Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens.

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [(3)H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR-mAChR agonists acetylcholine (ACh) and carbachol provoked [(3)H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 micromol/L, together with 3, 10, or 100 micromol/L (-)nicotine provoked synergistic effect on [(3)H]DA overflow. The [(3)H]DA overflow elicited by 100 micromol/L (-)nicotine plus 30 micromol/L oxotremorine was reduced by atropine down to the release produced by (-)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (-)nicotine/oxotremorine evoked [(3)H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (-)nicotine/oxotremorine. Similarly to (-)nicotine, the selective alpha4beta2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, alpha4beta2 nAChRs exert a permissive role on the releasing function of reportedly M(5) mAChRs co-existing on the same dopaminergic nerve endings.

Evidence that alpha7 nicotinic receptor modulates glutamate release from mouse neocortical gliosomes.

The presence of nicotinic receptors on astrocytes in human and rat brain has been previously demonstrated however their possible functional role is still poorly understood. In this study we investigated on the presence of nicotinic receptors on gliosomes, purified from mouse cortex, and on their role in eliciting glutamate release. Epibatidine significantly increased basal release of [3H]D-aspartate and of endogenous glutamate from mouse gliosomes but not from synaptosomes. This effect was prevented by methyllycaconitine, alpha-bungarotoxin and mecamylamine but not by dihydro-beta-erythroidine. Epibatidine provoked also a significant increase of calcium concentration in gliosomes but not in synaptosomes; the increase in [Ca2+]i induced by epibatidine and KCl in gliosomes was very similar to each other. The present results indicate that alpha7 nicotinic receptors exist on mouse cortical glial particles and stimulate glutamate release.

P2X(7) receptors exert a permissive role on the activation of release-enhancing presynaptic alpha7 nicotinic receptors co-existing on rat neocortex glutamatergic terminals.

Adenosine triphosphate (ATP) has been reported to enhance the release of glutamate by acting at P2X presynaptic receptors. Acetylcholine (ACh) can elicit glutamate release through presynaptic nicotinic cholinergic receptors (nAChRs) of the alpha7 subtype situated on glutamatergic axon terminals, provided that the terminal membrane is weakly depolarized. Considering that ATP and ACh are co-transmitters, we here investigate on the possibility that P2X and nAChRs co-exist and interact on the same glutamatergic nerve endings using purified rat neocortex synaptosomes in superfusion. ATP evoked Ca(2+)-dependent release of pre-accumulated D-[(3)H]aspartate ([(3)H]D-ASP) as well as of endogenous glutamate; (-)-nicotine, inactive on its own, potentiated the ATP-evoked release. The ATP analogue benzoylbenzoylATP (BzATP) behaved like ATP, but was approximately 30 times more potent; the potentiation of the BzATP-evoked release was blocked by methyllycaconitine or alpha-bungarotoxin. Adding inactive concentrations of (-)-nicotine, epibatidine or choline together with inactive concentrations of BzATP resulted in significant elevation of the [(3)H]D-ASP release mediated by alpha7 nAChRs. To conclude, P2X(7) receptors and alpha7 nAChRs seem to co-exist and interact on rat neocortex glutamatergic terminals; in particular, P2X(7) receptors exert a permissive role on the activation of alpha7 nAChRs, suggesting that ATP may not only evoke glutamate release on its own, but may also regulate the release of the amino acid elicited by ACh.

Nicotine has a permissive role on the activation of metabotropic glutamate 5 receptors coexisting with nicotinic receptors on rat hippocampal noradrenergic nerve terminals.

The existence of metabotropic glutamate receptors (mGluRs) on hippocampal noradrenergic nerve terminals and their interaction with coexisting nicotinic acetylcholine receptors (nAChRs) were investigated in superfused rat synaptosomes using [(3)H]-noradrenaline ([(3)H]-NA) release as a readout. The selective agonist of group I mGluRs, (S)-3,5-dihydroxyphenylglycine (DHPG), inactive on its own, acquired ability to release [(3)H]-NA when added together with (-)-nicotine. The effect of DHPG was prevented by 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of mGluR5, but not by 7-(hydroxyimino)cyclopropane[b]chromen-1-carboxylate ethyl ester (CPCCOEt), selective antagonist of mGluR1. The [(3)H]-NA release evoked by (-)-nicotine plus DHPG was totally abrogated by the nAChR antagonist mecamylamine. Veratrine mimicked the permissive role of (-)-nicotine on the activation of mGluR5 mediating [(3)H]-NA release. The mGluR5-mediated component of the [(3)H]-NA release provoked by DHPG plus (-)-nicotine was blocked by xestospongin C, a selective antagonist of inositoltrisphosphate (IP(3)) receptors. It can be concluded that (i) release-enhancing mGluRs of subtype 5 exist on hippocampal noradrenergic axon terminals; (ii) activation of mGluR5 to mediate IP(3)-dependent NA release requires activation of depolarizing nAChRs coexisting on the same terminals.

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.