Inhibition of capacitative Ca²⁺ entry by a Cl^- channel blocker in human endothelial cells

We have used the patch clamp technique in combination with intracellular calcium measurements to measure simultaneously Ca²⁺ entry and ionic currents activated by emptying of intracellular Ca²⁺ stores (capacitative Ca²⁺ entry and Ca²⁺ release-activated Ca²⁺ currents, CRAC) in human endothelial cells from umbilical veins. Intracellular stores were depleted of Ca²⁺ by preincubating endothelial cells for 20 minutes with 2 μM thapsigargin in Ca²⁺-free solution. Reapplication of 10 mM [Ca²⁺]_e evoked an increase in [Ca²⁺]_i indicating Ca²⁺ influx after the thapsigargin-induced store depletion (capacitative Ca²⁺ entry), however no measurable CRAC could be detected. The increase in [Ca²⁺]_i after [Ca²⁺]_e resubmission was substantially reduced in the presence of 50 μM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 ± 0.25 μM to 0.2 ± 0.06 μM (n = 6) at a holding potential of -40 mV. Estimates of the capacitative Ca²⁺ entry at various membrane potentials from the first time derivative of the Ca²⁺ transients showed a highly inwardly rectifying I-V curve with a Ca²⁺ inward current amplitude of 1.0 ± 0.3 pA (membrane capacitance 59 ± 9 pF, n = 8) at -80 mV. This current amplitude was decreased to 0.32 ± 0.12 pA(n = 6) in the presence of 50 μM NPPB. This corresponds to a decrease in the Ca²⁺ permeability of the endothelial cell membrane from 0.15·10^-8 cm/s (control) to 0.06·10^-8 cm/s (50 μM NPPB).