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HORMONES AND SIGNALING

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

Laboratory of Pharmacology, Department of Biomedical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Submitted 29 March 2005 ; accepted in final form 25 July 2005

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 P2X₂ receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X₂ 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.

enteric neuron; fura-2; patch clamp; purinoceptor

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