P-2 PURINOCEPTORS IN RAT CORTICAL ASTROCYTES - EXPRESSION, CALCIUM-IMAGING AND SIGNALING STUDIES

Extracellular ATP is known to activate intracellular enzymes in astroc ytes 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 cerebr al cortices were identified in receptor expression experiments, when a strocytic messenger RNA was injected into Xenopus oocytes and recombin ant P-2 purinoceptors were characterized pharmacologically. In messeng er RNA-injected oocytes, ATP evoked inward chloride currents (I-Cl,I-C a) typical of stimulating metabotropic receptors that release intracel lular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hi ll coefficient was 0.5, which indicated that ATP stimulated two subtyp es 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. Re sponses to ATP were antagonized by the P-2 purinoceptor antagonist (su ramin) but not by the P-1 purinoceptor blocker (sulphophenyltheophylli ne). Findings in expression studies were confirmed in assays of intrac ellular signalling systems using primary cultures of rat astrocytes. U TP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP o r 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as m easured by fura-2/AM luminescence) which, in cross-desensitization exp eriments, indicated the involvement of two subtypes of P-2 purinocepto rs. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, rais e intracellular Ca2+ and also stimulate mitogen-activated protein kina se. Copyright (C) 1996 IBRO. Published by Elsevier Science Ltd.