig. 1 we present the diagrammatic representation of the postulated mechanism of action of alcohol to suppress NMDA-stimulated LHRH release that we described above.
Effect of the active cannabinoid, delta-9tetrahydrocannabinol (THC) on LHRH release All previous studies including ours in several species (Ayalon et al., 1977; Almirea et al., 1983; Wenger et al., 1987) indicate that the inhibitory effect of THC on the reproductive axis is exerted mainly at the hypothalamic level with the inhibition mainly of LH secretion by the pituitary and consequent alteration of reproductive function. Studies performed by our group (Rettori et al., 1990) using a static incubation system to incubate medial basal hypothalamic (MBH) explants in the presence of different concentrations of THC (IO-” to IO-’ M) showed that THC was without effect on basal release of LHRH. Since it was reported previously (Negro-Vilar et al., 1979), that catecholamines stimulate LHRH release, we used this approach to study the effect of THC on stimulated LHRH release and found as expected, that norepinephrine (NE) (5 x 10e5) as well as dopamine (5 x lop5 M) stimulated significantly the release of LHRH and that it was inhibited by the addition of THC (10s8 M). Since it is also established that PGEz is stimulated by NE and is also part of the
secretory pathway of LHRH release, we measured the release of PGE2 from MBH in the presence of THC and found that THC (lop7 M) lowered significantly the release of PGE:! from MBH as measured by RIA. The inhibition of PGE2 release could also be due to an inhibition of COX activity. Therefore, we performed radio-conversion studies using 14Carachidonic acid and measured the eicosanoids that are formed by the action of COX and found that addition of THC to MBH incubated with 14C-arachidonic acid had a dramatic inhibitory effect on COX activity, since all the eicosanoids measured such as 6-keto Ft,, PGFzc(,PGE2 and TxBz were highly significantly inhibited by THC (10e8 and lop7 M) as compared to values in controls (Rettori et al., 1990). Our findings are in agreement with the known fact that THC and other cannabinoids inhibit adenylate cyclase in a reversible, dose-dependent and stereoselective manner (Bidau-Russell et al., 1990). These facts taken together (inhibition of PGE2 and PGEZ-stimulated CAMP release) explain the inhibition of LHRH release with the consequent lowering of plasma LH levels. The effects of THC as well as of other cannabinoids were believed to be due to a non-specific interaction with the membrane lipids, since cannabinoids are highly lipophilic molecules. But since the discovery of cannabinoid receptor CBl in the brain (Devane et al., 1988), its abundance and anatomical localization, together with the behavioral effects of THC provide the molecular basis for the action of cannabinoids. Till now, two cannabinoid receptors: CBl localized mostly in the brain (Herkenham et al., 1990) and CB2 receptor localized mostly in peripheral tissues and immune cells (Galiegue et al., 1995) have been described. Since then, complimentary DNAs have been cloned, the expression of their genes, and their functional domains have been described. The structure of the CBl receptor exhibits the basic structure of a G-protein-coupled receptor with a molecular weight of 64 kDA (Ameri, 1999). The distribution of CBl has been well described in rat brain by Herkenham et al. (1991) who found that these binding sites are not homogenously distributed. Although they present high density binding by radioautographic method in some areas such as hippocampus, the hypothalamus present sparse binding that is slightly elevated in the ventromedial nu-
-------__ -.__ GABAn
!
LHRH-n ,‘,A -
PGE,
! Lipid
cGMP
1
t GC ’
AC
k
LHRH
Fig. 1. Diagrammatic representation of the postulated mechanism of action of alcohol (EtOH) to suppress NMDA-stimulated LHRH release. For explanation, see text. b-End, beta-endorphin; ~LR, p-opioid receptor; GABA-n, GABA neuron; NO-n, NO-ergic neuron; NE-n, noradrenergic neuron; IX,,, ~11 adrenergic receptor; NMDA-r, NMDA receptor; Glut-n, glutamergic neuron; LHRHn, LHRH neuronal terminal; lipids, membrane phospholipids; PLA2, phospholipase As; GC, guanylate cyclase; AC, adenylate cyclase. Solid arrow indicates stimulation, Dashed arrow indicates inhibition. (From Lomniczi et al., 2000, Proc. Natl. Acad. Sci. USA, 97: 2337-2342.)
cleus (Mailleux and Vanderhaeghen, 1992). In order to find out if the CBl receptors in the hypothalamus are localized on LHRH neurons and terminals,
we performed immunohistochemical studies using an antiserum raised in rabbits against CBl receptors (anti-rat CB 1, working dilution 1 : 200, kindly do-
179
Fig. 2. Transversal sections (B) Detail of a neuron with
(20 pm) of rat hypothalamus CB 1 receptors.
immunostained
nated by Dr. K. Mackie, Dept. of Anesthesiology, Univ. of Washington, Seattle, WA, USA). We observed a scattered distribution in the hypothalamus (Fig. 2A and B). Although ir-CBl receptors were distributed in the hypothalamic area where LHRH neurons are found, using double immunohistochemistry techniques, we did not observed colocalization of CB 1 receptors with LHRH (data not shown). These studies suggest that THC most probably is acting on LHRH release by affecting neurotransmitters that are involved in the pathway of LHRH release (Murphy et al., 1998). There is evidence that cannabinoids can inhibit pre-synaptic release of glutamate in rat hippocampus (Shen et al., 1996).
Effect of endogenous cannabinoid, on LHRH release
anandamide,
The discovery of specific cannabinoid receptors mediating the effects of marihuana raised the possibility of the existence of endogenous ligands, similar
with
anti-CBl
receptor
(red).
(A) 3v: third
cerebral
ventricle.
to the endogenous ligands for opiate receptors, such as beta-endorphin. Endogenous substances that bind to cannabinoid receptors and mimic the action of THC isolated from nervous and peripheral tissues are amides and esters of eicosanoid-like fatty acids. The first such substance isolated from porcine brain by Devane et al., 1992 is N-arachidonylethanolamine, named ‘anandamide’ (‘ananda’ means ‘inner bliss’ in Sanskrit) and amide for chemical bonding. Anandamide possess all the properties of a cannabinoid agonist for CBl and CB2 receptors (Felder et al., 1993) and as THC causes inhibition of adenylate cyclase. Another endocannabinoid is 2arachidonylglycerol (Mechoulam et al., 1998). The pathway of anandamide formation is from the hydrolysis of Narachidonoyl-phophatidylethanolamine catalyzed by a phospholipase D-like enzyme (Di Marzo et al., 1994). This pathway suggests that anandamide is formed ‘on demand’ by stimulated cells and accounts for very low levels found in the brain. As with THC, anandamide was found to be able
180
to lower plasma LH levels (Wenger et al., 1995). Therefore, we studied the effect of different concentrations of anandamide on LHRH release from MBH in the in vitro system described here. As seen with THC, anandamide (10e9 to 10e6 M) did not modify significantly the basal release of LHRH from MBH incubated in vitro. When LHRH release was stimulated with NMDA (20 mM) the addition of anandamide together with NMDA inhibited the increase in LHRH release that was evoked by NMDA. This inhibition could be completely reversed by addition of GABA-A antagonist (bicuculline, 10m5 M) but not by the opioid antagonist naltrexone (lop6 M). Therefore we studied the effect of anandamide on GABA release from MBH in vitro. The addition of anandamide ( lop9 M) stimulated highly significantly GABA release from MBH. Studies on the effect of anandamide on LH secretion from pituitaries incubated in vitro, showed that only the concentration of lop9 M induced a significant decrease of LH secretion and did not modify the stimulatory response to exogenous LHRH. All these results indicate that anandamide lowers plasma LH mainly by inhibiting the pulsatility of LHRH (since basal release was not modified) and that the pathway is by increased GABA release, a well known inhibitory neurotransmitter that inhibits LHRH release. In conclusion, all these studies indicate that alcohol and plant derived cannabinoid (THC) as well as endogenous compound (anandamide) have a deleterious effect on reproduction in adult male rats, mainly by lowering plasma LH levels exerting its action mainly at the hypothalamic level by inhibiting LHRH release. Also, they share a common pathway by stimulating inhibitory neurotransmitters such as GABA and consequently inhibiting cyclooxygenase with consequent decrease of PGE2 release and inhibition of adenylate cyclase with inhibition of CAMP that is necessary for the extrusion of LHRH from its terminals into hypophyseal portal vessels in order to reach the pituitary gland and release LH from gonadotropes. Acknowledgements This work was supported by Ministery of Public Health ‘Carrillo-Ofiativia’ grant 2001 and BID 802/OC-AR PICDCT No. 5-6117.
References Almirea, R.G., Smith, C.G. and Asch, R.H. (1983) The effects of marihuana and delta-9-tetrahydroanabinol on luteal function in the rhesus monkey. Fert. StetiZ., 39: 212-217. Ameri, A. (1999) The effects of cannabinoids on the brain. Prog. Neurobiol., 58: 315-348. Ayalon, D., Nir, L., Cordova, T., Bauminger, S., Puder, M., Naor, Z., Kashi, B., Zor, U., Harrell, A. and Lindner, H.R. (1977) Acute effect of delta-9-tetrahydrocannabinol on hypothalamopituitary-ovarian axis in the rat. Neuroendocrinology, 23: 3142. Bidau-Russell, M., Devane, W.A. and Howlett, A.C. (1990) Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain. J. Neurochem., 55: 2 l-26. Canteros, G., Rettori, V., Franchi, A., Genaro, A., Cebral, E., Faletti, A., Gimeno, M. and McCann, SM. (1995) Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide. Proc. Narl. Acad. Sci. USA, 92: 3416-3420. Canteros, G., Rettori, V., Genaro, A., Suburo, A., Gimeno, M. and McCann, S.M. (1996) Nitric Oxide synthase content of hypothalamic explants: Increased by norepinephrine and inactivated by NO and cGMP. Proc. Natl. Acad. Sci. USA, 93: 4246-4250. Dees, W.L., Rettori, V., Kozzlowski, G.P. and McCann, S.M. (1985) Ethanol and the pulsatile release of luteinizing hormone, follicle stimulating hormone and prolactin in ovariectomized rats. Alcohol, 2: 641-646. Devane, W.A., Dysarz, EA., Johnson, M.R., Melvin, L.S. and Howlett, A.C. (1988) Determination and characterization of a cannabinoid receptor in rat brain. Mol. Pharmacol., 34: 605613. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor, Science, 258: 1946-1949. Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.C. and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature, 372: 686-69 1. Faletti, A., Mastronardi, CA., Lomniczi, A., Seilicovich, A., Gimeno, M., McCann, S.M. and Rettori, V. (1999) Betaendorphin blocks luteinizing hormone-releasing hormone release by inhibiting the nitric oxidergic pathway controlling its release. Proc. Natl. Acad. Sci. USA, 96: 1722-1726. Felder, C.C., Briley, E.M., Axelrod, J., Simpson, J.T., Mackie, K. and Devane, W.A. (1993) Anandamide, an endogenous cannabinomimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction. Proc. Natl. Acad. Sci. USA, 90: 7656-7660. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carridre, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G. and Casellas, P. (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eu,: J. Biochem., 232: 54-61.
181 Herkenham, M., Lynn, A.B., Little, M.D., Johnson, M.R., Melvin, LX, de Costa, B.R. and Rice, K.C. (1990) Cannabinoid receptor localization in brain. Pmt. N&Z. Acad. Sci. USA, 87: 1932-1936. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, LX, de Costa, B.R. and Rice, K.L. (1991) Characterization and localization of cannabinoid receptors in the rat brain: a quantitative in vitro autoradiographic study. J. Neurosci., 11: 563-583. Lomniczi, A., Mastronardi, C.A., Faletti, A.G., Seilicovich, A., De Laurentiis, A., McCann, SM. and Rettori, V. (2000) Inhibitory pathways and the inhibition of luteinizing hormonereleasing hormone release by alcohol. Proc. Natl. Acad. Sci. USA, 97: 2337-2342. Mailleux, P. and Vanderhaeghen, J.J. (1992) Distribution of the neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry. Neuxwcience, 48: 655-688. Masotto, C., Wisnieski, G. and Negro-Vilar, A. (1989) Different gamma-aminobutyric acid receptor subtypes are involved in the regulation of opiate-dependent and independent luteinizing hormone-releasing hormone secretion. Endocrinology, 125: 548-553. Mechoulam, R., Fide, E. and Di Marzo, V. (1998) Endocannabinoids. Em J. Phamacol., 359: I-18. Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev., 43: 109-l 15. Murphy, L.L., Muhoz, R.M., Adrian, B.A. and Villanua, M.A. (1998) Function of cannabinoid receptors in the neuroendocrine regulation of hormone secretion. Neurobiol. Dis., 5: 432-446. Negro-Vilar, A., Ojeda, S.R. and McCann, S.M. (1979) Catecholaminergic modulation of luteinizing hormone-releasing hormone release by median eminence terminals in vitro. Endocrinology, 104: 1749-1757. Ojeda, S.R., Negro-Vilar, A. and McCann, SM. (1979) Release of prostaglandin E2 by hypothalamic tissue: evidence for their involvement in catecholamine-induced luteinizing hormonereleasing hormone release. Endocrinology, 104: 617-624.
Rettori, V., Aguila, M.C., Gimeno, M.F., Franchi, A.M. and McCann, S.M. (1990) In vitro effect of delta-9-tetrahydrocannabinol to stimulate somatostatin release and block that of luteinizing hormone-releasing hormone by suppression of the release of prostaglandin Ez. Proc. Natl. Acad. Sci. USA, 87: 10063-10066. Rettori, V., Gimeno, M., Lyson, K. and McCann, S.M. (1992) Nitric oxide mediates norepinephrine-induced prostaglandin Es release from the hypothalamus. Proc. Natl. Acad. Sci. USA, 89: 11543-l 1546. Rettori, V., Belova, N., Dees, W.L., Lyson, K., Dees, W.L., Gimeno, M. and McCann, S.M. (1993) Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc. Natl. Acad. Sci. USA, 90: 1013010134. Rettori, V., Kamat, A. and McCann, S.M. (1994) Nitric oxide mediates the stimulation of luteinizing-hormone releasing hormone release induced by glutamic acid in vitro. Brain Rex Bull., 33: 501-503. Seilicovich, A., Duvilanski, B.H., Pisera, D., Thea, S., Gimeno, M., Rettori, V. and McCann, S.M. (1995) Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid. Proc. Natl. Acad. Sci. CJSA,92: 3421-3424. Shen, M., Piser, T.M., Seybold, VS. and Thayer, S.A. (1996) Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures. J. Neurosci., 16: 4322-4334. Wenger, T., Rettori, V., Snyder, G.D., Dalterio, S. and McCann, S.M. (1987) Effects of delta-9-tetrahydrocannabinol on the hypothalamic-pituitary control of luteinizing hormone and follicle-stimulating-hormone secretion in adult male rats. Neuroendocrinology, 46: 488-493. Wenger, T., Toth, B.E. and Martin, B.R. (1995) Effects of anandamide (endogen cannabinoid) on anterior pituitary hormone secretion in adult ovariectomized rats. Life Sci., 56: 20572063.