AB313. SPR-40 Spontaneous Ca(2+) waves in mouse urethral smooth muscle visualized with a genetically encoded Ca(2+) indicator in situ

OBJECTIVE: The urethra generates myogenic tone during bladder filling to maintain continence. There are few visual studies of urethra motor activity and the specific Ca(2+) 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 Ca(2+) imaging with a genetically encoded Ca(2+) reporter, GCaMP3. METHODS: Ca(2+) 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. Ca(2+) 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. RESULTS: Urethral smooth muscle cells fired spontaneous intracellular Ca(2+) waves. Ca(2+) waves did not propagate from cell to cell and activity in individual cells was not correlated with adjacent cells. Ca(2+) waves were significantly increased in frequency, amplitude, duration and spatial spread by the α1a agonist phenylephrine and abolished by the guanylate cyclase donor NONATE. Ca(2+) waves originated from both Ca(2+) influx and Ca(2+) release from intracellular stores as removal of extracellular Ca(2+) and blocking the sarcoplasmic Ca(2+)-ATPase abolished Ca(2+) waves. Ca(2+) waves were insensitive to the L-type Ca(2+) channel blocker nifedipine but were inhibited by blockers of T-type Ca(2+) channels (NNC 55-0396 and TTA-A2) as well as blockers of ryanodine and IP(3) receptors (tetracaine and 2-APB). qPCR revealed that urethral smooth muscle cells expressed the ER Ca(2+) release channels IP(3)R1,2 and RyR1-3 as well as the voltage dependent Ca(2+) influx channels Cacna1s, Cacna1c, Cacna1d, Cacna1f and Cacna1h. CONCLUSIONS: Urethral smooth muscle cells fire spontaneous Ca(2+) waves which are modulated by both adrenergic and nitrergic inputs. These Ca(2+) waves rely on Ca(2+) influx from a non-L type Ca(2+) influx mechanism and release of Ca(2+) from intracellular stores for their generation. FUNDING SOURCE(S): R01 DK-091336, P01 DK41315