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Abstract: GABAB receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABAB receptor antagonist CGP35348 and, in part, the compound CGP52432, are now found to inhibit on their own the K+-evoked exocytosis of glycine when added at low micromolar concentrations to superfused mouse glycinergic nerve endings prelabelled with [3H]glycine through GLYT2 transporters. CGP35348 inhibited [3H]glycine release both in spinal cord and in hippocampus, but was also able to prevent the inhibitory effect of (−)-baclofen; CGP52432 exhibited intrinsic activity only in the hippocampus; in spinal cord, it behaved exclusively as a silent orthosteric antagonist by blocking the release inhibition brought about by (−)-baclofen. The intrinsic activity of CGP35348 in spinal cord was not prevented by CGP52432, indicating that CGP35348 is not a partial GABAB agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABAB antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100–300μM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABAB1 (GABAB1−/− mice) or the GABAB2 (GABAB2−/− mice) subunit, the evoked exocytosis of [3H]glycine was no longer inhibited by (−)-baclofen, whereas the intrinsic activity of CGP35348 and CGP52432 was not decreased. Activation of unknown sites on glycinergic terminals is likely to be involved. These unexpected effects should not be ignored when interpreting results obtained with the above GABAB receptor antagonists. [Copyright &y& Elsevier]

Abstract: Glycine release and modulation of glycine exocytosis by presynaptic receptors have been rarely studied. We here investigate on the existence and the pharmacological profile of GABAB receptors regulating glycine release in mouse spinal cord and hippocampus. Synaptosomes were preincubated with [3H]glycine in presence of the glycine transporter 1 inhibitor NFPS, in order to study release from glycinergic terminals selectively labelled through the glycine transporter 2, and depolarized in superfusion with concentrations of KCl (12 and 15mM) previously found to elicit exocytosis of glycine. Exposure of synaptosomes to (−)-baclofen caused inhibition of [3H]glycine overflow: EC50 =0.62±0.08μM, in the spinal cord and EC50 =0.58±0.07μM, in the hippocampus. The effect of 3μM (−)-baclofen in the spinal cord was prevented by the GABAB receptor antagonists CGP 52432 (IC50 =22.6±2.15μM) and CGP 35348 (IC50 =5.99±1.21μM), whereas phaclofen was ineffective. In the hippocampus, the CGP antagonists were more potent than in the spinal cord, their IC50 values amounting to 0.81±0.08μM and to 0.94±0.09μM, respectively; phaclofen (100–300μM) was a weak antagonist. It is concluded that: (i) glycinergic nerve terminals in spinal cord and hippocampus possess GABAB receptors mediating inhibition of the evoked glycine exocytosis; (ii) the pharmacological profiles of the receptors are qualitatively similar in the two regions; (iii) low concentrations of GABAB antagonists appear able to preferentially inhibit the receptors located on hippocampal glycinergic nerve endings. [Copyright &y& Elsevier]