title: P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones.
abstract: 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 G protein-coupled P2Y2 and P2Y4 receptors. 3. Brief application of either 300 microM ATP or 300 microM 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 microM PPADS (a P2 purinoceptor antagonist) and partially inhibited by 100 microM suramin (a relatively non-specific purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (10 microM) 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 microM) 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 r 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+ store(s).
authors.affiliation: Department of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: D M
authors.initials: DM
authors.lastname: Liu
authors.forename: C
authors.initials: C
authors.lastname: Katnik
authors.forename: M
authors.initials: M
authors.lastname: Stafford
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005726:Ganglia, Parasympathetic / Q000166:cytology / Q000502:physiology; D006321:Heart / Q000294:innervation*; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008959:Models, Neurological; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012433:Ryanodine / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014544:Uridine Triphosphate / Q000378:metabolism / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; C546387:P2ry2 protein, rat; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C104749:purinoceptor P2Y4; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D012433:Ryanodine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D014544:Uridine Triphosphate; C000543:cyclopiazonic acid