CA2-TYPE PURINOCEPTORS IN CULTURED CEREBELLAR PURKINJE-CELLS( SIGNALSMEDIATED BY P2X)

We have studied [Ca2+](i) signals elicited by extracellular ATP in cul tured cells from postnatal day 7-8 rat cerebellum using single-cell fl uorescence microscopy and fura-2. Putative Purkinje cells selected und er phase contrast by size and characteristic cytoplasm appearance were uniquely identified by selective labeling with anti-calbindin antibod ies. Extracellularly applied ATP (50 mu M) evoked fast [Ca2+](i) rises revealed by a rapid and transient increase in fura-2 F-340/F-380 rati o in all Purkinje cells tested, whereas granule cells failed to show a response to ATP The mean [Ca2+](i) increase was similar to 400 nM, co mparable to that obtained after glutamate stimulation. The response to ATP was completely abolished by removal of extracellular Ca2+ with EG TA. Conversely, an increased extracellular Mn2+ entry pathway was acti vated by ATP stimulation. These results indicate that the effect of AT P is mediated by an ionotropic P2X receptor. The action of ATP was mim icked by the analog 2-methylthio-adenosine 5'-triphosphate with simila r efficacy but almost half its potency (EC50, 10.6 +/- 0.7 vs 21.7 +/- 1.9 mu M). Other purinergic compounds tested, such as adenosine(5')-t etraphospho-(5')adenosine adenosine(5')-pentaphospho-(5')adenosine, ad enosine 5'-(alpha,beta-methylene) triphosphate, UTP, and adenosine, we re completely inactive in eliciting [Ca2+](i) responses. The purinocep tor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disul phonic acid effectively blocked the responses elicited by ATP Our resu lts demonstrate for the first time the presence of functional ionotrop ic P2X purinoceptors in the cerebellar Purkinje cells and indicate tha t their pharmacology is similar to receptors formed by P2X(2) subunit oligomers.