Testosterone regulates urinary bladder smooth muscle function through a signaling mechanism involving direct activation of large conductance voltage‐ and Ca2+‐activated K+ channels
Authors: Hristov Kiril L , Parajuli Shankar P , Petkov Georgi V 
Published: November 2023

In addition to improving sexual function, testosterone replacement therapy has been reported to have beneficial effects in ameliorating lower urinary tract symptoms (LUTS) by increasing bladder capacity, compliance, and decreasing bladder pressure. The cellular mechanism by which testosterone regulates urinary bladder smooth muscle (UBSM) excitability have not been elucidated. Here, we provided a detailed investigation into the regulatory role of testosterone on UBSM excitability in freshly‐isolated guinea pig UBSM cells. We used amphotericin‐B perforated patch‐clamp, which preserves the native intracellular environment of UBSM cells intact, as well as single channel recordings in inside‐out excised membrane patches. Our patch‐clamp data showed that testosterone (100 nM) significantly increased the depolarization‐induced whole cell outward currents in freshly‐isolated guinea pig UBSM cells (n=14). The effect of testosterone was more pronounced at positive membrane potentials, and at +80 mV testosterone increased the current density by ~19% in comparison to control (n=14). The majority of the whole cell outward currents in UBSM cells are conducted by the large conductance voltage‐ and Ca2+‐activated K+ (BK) channels. These channels are key regulators of UBSM excitability and contractility, and modulating their activity could be a basis for the development of promising novel therapies for bladder dysfunction. Our data revealed that selective pharmacological inhibition of the BK channels with paxilline (1 μM) completely abolished the stimulatory effect of testosterone on the whole cell outward currents (n=6), suggesting that testosterone activates the whole‐cell currents due to BK channel stimulation rather than other mechanisms. In UBSM, localized Ca2+ release from sarcoplasmic reticulum activate BK channels and generate transient BK currents (TBKCs). Our data revealed that at holding potential of −20 mV, UBSM cells exhibit TBKC activity which was significantly increased by testosterone (100 nM) with ~27% in comparison to control (n=5). These results provided additional line of evidence for the stimulatory role of testosterone on BK channels in UBSM cells. Furthermore, our current‐clamp recordings revealed that testosterone (100 nM) hyperpolarized the UBSM cell resting membrane potential by ~2.5 mV, consistent with the reported hyperpolarizing effects of BK channel activators in this cell type (n=24). To investigate the intracellular signaling mechanism by which testosterone regulates BK channels, we performed a series of single BK channel recordings in excised membrane patches from UBSM cells. The data showed that testosterone (100 nM) increased the single BK channel open probability by more than 300% in comparison to control (n=9). These results support the concept that in UBSM cells testosterone can activate BK channels through a direct non‐genomic mechanism. In conclusion, we provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in guinea pig UBSM cells, and thus regulate UBSM excitability.Support or Funding InformationSupported by NIH grant R01 DK106964 to Georgi V. Petkov.