Abstract
Key points: Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca2+ influx by store-operated calcium entry was required to maintain Ca2+ release events. Ca2+ release and development of Ca2+ waves appear to be the primary source of Ca2+ for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca2+ entry via L-type or T-type channels was required for responses to α adrenergic responses. 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 α1 adrenoceptor agonist phenylephrine (10 μm) and to ATP (10 μm). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μm). 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 (100 μm) and xestospongin C (1 μm) reduced Ca2+ waves. Ca2+ waves were insensitive to L-type Ca2+ channel modulators nifedipine (1 μm), nicardipine (1 μm), isradipine (1 μm) and FPL 64176 (1 μm), and were unaffected by the T-type Ca2+ channel antagonists NNC-550396 (1 μm) and TTA-A2 (1 μm). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 μm) and by an Orai antagonist, GSK-7975A (1 μm). 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.