Abstract
The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.
Highlights
(f,h), in green) and CK7 (g,i), in red) in cultured urothelial cells
Cultured urothelial cells were transfected with vesicular nucleotide transporter (VNUT)-red fluorescent protein (RFP), followed by staining with MANT-ATP, a fluorescent ATP. (j) Labelling of urothelial cells with VNUT-RFP. (k) Urothelial cells incubated with MNAT-ATP. (l) Merged image, showing VNUT-RFP localized with vesicles where MANT-ATP accumulated. (m–o) Co-localization of VNUT-RFP with quinacrine
We show that VNUT is present and functional in the bladder urothelium and that it plays an essential role in the exocytosis of ATP during stimulation by weak stretch
Summary
(f,h), in green) and CK7 (g,i), in red) in cultured urothelial cells. Cultured urothelial cells were transfected with VNUT-RFP, followed by staining with MANT-ATP, a fluorescent ATP. Urothelial cells were transfected with VNUT-RFP followed by staining with quinacrine. (p) VNUT-positive staining (green) was localized in each urothelial cell layer. (q) Urothelial marker CK7-positive staining (red) in human bladder tissue. We have previously shown that cultured urothelial cells release ATP in response to mechanical stretch stimulation[15]. The mechanisms underlying urothelial ATP release during bladder filling remain unclear. We focused on VNUT in the bladder to clarify the mechanisms that underlie urothelial ATP release, along with the physiological consequences of this. Urothelial VNUT-dependent ATP exocytosis has an important role in urine storage mechanisms that promote bladder relaxation during the early stages of filling
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