Abstract: PD70-11
Sources of Funding: none

Introduction

We have previously reported that a peripheral circadian clock exists in the bladder and that connexin 43 (Cx43), a major gap junction protein, oscillates and is a regulator of circadian functional bladder capacity. On the other hand, it has been reported that Cx43 forms hemichannels involved in the release of adenosine triphosphate (ATP) in other cell types. Moreover, it has been also known that extracelluar ATP activates the afferent nerves, which leads to the sensation of bladder fullness. We thus hypothesized that the urothelium had circadian rhythm, which regulated the Cx43 oscillation and function as the hemichannels for ATP release to coordinate the diurnal urination rhythm.

Methods

We measured bioluminescence of cultured urothelium from Per2::luc clock gene reporter mice to identify whether the urothelium had circadian rhythms. We also quantified the expression levels of the major clock genes and Cx43 in the urothelium of 11-week-old C57BL/6 female mice at seven consecutive points every 4 hours by real-time polymerase chain reaction and immunoblotting methods. The concentrations of ATP released into the bladder were measured after bladder distention with 30 cm H2O for 10 minutes at zeitgeber time (ZT) 7, a sleeping phase, and ZT19, an active phase for mice. Additionally, we analyzed the circadian rhythm and Cx43 functions with immortalized human urothelial cells.

Results

The cultured urothelium from Per2::luc mice showed robust oscillations of bioluminescence, while such rhythms were completely lost in that from Per2::luc mice with Bmal1 knockout having a dysfunctional clock. Cx43 in the urothelium demonstrated a circadian rhythm in conjunction with major clock genes with a peak time at ZT19 and with a nadir time at ZT7. The concentration of ATP released into the bladder showed diurnal changes along with the Cx43 expression. In addition, the circadian rhythms of major clock genes and Cx43 were also observed in immortalized human urothelial cells. The concentration of mechanically-induced ATP release had oscillations in correlation with the Cx43 expression. Furthermore, it was significantly higher in the Cx43-overexpressed cells, whereas it was significantly lower in Cx43- knockdown ones or in the presence of GAP19 peptide, a selective Cx43 hemichannel blocker.

Conclusions

A functional circadian rhythm existed in the urothelium, and coordinated the Cx43 expression and function as hemichannels which provided a direct pathway of ATP release for mechanotransduction and signaling in the urothelium.

Funding

none