STUDIES ON THE MECHANISM OF SHORT-TERM REGULATION OF PULMONARY-ARTERYENDOTHELIAL-CELL NA K PUMP ACTIVITY/

The Na/K pump is critically important in maintenance of cell homeostas is in the face of injury, Little is known about the regulation of endo thelial cell Na/K-pump activity, We previously reported that short-ter m (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery end othelial cells (BPAECs), In this study we investigated the mechanism o f oxidant-induced increases in endothelial Na/K-pump activity, focusin g on short-term modulation of alpha(1)-pump subunit, By using immunofl uorescence microscopy and confocal scanning laser microscopy, we found alpha(1) subunit on both apical and basal aspects of BPAECs without p olarized distribution, Short-term (30-minute) incubation of PAEC monol ayers with H2O2 (1 mmol/L) did not change the relative amounts of alph a(1) subunit in membrane fractions, as assessed by immunoblotting. Pho sphorylation of the alpha(1) subunit also was not affected by H2O2 tre atment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to incr ease PKC activity of endothelial cells, we determined the effects of i nhibition and activation of protein kinase C (PKC) on Na/K-pump activi ty quantitated as ouabain-inhibitable uptake of Rb-86. We also determi ned the effects of PKC activation and inhibition on H2O2-induced incre ases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump acti vity over a 30-minute period in intact monolayers. Inhibition or deple tion of PKC did not prevent H2O2-induced increases in pump activity, T hese results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2 O2 are not mediated by activation of PKC or by changes in the expressi on or phosphorylation of alpha(1) subunit.