Increase of intracellular Ca 2+ by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat

Abstract

Astrocytes express purinergic receptors that are involved in glial–neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed “astroglia” throughout. Astroglia expressed predominantly P2X 4,6,7 as well as P2Y 1,2 receptor-subtypes. Less intensive immunostaining was also found for P2X 5 and P2Y 4,6,13,14 receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca 2+ ([Ca 2+] i). Of the agonists tested, only the P2X 1,3 receptor-selective α,β-methylene ATP was ineffective. Experiments with Ca 2+-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca 2+-ATPase, indicated that the [Ca 2+] i response to most nucleotides, except for ATP and 2′,3′- O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca 2+ from intracellular stores. A Gprotein–mediated release of Ca 2+ is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y 1,2,6,14 and probably also P2Y 4) receptors. Several unidentified P2X receptors, including P2X 7, may also be present, although they appear to only moderately participate in the regulation of [Ca 2+] i. The rise of [Ca 2+] i is due in this case to the transmembrane flux of Ca 2+ via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca 2+] i of cultured astroglia and thereby may play an important role in cell-to-cell signaling.