P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones
1. The mobilization of Ca2+ by purinoceptor activation and the relative contributions of intra- and extracellular sources of Ca2+ were investigated using microfluorimetric measurements of fura-2 loaded in cultured neurones from rat intracardiac ganglia. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of mRNA for the protein-coupled P2Y2 and P2Y4 receptors. 3. Brief application of either 300 μM ATP or 300 μM UTP caused transient increases in [Ca2+](i) of 277 ± 22 nM and 267 ± 39 nM, respectively. Removal of external Ca2+ did not significantly reduce these [Ca2+](i) responses. 4. The order of purinoceptor agonist potency for [Ca2+](i) increases was ATP = UTP > 2-MeSATP > ADP >> adenosine, consistent with the profile for P2Y2 purinoceptors. ATP- and UTP-induced rises in [Ca2+](i) were completely and reversibly blocked by 10 μM PPADS (a P2 purinoceptor antagonist) and partially inhibited by 100 μM suramin (a relatively non-specific purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+ -ATPase inhibitor cyclopiazonic acid (10 μM) in Ca2+ -free media, the [Ca2+](i) responses evoked by ATP were progressively decreased and abolished. 6. ATP- and UTP-induced [Ca2+](i) rises were insensitive to pertussis toxin, caffeine (5 nM) and ryanodine (10 μM) but were significantly reduced by U-73122, a phospholipase C (PLC) inhibitor. 7. In fura-2-loaded cells, perforated patch whole-cell recordings show that ATP and UTP evoked slow outward currents at -60 mV, concomitant with the rise in [Ca2+](i), in approximately 30% of rat intracardiac neurones. 8. In conclusion, these results suggest that in rat intracardiac neurones, ATP binds to P2Y2 purinoceptors to transiently raise [Ca2+](i) and activate an outward current. The signalling pathway appears to involve a PTX-insensitive G protein coupled to PLC generation of IP3 which triggers the release of Ca2+ from a ryanodine-insensitive Ca2+ stores(s).