Ca2+ signals mediated by P2X-type purinoceptors in cultured cerebellar Purkinje cells

Abstract

We have studied [Ca2+](i) signals elicited by extracellular ATP in cultured cells from postnatal day 7-8 rat cerebellum using single-cell fluorescence microscopy and fura-2. Putative Purkinje cells selected under phase contrast by size and characteristic cytoplasm appearance were uniquely identified by selective labeling with anti-calbindin antibodies. Extracellularly applied ATP (50 μM) evoked fast [Ca2+](i) rises revealed by a rapid and transient increase in fura-2 F340/F380 ratio in all Purkinje cells tested, whereas granule cells failed to show a response to ATP. The mean [Ca2+](i) increase was ~400 nM, comparable to that obtained after glutamate stimulation. The response to ATP was completely abolished by removal of extracellular Ca2+ with EGTA. Conversely, an increased extracellular Mn2+ entry pathway was activated by ATP stimulation. These results indicate that the effect of ATP is mediated by an ionotropic P2X receptor. The action of ATP was mimicked by the analog 2-methylthio-adenosine 5'-triphosphate with similar efficacy but almost half its potency (EC50, 10.6 ± 0.7 vs 21.7 ± 1.9 μM). Other purinergic compounds tested, such as adenosine(5')-tetraphospho-(5')adenosine, adenosine(5')-pentaphospho- (5')adenosine, adenosine 5'-(α,β-methylene) triphosphate, UTP, and adenosine, were completely inactive in eliciting [Ca2+](i) responses. The purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'- disulphonic acid effectively blocked the responses elicited by ATP. Our results demonstrate for the first time the presence of functional ionotropic P2X purinoceptors in the cerebellar Purkinje cells and indicate that their pharmacology is similar to receptors formed by P2X2 subunit oligomers.