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

Methods: Ca²⁺ 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.

Results: Urethral 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.

Conclusions: Urethral 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

Keywords: Urethra; calcium imaging; gene expression; smooth muscle; fluorescence-activated cell sorting