P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+](i)) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+](i) responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+](i) that was greatly decreased by the removal of extracellular Ca2+ concentration ([ Ca2+](o)). The amplitude of the [Ca2+](i) response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+](o)-free solution, ATP produced a small transient rise in [Ca2+](i) similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+](i) was ATP >= adenosine 5'-O-3-triphosphate >= CTP >= 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X(2) receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X(2) receptors, resulting in a [Ca2+](i) increase dependent on [Ca2+](o) in rat myenteric neurons. A small part of the ATP-induced [Ca2+](i) increase may be also mediated via a P2Y receptor-related mechanism.