Work

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

Year: 2018

Type: article

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 activein 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 USMCsin 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.

Source: The Journal of Physiology

DOI: 10.1113/jp275719