Introduction

Urethral smooth muscle (USM) contractions contribute to urethral closure pressure, and are modulated by agonists that liberate Ca²⁺ from intracellular stores via IP₃ receptors (IP₃Rs), such as phenylephrine (PE), or arginine vasopressin (AVP). Voltage dependent L-type Ca²⁺ channels (LTCC) contribute to USM contractility in numerous species, however a previous study in mice failed to detect any effect of LTCC inhibitors (nifedipine, nicardipine) on male USM contractions induced by PE (Drumm et al., 2018). Conversely, inhibiting store-operated Ca²⁺ entry (SOCE) with Orai channel inhibitors markedly reduced PE responses. However, this study was limited in that only high PE concentrations (10 mM) were tested on USM responses and contractions evoked by nerve stimulation were not assessed.

Hypothesis/Aims

We hypothesised that contributions of LTCC may be apparent at lower agonist concentrations or during nerve stimulation, rationalizing exclusively high agonist doses would cause exaggerated Ca²⁺ release from internal stores and activate SOCE, masking possible contributions of LTCC to lower magnitude responses, thus importance of LTCC in mouse USM contractility under a range of experimental conditions may be underestimated. To test this, we carefully examined SOCE and LTCC contribution to murine USM contractile behaviours.

Results

qPCR revealed male and female USM expressed transcripts for LTCC (Cacna1a,c,d,f) and Orai1-3 paralogs. Orai1-3 expression was 4-5x greater in both sexes compared to LTCC (n=3). In male USM organ bath experiments, a range of PE concentrations (0.1-30 mM) or electrical field stimulation (EFS) at 1, 2, 5, 10 Hz  (30 sec), led to dose-dependent and frequency dependent increases in contractile area and amplitude respectively. Nifedipine failed to affect PE or EFS evoked contractions at any agonist concentration (n=6) or EFS frequency (n=6). Sustained female USM contractions in response to 10 nM AVP were unaffected by nifedipine (n=5). FPL64176 (LTCC agonist) slightly increased EFS response amplitude by ~10% at frequencies >5Hz, and this was reversed by nifedipine (n=6). FPL64176 slightly increased (~10%) PE evoked contractile area of male USM at agonist concentrations of 0.3-30 mM (n=6), but did not affect AVP responses in female USM (n=5). All responses to PE, EFS and AVP were abolished by the IP₃R antagonist 2-APB, highlighting dependence on sarcoplasmic reticulum (SR) Ca²⁺ release. Inhibition of SOCE with the Orai antagonist GSK 7975A (10 mM) significantly reduced PE induced contractile area of male USM (>50%, P<0.001) across agonist concentrations 0.1-30 mM (n=3). Amplitudes of EFS evoked contractions in male USM were significantly reduced (~50%) by GSK 7975A (n=13, P-0.002-<0.001), and Synta66 (Orai antagonist, 10 mM, P=0.008-<0.001, n=6). When EFS responses were reduced by either Orai antagonist, further addition of nifedipine caused a 5-20% decrease in amplitude (n=13, P<0.001 (GSK), (n=6, P=0.001, Synta66). Both Orai inhibitors significantly reduced sustained contractions evoked by continuous application of 1 mM PE in males (P<0.01, n=5) or 10 nM AVP in females (P<0.0001, n=6), and subsequent application of nifedipine had no effect.

Conclusions

We conclude that in mouse USM, Ca²⁺ influx via SOCE through Orai channels, and not via LTCC, is a key Ca²⁺ source required to sustain agonist and nerve evoked contractions.