We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca(2+) concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y(1)-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that all single astrocytes 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 selective for UDP coupled to pertussis-toxin insensitive G protein.