Ca2+ signalling in mouse urethral smooth muscle in situ: role of Ca2+ stores and Ca2+ influx mechanisms. (15th April 2018)

• Record Type: Journal Article
• Authors: Drumm, Bernard T.; Rembetski, Benjamin E.; Cobine, Caroline A.; Baker, Salah A.; Sergeant, Gerard P.; Hollywood, Mark A.; Thornbury, Keith D.; Sanders, Kenton M.
• Is Part Of: Journal of physiology. Volume 596:Number 8(2018)
• Page Start: 1433
• Page End: 1466
• Publication Date: 2018-04-15
• Subjects: Ca2+ imaging -- urinary continence -- lower urinary tract -- optogenetics -- store‐operated Ca2+ entry; Physiology -- Periodicals; 612.005
• DOI: 10.1113/JP275719
• Languages: English
• ISSNs: 0022-3751

Abstract

Abstract : Key points: Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca 2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca 2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca 2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca 2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca 2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca 2+ influx by store‐operated calcium entry was required to maintain Ca 2+ release events. Ca 2+ release and development of Ca 2+ waves appear to be the primary source of Ca 2+ for excitation–contraction coupling in the mouse urethra, and no evidence was found that voltage‐dependent Ca 2+ 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 2+ signalling in USMCs in situ, and therefore little is known about the source(s) of Ca 2+ required for excitation–contraction coupling. We characterized Ca 2+ signalling in USMCs within intact urethral muscles using a genetically encoded Ca 2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca 2+ waves that did not propagate cell‐to‐cell across muscle bundles. Ca 2+ waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 μm ) and to ATP (10 μm ). Ca 2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μm ). Ca 2+ influx and release from sarcoplasmic reticulum stores contributed to Ca 2+ waves, as Ca 2+ free bathing solution and blocking the sarcoplasmic Ca 2+ ‐ATPase abolished activity. Intracellular Ca 2+ release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 μm ) and xestospongin C (1 μm ) reduced Ca 2+ waves. Ca 2+ waves were insensitive to L‐type Ca 2+ channel modulators nifedipine (1 μm ), nicardipine (1 μm ), isradipine (1 μm ) and FPL 64176 (1 μm ), and were unaffected by the T‐type Ca 2+ channel antagonists NNC‐550396 (1 μm ) and TTA‐A2 (1 μm ). Ca 2+ waves were reduced by the store operated Ca 2+ 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 2+ waves in mouse USMCs are a source of Ca 2+ for excitation–contraction coupling in urethral muscles. Key points: Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca 2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca 2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca 2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca 2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca 2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca 2+ influx by store‐operated calcium entry was required to maintain Ca 2+ release events. Ca 2+ release and development of Ca 2+ waves appear to be the primary source of Ca 2+ for excitation–contraction coupling in the mouse urethra, and no evidence was found that voltage‐dependent Ca 2+ entry via L‐type or T‐type channels was required for responses to α adrenergic responses.