OXIDANT STRESS ACTIVATES A NONSELECTIVE CATION CHANNEL RESPONSIBLE FOR MEMBRANE DEPOLARIZATION IN CALF VASCULAR ENDOTHELIAL-CELLS

1. In vascular endothelial cells, oxidant stress increases cell Na+ co ntent and inhibits the agonist-stimulated influx of external Ca2+. Fur ther, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Naf and Ca2+ homeostasis, the present study examined the effect of ox idant stress on ionic current and channel activity in calf pulmonary a rtery endothelial cells. 2. Voltage-clamped control cells had a zero-c urrent potential of -60 mV. Incubation of cells with the oxidant tert- butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to -4 m V and activation of ionic current equally selective for Na+ and K+. 3. Cell-attached membrane patches made on tBuOOH-treated cells contained ion channels that had a bidirectional conductance of 30 pS and that w ere not present in patches from control cells. Inside-out patches exci sed from oxidant-treated cells showed the channel to be equally select ive for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant-activa ted channels were observed to display two gating modalities that were further evident during analysis of single-channel open probability. Ne ither modality was significantly affected by altering internal [Ca2+] (1 mu M-10 nM). 5. Activation of non-selective channels provides a pos sible mechanism by which oxidants may increase endothelial cell Na+ co ntent. Channel permeability to Ca2+ may account in part for the elevat ion of cytosolic free [Ca2+] that occurs in oxidant-treated cells. 6. Channel activation is associated with membrane depolarization, a mecha nism that may contribute to oxidant inhibition of the agonist-stimulat ed Ca2+ influx pathway.