Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, PC25
ATP inhibits IP3-mediated Ca2+ release in smooth muscle via P2Y1 receptors
D. MacMillan1, C. Kennedy1, J. G. McCarron1
1. SIPBS, University of Strathclyde, Glasgow, United Kingdom.
Introduction The inhibitory neurotransmitter, ATP, is pivotal in regulating smooth muscle activity. The underlying mechanism(s) by which ATP evokes relaxation remain unclear, but have been proposed to involve phospholipase C (PLC)-mediated IP3 production, to evoke Ca2+ release from the internal store and stimulation of Ca2+-activated potassium (KCa) channels to cause membrane hyperpolarization. ATP-induced Ca2+ rises have been reported in smooth muscle, but the frequency with which they are observed are often varied. Indeed, Ca2+ rises to ATP may be either limited or not observed at all. To investigate the role of Ca2+ release in ATP-evoked smooth muscle relaxation, the effect of ATP on IP3-mediated Ca2+ release from the store was examined. Methods Single voltage-clamped myocytes were dissociated  from the colon of guinea-pigs (sacrificed by intraperitoneal injection of euthatal (200 mg/kg) followed by exsanguination). [Ca2+]i was measured as fluorescence using fluo-3. Intracellular Ca2+ release was evoked either by activation of IP3 receptors (IP3R; by carbachol or photolysis of caged IP3) or ryanodine receptors (by caffeine). Results ATP transiently increased [Ca2+]i in only 10% of voltage-clamped single smooth muscle cells. Interestingly, ATP inhibited IP3R-mediated Ca2+ release in cells that did not show a Ca2+ rise in response to purinergic stimulation (1mM, n=5). The reduction in IP3R-mediated Ca2+ release was mimicked by its metabolite, ADP (1mM, n=4), but not by adenosine (1mM, n=4). Furthermore, the inhibitory response to ATP was blocked by the P2Y1R antagonist, MRS2179 (10μM, n=5), and the G-protein antagonist, GDPβS (1mM, n=8), but not by the PLC inhibitor, edelfosine (10μM, n=4). Discussion Since ATP did not release Ca2+, neither activation of KCa channels nor depletion of the store of Ca2+ can explain the inhibitory response to ATP. Neither can the inhibitory response be attributed to breakdown of ATP to adenosine since the nucleoside was without effect on IP3R-mediated Ca2+ release. ADP, however, inhibited Ca2+ release, indicating that the reduction is mediated by P2YR. The inhibitory response to ATP was blocked by MRS2179 and by GDPβS, confirming a role of G-protein coupled P2Y1R in this response. ATP remained effective in inhibiting IP3R-mediated Ca2+ release in the presence of edelfosine, thus excluding a role of PLC. The study reveals, for the first time, an inhibitory effect of P2Y1R activation on IP3R-mediated Ca2+ release, such that purinergic stimulation acts to prevent the excitatory influence of IP3 on smooth muscle and promote relaxation. The observed inhibitory response to ATP appears to be independent of PLC.
Where applicable, experiments conform with Society ethical requirements