Smooth muscle cells are able to sense changes in mechanical stress due to a number of mechanoreceptors and mechano-gated ion channels, many of which are localized within caveolae. Caveolae are cholesterol enriched membrane microdomains that facilitate or inhibit a myriad of cell-surface receptors and ion channels.  In the bladder, where the morphology of smooth muscle cell membrane changes significantly during distension, bladder smooth muscle (BSM) caveolae may play an important role in mechanotransduction by regulating stretch-activated receptors. BSM expresses the transient receptor potential vanilloid TRPV4. Depending on the cell type, TRPV4 responds to a variety of stimuli including cell stretch. However, neither its physiologic role nor its regulation has been fully elucidated in BSM. In other smooth muscle systems, the activation of TRPV4 triggers the release of adenosine triphosphate (ATP) [1]. Moreover, isolated BSM cells have been shown to release ATP upon stretch [2]. The purpose of this study was to investigate the molecular and functional relationship between TRPV4 and caveolin proteins and determine whether TRPV4 activation in BSM was associated with purinergic signaling.

BSM tissue was procured from C57 adult mice, and the mucosa (uroepithelium and lamina propria) was removed from all preparations. The expression of TRPV4 and its potential co-localization with caveolin-1 (Cav-1) protein was investigated by confocal microscopy immunofluorescence.  The protein interaction between TRPV4 and Cav-1 was carried out by co-immunoprecipitation (Co-IP) and western blotting. For functional studies, the activation of TRPV4 was investigated by ex vivo tissue myography. Mouse BSM tissue was stimulated in organ bath with the TRPV4 selective agonist GSK1016790A (1μM). In separate tissue, BSM responses to GSK1016790A were generated in the presence of apyrase (1µM), or purinergic receptor antagonists (BX430, 50µM) or after the experimental depletion of caveolae [achieved by the cholesterol depleting agent methyl-β-cyclodextrin (mβCD; 10mM, 1hr)]. Functional BSM responses were calculated as mean ± SE and differences between conditions were analyzed by unpair Student T-test. p˂0.05 was considered statistically significant.

Immunostaining of mouse bladder tissue with TRPV4 antibody yielded abundant immunoreactivity within the bundles of detrusor muscle. Double labelling with Cav-1 antibody revealed co-localization between TRPV4 and Cav-1.  The partial interaction between TRPV1 and Cav-1 proteins was confirmed by Co-IP experiments. For functional studies, administration of GSK1016790A induced a tonic contraction of BSM tissue that was completely abolished in BSM tissue pre-treated with the ATP-degrading enzyme apyrase. Moreover, the contractile response generated by GSK1016790A was significantly reduced in the presence of P2X4R antagonist BX430, as well as after the depletion of caveolae with mβCD (figure 1).

The expression of TRPV4 on BSM along with the contractile response elicited by its selective activation are consistent with a physiologic role for this mechanoreceptor in bladder function. The effect of both apyrase and P2X4R antagonist in decreasing BSM response to GSK1016790A suggest that the activation of TRPV4 is associated with the release of ATP acting on an unidentified cell type that expresses P2X4R. The interaction between TRPV4 and Cav-1 together with the effect of mβCD in preventing TRPV4 activation are consistent with a positive regulation of this mechanoreceptor by caveolae.

Our study supports the importance of caveolae integrity for the regulation of specific mechanoreceptor-mediated signaling. Therefore, in conditions associated with a loss in caveolar density (such as overactive bladder or the aging bladder), altered mechanotransduction might be a potential contributor to the changes in BSM contractility.

Takahara N, Ito S, Furuya K, Naruse K, Aso H, Kondo M, Sokabe M, Yoshinori Hasegawa Y. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells Am J Respir Cell Mol Biol. 2014;51(6):772-82.
Cheng Y, Mansfield KJ, Sandow SL, Sadananda P, Burcher E, Moore KH. Porcine bladder urothelial, myofibroblast, and detrusor muscle cells: characterization and ATP release. Front Pharmacol. 2011 Jun 7:2:27. doi: 10.3389/fphar.2011.00027. eCollection 2011.

Continence 19S (2026) 102655
DOI: 10.1016/j.cont.2026.102655