Calcium sparks activate calcium-dependent Cl− current in rat corpus cavernosum smooth muscle cells

Abstract

Spontaneous transient currents, due to activation of Ca(2+)-dependent K(+) and Cl(-) channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca(2+) events responsible for triggering Ca(2+)-dependent Cl(-) channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca(2+) events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca(2+) (Ca(2+) sparks) in 57% of cells. There was an average of 6.4 +/- 0.5 release sites/cell with a frequency of 0.9 +/- 1 Hz/cell and peak amplitude DeltaF/F(o) of 67 +/- 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP(3)) receptors. Caffeine caused either a global Ca(2+) rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP(3) receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca(2+) sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca(2+) channels, consistent with Ca(2+) influx regulating Ca(2+) release from stores. We establish for the first time that Ca(2+) sparks occur in CCSMC and arise from Ca(2+) release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca(2+) influx in CCSMC.