Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP3

Many cellular functions are regulated by agonist-induced InsP(3)-evoked Ca2+ release from the internal store. In non-excitable cells, predominantly, the initial Ca2+ release from the store by InSP3 is followed by a more sustained elevation in [Ca2+]i via store-operated Ca2+ channels as a consequence of depletion of the store. Here, in smooth muscle, we report that the initial transient increase in Ca2+, from the internal store, is followed by a sustained response also as a consequence of depletion of the store (by InsP(3)), but, influx occurs via voltage-dependent Ca2+ channels. Contractions were measured in pieces of whole distal colon and membrane currents and [Ca2+]; in single colonic myocytes. Carbachol evoked phasic and tonic contractions; only the latter were abolished in Ca2+-free solution. The tonic component was blocked by the voltage-dependent Ca2+ channel blocker nimodipine but not by the store-operated channel blocker SKIT 96365. InsP(3) receptor inhibition, with 2-APB, attenuated both the phasic and tonic components. InsP(3) may regulate tonic contractions via sarcolemma Ca2+ entry. In single cells, depolarisation (to similar to-20 mV) elevated [Ca2+]i and activated spontaneous transient outward currents (STOCs). CCh suppressed STOCs, as did caffeine and InSP3. InSP3 receptor blockade by 2-APB or heparin prevented CCh suppression of STOCs; protein kinase inhibition by H-7 or PKC19-36 did not. InsP(3) suppressed STOCs by depleting a Ca2+ store accessed separately by the ryanodine receptor (RyR). Thus depletion of the store by RyR activators abolished the InSSP3-evoked Ca2+ transient. RyR inhibition (by tetracaine) reduced only STOCs but not the InsP(3) transient. InsP(3) contributes to both phasic and tonic contractions. In the former, muscarinic receptor-evoked InSP3 releases Ca2+ from an internal store accessed by both InSP3 and RyR. Depletion of this store by InsP(3) alone suppresses STOCs, depolarises the sarcolemma and permits entry of Ca2+ to generate the tonic component. Therefore, by lowering the internal store Ca2+ content, InsP(3) may generate a sustained smooth muscle contraction. These results provide a mechanism to account for phasic and tonic smooth muscle contraction following receptor activation.