ATP and nitric oxide modulate a Ca²⁺-activated non-selective cation current in macrovascular endothelial cells

We have studied the properties of a non-selective cation current (NSC_Ca) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca²⁺]_i relationships were fitted to a Ca²⁺ binding site model with a concentration for half-maximal activation of 417±76 nM, a Hill coefficient of 2.3±0.8 and a maximum current of -23.9±2.7 pA/pF at -50 mV. The Ca²⁺-activated channel is more permeable to Na⁺ than for Cs⁺ (P_Cs/P_Na=0.58, n=7), but virtually impermeable to Ca²⁺. Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca²⁺]_i were smaller than in the absence of ATP, but were not significantly different at 2 μM. The intracellular Ca²⁺ concentration for half-maximal activation of the Ca²⁺-activated current was shifted to 811±12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylim-idodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC_Ca in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC_Ca. In contrast, nitro-L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC_Ca, whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC_Ca. It is concluded that the Ca²⁺-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.