Introduction

Myogenic tone in vascular smooth muscle cells (VSMC) relies on Ca²⁺ influx via voltage-dependent L-type Ca²⁺ channels (LTCC), and LTCC activity is tightly regulated by concurrent hyperpolarizing braking mechanisms of voltage-gated K_v7 channels and large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels activated by sarcoplasmic reticulum (SR) Ca²⁺ sparks (Ma et al., 2020; Dopico et a., 2018). Whether this negative feedback signal is a universal feature shared by visceral smooth muscle remains poorly understood. In murine urethral smooth muscle cells (USMC), LTCCs are expressed but surprisingly fail to contribute to basal tone or contractile responses (Gupta et al., 2023). Drawing a direct comparative parallel to VSMC dynamics, we hypothesized that robust basal activation of K_v7 and/or BK_Ca channels in the mouse urethra exerts a powerful hyperpolarizing brake that completely silences LTCC contribution to contractility, hiding a conserved mechanism between urinary and vascular systems.

Results

All experimental procedures were approved by the DkIT Ethics Committee and conformed to EU guidelines on the care and use of animals in research. In isometric tension experiments, when urethral rings were pre-contracted by phenylephrine (PE, 1 μM), or by electrical field stimulation (EFS), neither XE991 (10 μM, K_v7 inhibitor) nor iberiotoxin (300 nM, BK_Ca inhibitor) had any effect when applied in isolation (n=6, P>0.05). However, joint application of iberiotoxin and XE991 induced robust phasic contractions superimposed on PE responses (n=12, P<0.001) and enhanced EFS-evoked contractions (n=7, P<0.01), both of which were fully reversed by the LTCC inhibitor nifedipine. During in situ Ca²⁺ imaging of USMC, combined application of XE991 and iberiotoxin converted asynchronous, localized intracellular Ca²⁺ signals into coordinated, propagating intercellular waves abolished by nifedipine (n=5). Crucially, defining a key divergence from classic vascular mechanisms, pre-contracted urethral rings were relaxed by the Orai channel inhibitors GSK-7975A (n=6) or Synta 66 (n=5). Under Orai inhibition, XE991 alone successfully evoked nifedipine-sensitive phasic activity, whereas subsequent iberiotoxin addition had no additional effect (n=6, P>0.05), indicating that BK_Ca channel activation in the urethra depends, at least in part, on upstream Orai-mediated Ca²⁺ influx.

Conclusion

These data demonstrate a fundamental similarity between vascular and visceral tissues: both utilize a tandem K_v7/BK_Ca hyperpolarizing brake to dampen LTCC activity. However, while vascular BK_Ca activation typically relies on SR mediated Ca²⁺ sparks, mouse USMC relies on Ca²⁺ influx via Orai channels to drive some element of BK_Ca activity. This comparative insight reveals that while the downstream regulators of contractility control is conserved across smooth muscle types, the architecture of coordination between these players is tissue-specific.