Nonselective Cation Channels in Endothelial Cells Derived from Human Umbilical Vein

Abstract.

(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.