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− ( IClCa) 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 IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa. T16Ainh-A01 and CaCCinh-A01 (10 μM) reduced IClCa by ~85%, while 30 μM abolished it. L-type Ca2+ current was unaffected by 10 μM CaCCinh-A01 but was reduced by 50% at 30 μM CaCCinh-A01, 46% at 10 μM T16Ainh-A01, and 78% at 30 μM T16Ainh-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, CaCCinh-A01; 14 μM, T16Ainh-A01). CaCCinh-A01 at 10 μM had little effect on 60 mM KCl contractures, though they were reduced by 30 μM CaCCinh-A01 and T16Ainh-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 IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 μM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.