A new function for glycine GlyT2 transporters: Stimulation of γ-aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca(2+)-dependent anion channels.

Glycine GlyT2 transporters are localized on glycine-storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ-aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule-associated protein-2-positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [3H]GABA and exposed to glycine. Glycine stimulated [3H]GABA release in a concentration-dependent manner. The glycine effect was insensitive to strychnine or to 5,7-dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca2+ entered by reversal of plasmalemmal Na+/Ca2+ exchangers. A significant portion of the GlyT2-mediated release of [3H]GABA (about 50% of the external Ca(2+)-independent release) occurred by reversal of GABA GAT1 transporters. Na+ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na+/Ca2+ exchangers, causing an increase in cytosolic Ca2+, which in turn triggered a Ca(2+)-induced Ca2+ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca2+ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels.

Milanese M ，Romei C ，Usai C ，Oliveri M ，Raiteri L ... -  《-》

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels.

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.

Romei C ，Raiteri M ，Raiteri L 《-》

GABA release provoked by disturbed Na(+), K(+) and Ca(2+) homeostasis in cerebellar nerve endings: roles of Ca(2+) channels, Na(+)/Ca(2+) exchangers and GAT1 transporter reversal.

GABA release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in purified cerebellar synaptosomes pre-labeled with [(3)H]GABA and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were almost totally external Ca(2+)-dependent. Higher concentrations of KCl or veratridine, but not 4-AP, involved also external Ca(2+)-independent mechanisms. The GABA overflows evoked by veratridine and, less so, the overflow evoked by high K(+), occurred in part by reversal of the GAT1 transporter. None of the depolarizing agents activated store-operated or transient receptor potential or L-type Ca(2+) channels. Only the overflow caused by 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca(2+)-dependent overflows evoked by the three releasers involved reversal of plasmalemmal Na(+)/Ca(2+) exchangers. The overflows evoked by high K(+) or veratridine, but not by 4-AP were evoked by Ca(2+) originated through mitochondrial Na(+)/Ca(2+) exchangers. Ca(2+)-induced Ca(2+) release mediated by inositoltrisphosphate receptors participated exclusively in the GABA release stimulated by high KCl which also occurred in a modest portion through anion channels. Important differences could be observed between the release mechanisms of GABA here described and those previously reported for glycine, in spite of the abundant vesicular co-localization of the two transmitters in cerebellar interneurons.

Romei C ，Sabolla C ，Raiteri L 《-》

Glycine release provoked by disturbed Na⁺, Na⁺ and Ca²⁺ homeostasis in cerebellar nerve endings: roles of Ca²⁺ channels, Na⁺/Ca²⁺ exchangers and GlyT2 transporter reversal.

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [³H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca²⁺-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 μM), but not of 4-AP (1 mM), involved also external Ca²⁺-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca²⁺-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca²⁺ channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca²⁺-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na⁺/Ca²⁺ exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca²⁺ originated through mitochondrial Na⁺/Ca²⁺ exchangers. Ca²⁺-induced Ca²⁺ release (CICR) mediated by inositoltrisphosphate receptors (InsP₃Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP₃R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets.

Romei C ，Di Prisco S ，Raiteri M ，Raiteri L ... -  《-》

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal.

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.

Raiteri L ，Stigliani S ，Patti L ，Usai C ，Bucci G ，Diaspro A ，Raiteri M ，Bonanno G ... -  《JOURNAL OF NEUROSCIENCE RESEARCH》