Nonselective cation channels in endothelial cells derived from human umbilical vein.
(i) We have used a combined patch-clamp and fura-2 fluorescence technique to characterize a nonselective cation channel (NSC) in Ea. hy926 (EA) cells, an endothelial cell line derived from human umbilical vein. (ii) Stimulation with ATP, histamine and bradykinin activated slowly and with a long delay after application of the agonist, a nonselective cation current (INSC) which is time- and voltage-independent. The permeability sequence for cations was PNa > PCs >> PNMDG, PCa. In the absence of external Ca2+ and at rather high concentrations, La3+ and Gd3+ blocked INSC. (iii) Single channel analysis revealed that ATP activates in the cell-attached configuration a nonselective cation channel with a conductance of approximately 24 pS and a permeation sequence identical to that of the macroscopic current. The channel activity disappeared after membrane excision. (iv) Activation of NSC required physiological intracellular Ca2+ levels (100 nm or higher). All agonists failed to activate NSC if cytosolic Ca2+ ([Ca2+]i) was lowered by 10 mm BAPTA. Clamping internal Ca2+ at 1 microm sometimes (8 out of 17 cells) spontaneously activated INSC in the absence of any additional stimulus. (v) Application of 2,5-di-tert-butylhydroquinone and internal perfusion of inositol 1,4,5-trisphosphate also activated INSC. The phospholipase C inhibitor, U-73122 inhibited INSC and the sustained Ca2+ plateau during agonist stimulation whereas the inactive analogue, U-73343 had no effect. (vi) These results indicate NSC may act as a Ca2+ entry pathway in endothelium. [Ca2+]i and inositol 1,4,5-trisphosphate play a role in the activation cascade of NSC, and possibly also store depletion.
Kamouchi
M
M
Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium.
Mamin
A
A
Droogmans
G
G
Nilius
B
B
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000502:physiology*; D016257:Fura-2 / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D018408:Patch-Clamp Techniques
D007473:Ion Channels; D006632:Histamine; D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2