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 con tributions of intra-and extracellular sources of Ca2+ were investigated usi ng microfluorimetric measurements of fura-2 loaded in cultured neurones fro m rat intracardiac ganglia. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expres sion of mRNA for the G protein-coupled P2Y(2) and P2Y(4) receptors. 3. Brief application of either 300 mu M ATP or 300 mu M UTP caused transien t increases in [Ca2+], of 277+/-22nM and 267+/-39nM, 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 AT P = UTP > 2-MeSATP > ADP much greater than adenosine, consistent with the p rofile fur P2Y(2) purinoceptors. ATP-and UTP-induced rises in [Ca2+](i) wer e completely and reversibly blocked by 10 mu M PPADS (a P2 Purinoceptor ant agonist) and partially inhibited by 100 mu M suramin (a relatively nonspeci fic purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor cyclo piazonic acid (10 mu M) 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 mM) and ryanodine (10 par) 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 r ise in [Ca2+](i), in approximately 30 % of rat intracardiac neurones. 8. In conclusion, these results suggest that in rat intracardiac neurones, ATP binds to P2Y(2) purinoceptors to transiently raise [Ca2+](i) and activa te an outward current. The signalling pathway appears to involve a PTX inse nsitive G protein coupled to PLC generation of IF, which triggers the relea se of Ca2+ from a ryanodine insensitive Ca2+ store(s).