The Journal of Neurophysiology Vol. 79 No. 5 May 1998, pp. 2513-2521
Copyright ©1998 The American Physiological Society

Diverse Signal Transduction Pathways Mediated by Endogenous P₂ Receptors in Cultured Rat Cerebral Cortical Neurons

Tomoyuki Nishizaki and Masahiro Mori

Department of Physiology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan

Nishizaki, Tomoyuki and Masahiro Mori. Diverse signal transduction pathways mediated by endogenous P₂ receptors in cultured rat cerebral cortical neurons. J. Neurophysiol. 79: 2513-2521, 1998. The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPS) but not by the G_i/_o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca²⁺ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP₃) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca²⁺ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTPS induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein subunits and increased [Ca²⁺]_i by a mechanism independent of phospholipase C stimulation and IP₃ production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca²⁺ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P₂ receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P₂ receptors.

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