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

Abstract

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 (⩾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 μM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 μM).

3. Lower [ET-1] (⩽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 μM 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⁺.

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