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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]

Glycine release has been rarely studied. The aim of this work was to characterize the release of the amino acid from spinal cord glycinergic nerve endings selectively pre-labeled through glycine transporters of the GLYT2 type. Purified mouse spinal cord synaptosomes were incubated with [3H]glycine in the presence of the GLYT1 blocker N-[(3R)-3-([1,1′-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]- N-methylglycine hydrochloride and exposed in superfusion to varying concentrations of KCl, 4-aminopyridine (4-AP), or veratridine. KCl (≤ 15 mmol/L), 4-AP (up to 1 mmol/L), and veratridine (≤ 0.3 μmol/L)-provoked [3H]glycine release by external Ca2+-dependent, botulinum toxin C1-sensitive, exocytosis. The overflows evoked by higher concentrations of K+ or veratridine involved external Ca2+-independent mechanisms of different nature. Only the overflow evoked by 3 or 10 μmol/L veratridine occurred totally (3 μmol/L) or in part (10 μmol/L) by transporter reversal, being sensitive to the GLYT2 blockers 4-benzyloxy-3,5-dimethoxy- N-[1-(dimethylaminociclopentyl)-methyl] benzamide or O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-l-serine; in contrast, the external Ca2+-independent [3H]glycine overflow provoked by 50 mmol/L K+ was transporter-independent. This component of K+-evoked overflow and the GLYT2-independent portion of the 10 μmol/L veratridine-evoked overflow, were largely sensitive to the vesicle depletor bafilomycin or BAPTA-AM and were prevented by blocking the mitochondrial Na+/Ca2+ exchanger with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3 H)-one, indicating the involvement of exocytosis triggered by intraterminal mitochondrial Ca2+ ions. [ABSTRACT FROM AUTHOR]

GABA(B) receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABA(B) 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 [(3)H]glycine through GLYT2 transporters. CGP35348 inhibited [(3)H]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 GABA(B) agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABA(B) antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100-300 microM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABA(B1) (GABA(B1-/-) mice) or the GABA(B2) (GABA(B2-/-) mice) subunit, the evoked exocytosis of [(3)H]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 GABA(B) receptor antagonists., ((c) 2010 Elsevier Ltd. All rights reserved.)