Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1 In A7r5 cells loaded with the Ca2+ indicator fura-2, we examined the effect of a Ca2+ channel blocker SK&F 96365 on increases in intracellular free Ca2+ concentrations ([Ca2+](i)) and Mn2+ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed. 2 Higher concentrations (greater than or equal to 10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca2+](i): removal of extracellular Ca2+ abolished only the latter. A blocker of L-type voltage-operated Ca2+ channel (VOC) nifedipine at 1 mu M reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 mu M). 3 Lower [ET-1] (less than or equal to 1 nM) evoked only a sustained elevation in [Ca2+](i) which depends on extracellular Ca2+. The elevation was partly sensitive to nifedipine but not SK&F 96365. 4 In the presence of 1 mu M nifedipine, higher [ET-1] increased the rate of Mn2+ quenching but lower [ET-1] had little effect. 5 In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of -60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca2+ in the bath solution with Mn2+. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365. 6 In a bath solution containing only Ca2+ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn2+, whereas that at higher [ET-1] was partly sensitive to SK&F 96365. 7 These results indicate that in addition to VOC, ET-1 activates two types of Ca2+-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn2+.