Central-type benzodiazepines and the octadecaneuropeptide modulate the effects of GABA on the release of α-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro

Abstract

The involvement of the GABA-benzodiazepine receptor complex in the regulation of melanotropin secretion has been investigated using perfused frog neurointermediate lobes. The GABA A agonist 3-amino-1 propane sulfonic acid mimicked the biphasic effect of GABA on α-melanocyte-stimulating hormone secretion: a brief stimulation followed by an inhibition of melanotropin secretion. The GABA A antagonist SR 95531 (10 −4 M) inhibited both stimulation and inhibition of α-melanocyte-stimulatmg hormone release induced by GABA (10 −4 M). Since the inhibitory effect of baclofen (10 −4 M) was partially antagonized by SR 95531 (10 −4 M). it appears that the GABAergic control of α-melanocyte-stimulating hormone release is mainly achieved through activation of GABA A receptors. GABA-induced stimulation of α-melanocyte-stimulating hormone release was inhibited by tetrodotoxin (10 −5 M), an Na --channel blocker, or nifedipine (10 −5 M). a voltage-dependent Ca 2+-channel blocker, suggesting that Na + and Ca 2+ ions are involved in the stimulatory phase of GABA action. Only central-type benzodiazepine binding site agonists such as clonazepam (10 −4 M) modified α-melanocyte-stimulating hormone release. In fact, clonazepam (10 −5 to 10 −5 M) led to a dose-dependent potentiation of both GABA-induced stimulation and inhibition of α-melanocyte-stimulating hormone release. This potentiating effect was antagonized by the GABA A antagonist SR 95531 (10 −4 M) or by the central-type benzodiazepine binding site antagonist flumazenil (10 −4 M). whereas picrotoxin (10 −4 M) abolished only the stimulatory phase. In contrast, the peripheral-type benzodiazepine binding site agonist Ro 5-4864 (10 −4 M) was unable to potentiate the effects of GABA on α-melanocyte-stimulating hormone secretion. To investigate the possible involvement of endogenous benzodiazepine binding site ligands in the control of α-melanocyte-stimulating hormone secretion, a radioimmunoassay for the octadecaneuropeptide, a putative endogenous benzodiazepine binding site inhibitor, has been developed. High concentrations of octadecaneuropeptide-like material were found in neurointermediate lobe extracts, suggesting that this peptide may act as a regulator of the GABA-benzodiazepine receptor complex. The administration of synthetic octadecaneuropeptide (10 −6 to 10 −4 M) to perifused neurointermediate lobes markedly reduced the effects of GABA on α-melanocyte-stimulatmg hormone release. In addition, flumazenil (10 −4 M) reversed the inhibitory action of octadecaneuropeptide on GABA-induced modulation of α-melanocyte-stimulating hormone release. Taken together, these results indicate that GABA acts on frog melanotrophs through a GABA receptor complex including a GABA A recognition site, a Cl - channel and a central-type benzodiazepine binding site. Activation of benzodiazepine receptors has the potential to modulate the effects of GABA on α-melanocyte-stimulating hormone secretion, the final response depending on the properties of the benzodiazepine-receptor ligand used: central-type benzodiazepine binding site agonists cause an enhancement of the response to GABA, whereas the endogenous peptidergic ligand octadecaneuropeptide attenuates the action of GABA.