Ca signalling in mouse urethral smooth muscle in situ: role of Ca stores and Ca influx mechanisms.

KEY POINTS

Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca influx by store-operated calcium entry was required to maintain Ca release events. Ca release and development of Ca waves appear to be the primary source of Ca for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca 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 Ca signalling in USMCs in situ, and therefore little is known about the source(s) of Ca required for excitation-contraction coupling. We characterized Ca signalling in USMCs within intact urethral muscles using a genetically encoded Ca sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca waves that did not propagate cell-to-cell across muscle bundles. Ca waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 μm) and to ATP (10 μm). Ca waves were inhibited by the nitric oxide donor DEA NONOate (10 μm). Ca influx and release from sarcoplasmic reticulum stores contributed to Ca waves, as Ca free bathing solution and blocking the sarcoplasmic Ca -ATPase abolished activity. Intracellular Ca release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 μm) and xestospongin C (1 μm) reduced Ca waves. Ca waves were insensitive to L-type Ca channel modulators nifedipine (1 μm), nicardipine (1 μm), isradipine (1 μm) and FPL 64176 (1 μm), and were unaffected by the T-type Ca channel antagonists NNC-550396 (1 μm) and TTA-A2 (1 μm). Ca waves were reduced by the store operated Ca 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, Ca waves in mouse USMCs are a source of Ca for excitation-contraction coupling in urethral muscles.