Modulation by GABAB receptor ligands of glycine exocytosis from mouse CNS nerve endings

Luca Raiteri1,2, Elisa Luccini1, Cristina Romei1. 1Department of Experimental Medicine, Pharmacology and Toxicology Section, University of Genoa, Genoa, Italy, 2Center of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy.

Mechanisms of glycine release and its modulation by presynaptic receptors have been rarely studied. We here investigated on the existence and the pharmacological profile of GABAB presynaptic receptors regulating glycine exocytosis in mouse spinal cord and hippocampus. Purified synaptosomes were prelabelled with [3H]glycine in the presence of the GLYT1 transporter blocker NFPS, to selectively label glycinergic terminals through GLYT2, and depolarized in superfusion with 12-15 mM KCl (see 1; 2). The GABAB agonist (-)baclofen inhibited depolarization-evoked [3H]glycine overflow [EC50 = 0.62 ± 0.08 μM (n = 5) in the spinal cord; EC50 = 0.58 ± 0.07 μM (n = 5) in the hippocampus; Emax ∼ 60%]. The effect of 3 μM (-)baclofen in spinal cord percent inhibition was prevented by the GABAB antagonists CGP52432 [IC50 = 22.61 ± 2.15 μM (n = 3)] and CGP35348 [IC50 = 5.99 ± 1.20 μM (n = 3)], whereas phaclofen was ineffective. In the hippocampus, CGP antagonists were more potent CGP52432 and phaclofen (100-300 μM) was a weak antagonist. During these studies it was found, unexpectedly, that CGP35348 inhibited glycine exocytosis on its own, exhibiting similar affinity [EC50 = 1.61 ± 0.19 μM (n = 5) in the spinal cord; EC50 = 0.80 ± 0.09 μM (n = 5) in the hippocampus] but lower efficacy (Emax ∼ 30%) with respect to (-)baclofen; CGP52432 (3-10 μM) also exhibited intrinsic activity in the hippocampus (∼ 35% inhibition), while in the spinal cord it behaved as a silent antagonist. In mice lacking either GABAB1 or GABAB2 subunit, [3H]glycine exocytosis was almost insensitive to (-)baclofen, whereas the effects of CGP compounds were maintained. Activation of unknown sites on glycinergic terminals appears likely, although interactions with allosteric sites on GABAB receptors can not be excluded. To conclude, (i) glycinergic nerve terminals in spinal cord and hippocampus possess release-inhibiting GABAB receptors ; (ii) the pharmacological profiles of these receptors are qualitatively similar in the two regions; (iii) hippocampal receptors appear more sensitive to GABAB antagonists; (iv) the intrinsic effects of CGP compounds on glycine exocytosis should not be ignored during interpretation of results obtained with GABAB antagonists.

(1) Luccini E, Raiteri L J Neurochem 2007; 103(6):2439-48.
(2) Luccini E, Romei C, Raiteri L J Neurochem 2008; 105:2179-89.

This work was supported by grants from the Italian MIUR.