P-2 purinoceptors in rat cortical astrocytes: Expression, calcium-imaging and signalling studies

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P-2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P-2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P-2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (I-Cl,I-Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P-2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P-2 purinoceptors. These results indicated that the two P-2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P-2 purinoceptor antagonist (suramin) but not by the P-1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P-2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase. Copyright (C) 1996 IBRO. Published by Elsevier Science Ltd.