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

摘要

1 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. 2 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. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA_2 values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. 4 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~+. 5 In the presence of extracellular Mn~(2+), the response of the mixed cultures to 30 mM K~+ and 20μM 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. 6 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. 7 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.