The role of Ca²⁺-activated Cl⁻current in tone generation in the rabbit corpus cavernosum

Abstract

Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca²⁺-activated Cl^- (<i>I</i>_ClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation <i>I</i>_ClCa blockers, T16A_inh-A01 and CaCC_inh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca²⁺ current and <i>I</i>_ClCa T16A_inh-A01 and CaCC_inh-A01 (10 μM) reduced <i>I</i>_ClCa by ~85%, while 30 μM abolished it. L-type Ca²⁺ current was unaffected by 10 μM CaCC_inh-A01 but was reduced by 50% at 30 μM CaCC_inh-A01, 46% at 10 μM T16A_inh-A01, and 78% at 30 μM T16A_inh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 μM and >90% at 30 μM. Phenylephrine (PE)-enhanced tension was also reduced (ED₅₀ = 3 μM, CaCC_inh-A01; 14 μM, T16A_inh-A01). CaCC_inh-A01 at 10 μM had little effect on 60 mM KCl contractures, though they were reduced by 30 μM CaCC_inh-A01 and T16A_inh-A01 (10 μM and 30 μM) consistent with their effects on L-type Ca²⁺ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca²⁺ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of <i>I</i>_ClCa, the effect of T16A_inh-A01 on L-type Ca²⁺ current precludes its use for evaluating the role of <i>I</i>_ClCa in tone generation. However, 10 μM CaCC_inh-A01 selectively blocked <i>I</i>_ClCa versus L-type Ca²⁺ current and reduced spontaneous and PE-induced tone, suggesting that <i>I</i>_ClCa is important in maintaining penile detumescence.