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The Journal of Neuroscience, March 1, 1998, 18(5):1704-1712

Ca²⁺ Signals Mediated by P2X-Type Purinoceptors in Cultured Cerebellar Purkinje Cells

Jesús Mateo, Marta García-Lecea, M^a Teresa Miras-Portugal, and Enrique Castro

Departamento Bioquímica, Facultad Veterinaria, Universidad Complutense de Madrid, E-28040 Madrid, Spain

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'-(,-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.

Key words: ATP; ATP receptors; Purkinje cells; cell culture; fura-2 microfluorescence; suramin; PPADS; cerebellum; rat

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Copyright © 1998 Society for Neuroscience  0270-6474/98/1851704-09$05.00/0

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