AB313. SPR-40 Spontaneous Ca2+ waves in mouse urethral smooth muscle visualized with a genetically encoded Ca2+ indicator in situ

Abstract

ObjectiveThe urethra generates myogenic tone during bladder filling to maintain continence. There are few visual studies of urethra motor activity and the specific Ca2+ signalling of urethral smooth muscle is relatively unknown. In the present study, we sought to characterize the pattern of smooth muscle cell activation in urethra utilizing Ca2+ imaging with a genetically encoded Ca2+ reporter, GCaMP3.MethodsCa2+ signals in mouse urethra were imaged using GCaMP3, activated by a smooth muscle heavy chain promoter. The urethra was excised and urothelium layer removed by sharp dissection. Ca2+ signals were recorded in situ using an upright fluorescent microscope. Enzymatically dispersed mouse urethral smooth muscle cells with an eGFP tag were purified for qPCR analysis using fluorescence-activated cell sorting.ResultsUrethral smooth muscle cells fired spontaneous intracellular Ca2+ waves. Ca2+ waves did not propagate from cell to cell and activity in individual cells was not correlated with adjacent cells. Ca2+ waves were significantly increased in frequency, amplitude, duration and spatial spread by the α1a agonist phenylephrine and abolished by the guanylate cyclase donor NONATE. Ca2+ waves originated from both Ca2+ influx and Ca2+ release from intracellular stores as removal of extracellular Ca2+ and blocking the sarcoplasmic Ca2+-ATPase abolished Ca2+ waves. Ca2+ waves were insensitive to the L-type Ca2+ channel blocker nifedipine but were inhibited by blockers of T-type Ca2+ channels (NNC 55-0396 and TTA-A2) as well as blockers of ryanodine and IP3 receptors (tetracaine and 2-APB). qPCR revealed that urethral smooth muscle cells expressed the ER Ca2+ release channels IP3R1,2 and RyR1-3 as well as the voltage dependent Ca2+ influx channels Cacna1s, Cacna1c, Cacna1d, Cacna1f and Cacna1h.ConclusionsUrethral smooth muscle cells fire spontaneous Ca2+ waves which are modulated by both adrenergic and nitrergic inputs. These Ca2+ waves rely on Ca2+ influx from a non-L type Ca2+ influx mechanism and release of Ca2+ from intracellular stores for their generation.Funding Source(s)R01 DK-091336, P01 DK41315