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

Abstract

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.