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Abstract: 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 α7 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 Ca2+-dependent release of preaccumulated d-[3H]aspartate ([3H]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 ∼30 times more potent; the potentiation of the BzATP-evoked release was blocked by methyllycaconitine or α-bungarotoxin. Adding inactive concentrations of (−)-nicotine, epibatidine or choline together with inactive concentrations of BzATP resulted in significant elevation of the [3H]d-ASP release mediated by α7 nAChRs. To conclude, P2X7 receptors and α7 nAChRs seem to co-exist and interact on rat neocortex glutamatergic terminals; in particular, P2X7 receptors exert a permissive role on the activation of α7 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. [Copyright &y& Elsevier]

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 μM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A2A receptor antagonist SCH 58261 (1 μM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A2A receptors that enhance vesicular-like glutamate release during ischemia; A2A receptor antagonists would deserve consideration for their neuroprotective potential. [Copyright &y& Elsevier]

We have investigated the functional relationships between NMDA receptors and the NOS/sGC system in the rat pre-frontal cortex in vivo by microdialysis. cGMP basal levels were sensitive to NOS or sGC inhibitors (l-NARG or ODQ) or NO donors (SNAP) when enzymatic breakdown was blocked by the phosphodiesterase inhibitor IBMX, indicating that basal cGMP production derives, at least in part, from the NOS/sGC pathway activity and that the pre-frontal cortex possesses a very efficient degradation system for cGMP. The glutamate receptor agonist NMDA did not alter extracellular cGMP either in absence or presence of IBMX. cGMP was not augmented when NMDA was co-infused with the NOS substrate l-arginine, the glycine site agonist d-serine or the glutamate receptor agonist AMPA. Interestingly, the selective GABAA receptor antagonist bicuculline enhanced cGMP production, revealing that the cortical NOS/sGC system is tonically inhibited by endogenous GABA. However, in the presence of bicuculline, NMDA did not increase extracellular cGMP. In the presence of bicuculline, blockade of 5-HT1/2 receptors, known to inhibit the NMDA/NOS/sGC pathway, with the antagonist methiothepin did not unmask cGMP elevations by NMDA. Thus, it would seem that NMDA receptors do not regulate cortical NOS/sGC activity that, on the other hand, is modulated by endogenous GABA acting at GABAA receptors. [Copyright &y& Elsevier]

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A1 and A2A receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A1 and A2A receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [3H]d-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca2+ ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [3H]d-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor dl-TBOA prevented the ischemia-evoked efflux of [3H]d-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [3H]d-aspartate or glutamate was increased by the A1 receptor antagonist DPCPX. The A2A antagonist SCH 58261 decreased [3H]d-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC50: 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A1 or the A2A receptor antagonist was found on the ischemia-evoked efflux of [3H]d-aspartate in Ca2+-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A1 and stimulatory A2A receptors that down- or up-regulate the vesicular-like component of glutamate release. [Copyright &y& Elsevier]

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.