(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 (I(NSC)) which is time- and voltage-independent. The permeability sequence for cations was P(Na) > P(Cs) >> P(NMDG), P(Ca). In the absence of external Ca2+ and at rather high concentrations, La3+ and Gd3+ blocked I(NSC). (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 μm sometimes (8 out of 17 cells) spontaneously activated I(NSC) 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 I(NSC). The phospholipase C inhibitor, U-73122 inhibited I(NSC) 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.
All Science Journal Classification (ASJC) codes
- Cell Biology