P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia

We have investigated increases in cytosolic Ca 2+ in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron‐free glial cultures. In both cultures, the agonist‐response profile was 2‐methylthioADP(2MeSADP)>2‐methylthioATP(2MeSATP)>ADP>ATP>adenosine 5′‐ O ‐(3‐thiotriphosphate), consistent with a P2Y 1 receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. Suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent p A 2 values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. Single cell imaging divided the cells from the mixed neuronal–glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K + ]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K + . In the presence of extracellular Mn 2+ , the response of the mixed cultures to 30 m M K + and 20 μ M Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn 2+ . This further indicated that the 2MeSADP response was not in the neurons. Reverse transcriptase–polymerase chain reaction studies detected transcripts for P2Y 1 , P2Y 4 and P2Y 6 in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y 2 transcripts were not detected in the embryonic cortex. Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca 2+ in glial cells and presynaptic sites on neurons.British Journal of Pharmacology (2003) 139 , 279–288. doi: 10.1038/sj.bjp.0705242