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 microM thapsigargin in Ca(2+)-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 microM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 +/- 0.25 microM to 0.2 +/- 0.06 microM (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 microM NPPB. This corresponds to a decrease in the Ca2+ permeability of the endothelial cell membrane from 0.15 x 10(-8) cm/s (control) to 0.06 x 10(-8) cm/s (50 microM NPPB).
Gericke
M
M
KU Leuven, Belgium.
Oike
M
M
Droogmans
G
G
Nilius
B
B
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002463:Cell Membrane Permeability / Q000187:drug effects; D018118:Chloride Channels / Q000037:antagonists & inhibitors*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D009579:Nitrobenzoates / Q000378:metabolism / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D013729:Terpenes / Q000378:metabolism / Q000494:pharmacology*; D019284:Thapsigargin; D014471:Umbilical Veins
D018118:Chloride Channels; D009579:Nitrobenzoates; D013729:Terpenes; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium