Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA342

Poster Communications

The contribution of Ca2+-activated chloride channels to corpus cavernosum activity

K. Hannigan1, G. P. Sergeant1, M. Hollywood1, K. D. Thornbury1

1. Dundalk Institute of Technology, Dundalk, Ireland.

Relaxation of smooth muscle present in the corpus cavernosum (CCSM) is responsible for the increase in blood volume required for erection. Ca2+-activated chloride (ClCa) currents have been described in rat and rabbit CCSM cells (1, 2), however a lack of specific ClCa channel blockers has hindered the investigation for the role of these channels. Recently, novel compounds have been designed to specifically target TMEM16A, the proposed molecular structure of the ClCa channel (3). This study aims to investigate the contribution of ClCa to CCSM activity using these novel compounds. Rabbits were euthanised with pentobarbitone and the corpus cavernosum extracted. ClCa and VDCC currents were examined using the perforated patch clamp technique (2, 4). Tension recordings were made from CCSM strips. Ca2+ imaging was carried out using Fluo-4 AM (5). The effects of CaCCinhA01 and T16ainhA01 were examined on ClCa current. 10 mM CaCCinhA01 reduced peak inward ClCa current from -436 ± 146 pA in control, to -14 ± 6 pA (p<0.05, n=7). T16ainhA01 (10 mM) reduced peak inward ClCa current from -268 ± 50 pA to -48 ±28 pA (p<0.05, n=5). The effects of both compounds were examined on voltage-dependent Ca2+ current (VDCC). In the presence of CaCCinhA01 (10 mM) VDCC remained unaffected, however, peak VDCC was reduced in the presence of 10 mM T16ainhA01 from -143 ± 33 pA to -79 ± 23 pA (p<0.05, n=5). At a concentraiton of 30 mM, both compounds reduced VDCC. 30 mM CaCCinhA01 reduced peak VDCC from -154 ± 34 pA to -73 ± 12 pA (p<0.05, n=5), whereas 30 mM T16ainhA01 reduced peak VDCC from -146 ± 34 pA to -33 ± 14 pA (p<0.05, n=5). To investigate if there were non-specific effects of both compounds on tension, effects on KCl contractions were examined. 10 mM CaCCinhA01 reduced KCl-induced contraction to 84% ± 4.6% of the total KCl contracture. 10 mM T16ainhA01 reduced KCl-induced contraction to 64.7% ± 8%. At concentrations of 30 mM, both CaCCinhA01 and T16ainhA01 reduced the KCl-induced contraction to 45% and 46% respectively. As a result of these findings, CaCCinhA01 at a concentration of 10 mM was chosen for further study. CaCCinhA01 reduced both spontaneous and phenylephrine (PE)-induced contractions in a concentration-dependent manner (IC50= 2.9 mM, 95% confidence intervals: 2.2-4 mM). Increases in Ca2+ wave frequency induced by PE were reduced by CaCCinhA01, from 27 ± 4 to 9 ± 3 (p<0.005, n=6). Caffeine-evoked responses remained unaffected by CaCCinhA01, suggesting that it does not affect the Ca2+ store. To determine the effect of VDCC on Ca2+ wave activity, the effect of nifedipine (300 nM) was examined as described above. Nifedipine slowed down PE-induced wave activity, but did not fully abolish it, suggesting that PE-induced Ca2+ wave activity was modulated by Ca2+ influx. These data suggest a role for ClCa channels in the modulation of electrical, mechanical and Ca2+ activity in rabbit CCSM.

Where applicable, experiments conform with Society ethical requirements