Abstract
We previously showed that bladder functions are controlled by clock genes with circadian rhythm. The sensation of bladder fullness (SBF) is sensed by mechano-sensor such as Piezo1 and TRPV4 in the mouse bladder urothelium. However, functional circadian rhythms of such mechano-sensors remain unknown. To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+]i) using mouse primary cultured urothelial cells (MPCUCs). Using Ca2+ imaging, stretch-evoked [Ca2+]i was quantified every 4 h in MPCUCs derived from wild-type (WT) and ClockΔ19/Δ19 mice, which showed a nocturia phenotype. Furthermore, a Piezo1 inhibitor GsMTx4 and a TRPV4 inhibitor Ruthenium Red were applied and stretch-evoked [Ca2+]i in MPCUCs was measured to investigate their contribution to SBF. Stretch-evoked [Ca2+]i showed a circadian rhythm in the WT mice. In contrast, ClockΔ19/Δ19 mice showed disrupted circadian rhythm. The administration of both GsMTx4 and Ruthenium Red eliminated the circadian rhythm of stretch-evoked [Ca2+]i in WT mice. We conclude that SBF may have a circadian rhythm, which is created by functional circadian changes of Piezo1 and TRPV4 being controlled by clock genes to be active during wakefulness and inactive during sleep. Abnormalities of clock genes disrupt SBF, and induce nocturia.
Highlights
To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+]i) using mouse primary cultured urothelial cells (MPCUCs)
We revealed that clock genes function to create circadian expressions of mechano-sensor genes such as Piezo[1] and TRPV4 in the mouse bladder urothelium both ex vivo and in vitro[9,10]
We investigated the effect of various conditions on the circadian change of average [Ca2+]i at post-stretch as follows: (N) normal condition; extracted data from Fig. 3, left, at 0 h and 12 h; (a) absence of extracellular Ca2+ [Ca2+ free balanced salt solution (BSS)], (b) treatment with 10 μM GsMTx4, an inhibitor of Piezo[17,11], and (c) treatment with 10 μM Ruthenium Red (RR), an inhibitor of cation channels, including TRPV411 (Figs 4 and 5)
Summary
To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+]i) using mouse primary cultured urothelial cells (MPCUCs). It has been reported that lower urinary tract functions involve a circadian rhythm and Clock mutant (ClockΔ19/Δ19) mice urinated frequently in the sleep phase[2,3,4]. These reports suggested that malfunction of clock genes contributed to nocturia. We checked whether the circadian rhythm of clock genes in the urothelium regulates bladder fullness through the circadian rhythm of [Ca2+]i in the mouse primary cultured urothelial cells (MPCUCs)
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