Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes

We have examined the expression of mRNA for several P2Y nucleotide receptor s by northern blot analysis in purified type 1 cerebellar astrocyte culture s. These results suggest that different P2Y subtypes could be responsible f or ATP metabotropic calcium responses in single type 1 astrocytes. To ident ify these subtypes we have studied the pharmacological profile of ATP calci um responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astro cytes also responded to 2-methylthioATP and ADP challenges. The agonist pot ency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization exp eriments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methy lthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2 Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not b lock UTP responses, and UDP elicited clear intracellular Ca2+ concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium resp onses exhibited different sensitivity to pertussis toxin and different inhi bition patterns in response to P2 antagonists. The P2Y(1)-specific antagoni st N-G-methyl-2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthioATP responses. We can conclude that all single astr ocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y(1) and either P2Y(2) or P2Y(4) receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y(6) subtype, highly se lective for UDP coupled to pertussis-toxin insensitive G protein.