Ca2+ signalling in mouse urethral smooth muscle in situ: role of Ca2+ stores and Ca2+ influx mechanisms

Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca2+ signalling in USMCs in situ, and therefore little is known about the source(s) of Ca2+ required for excitation-contraction coupling. We characterized Ca2+ signalling in USMCs within intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca2+ waves that did not propagate cell-to-cell across muscle bundles. Ca2+ waves increased dramatically in response to the 1 adrenoceptor agonist phenylephrine (10m) and to ATP (10m). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10m). Ca2+ influx and release from sarcoplasmic reticulum stores contributed to Ca2+ waves, as Ca2+ free bathing solution and blocking the sarcoplasmic Ca2+-ATPase abolished activity. Intracellular Ca2+ release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100m) and xestospongin C (1m) reduced Ca2+ waves. Ca2+ waves were insensitive to L-type Ca2+ channel modulators nifedipine (1m), nicardipine (1m), isradipine (1m) and FPL 64176 (1m), and were unaffected by the T-type Ca2+ channel antagonists NNC-550396 (1m) and TTA-A2 (1m). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10m) and by an Orai antagonist, GSK-7975A (1m). The latter also reduced urethral contractions induced by phenylephrine, suggesting that Orai can function effectively as a receptor-operated channel. In conclusion, Ca2+ waves in mouse USMCs are a source of Ca2+ for excitation-contraction coupling in urethral muscles.