Effects of new-generation TMEM16A inhibitors on calcium-activated chloride currents in rabbit urethral interstitial cells of Cajal

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca²⁺-activated Cl^- currents (I _ClCa) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACC_inh-A01 and T16A_inh-A01, on I _ClCa recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I _ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACC_inh-A01 and T16A_inh-A01 with IC₅₀ values of 1.2 and 3.4 μM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60 mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACC_inh-A01 and T16A_inh-A01. In contrast, spontaneous Ca²⁺ waves in isolated RUICC were only partially reduced by CACC_inh-A01 and T16A_inh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACC_inh-A01 and T16A_inh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.