title: P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons.
abstract: 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 > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 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.
authors.affiliation: Laboratory of Pharmacology, Dept. of Biomedical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. tohta@vetmed.hokudai.ac.jp
authors.forename: Toshio
authors.initials: T
authors.lastname: Ohta
authors.forename: Akane
authors.initials: A
authors.lastname: Kubota
authors.forename: Matsuka
authors.initials: M
authors.lastname: Murakami
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Otsuguro
authors.forename: Shigeo
authors.initials: S
authors.lastname: Ito
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology* / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D009197:Myenteric Plexus / Q000166:cytology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2
chemical_list: C546403:P2rx2 protein, rat; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D012313:RNA; D000255:Adenosine Triphosphate; D002118:Calcium