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

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.

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.

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