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Transient Ca²⁺-Activated Cl^- Currents with Endothelin in Isolated Arteriolar Smooth Muscle Cells of the Choroid

From the Smooth Muscle Group, Physiology Department, Queens University, Belfast, United Kingdom.

PURPOSE. To characterize the effects of endothelin (ET)-1 on the Ca²⁺-activated Cl^- conductance of choroidal arteriolar smooth muscle.

METHODS. Microvascular smooth muscle cells were enzymatically isolated from choroidal arterioles from the eyes of freshly killed rabbits. Cells were voltage-clamped at -60 mV using the whole-cell perforated patch-clamp technique. Internal pipette solutions were K⁺ based and contained amphotericin B (200 µg/ml). The cells were bathed in a 20 mM tetraethyl–ammonium solution to block outward K⁺ currents.

RESULTS. Within 2 to 5 seconds of adding ET-1 (10 nM), inward current pulses were generated at a frequency of around 1 Hz. These evoked transient inward currents were blocked by niflumic acid (10 µM) or anthracene-9-carboxylic acid (1 mM). They were increased 2.4 ± 0.1-fold when Cl^- was replaced by I^- in the bathing medium and lost within 4 minutes when external Cl^- was reduced from 151.6 to 20 mM. The reversal potential was -1 ± 2 mV with 135 mM Cl^- in the recording pipette and with 54 mM Cl it was -18 ± 4 mV. When gramicidin D (100 µg/ml), which maintains [Cl^-]_i, was used instead of amphotericin B, the reversal potential was -18 ± 1 mV. Ca²⁺ release by caffeine (10 mM) produced a single transient inward current. Endothelin-evoked transient inward currents were slowly reduced and eventually abolished in Ca²⁺-free solution (~2 to 3 minutes) and were eliminated after ~30 seconds by the sarcoplasmic reticulum Ca²⁺-uptake inhibitor cyclopiazonic acid (5 µM). The ET_A receptor antagonist BQ123 (1 µM) prevented an effect by endothelin but did not inhibit the current oscillations once they had been triggered.

CONCLUSIONS. In choroidal arteriolar smooth muscle ET-1 evokes transient inward Ca²⁺-activated Cl^- currents induced through the cyclical release and re-uptake of Ca²⁺ from intracellular stores after ET_A receptor stimulation.

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