Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity.
The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of alpha1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found alpha1 subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H2O2 (1 mmol/L) did not change the relative amounts of alpha1 subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the alpha1 subunit also was not affected by H2O2 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H2O2-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H2O2-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2O2 are not mediated by activation of PKC or by changes in the expression or phosphorylation of alpha1 subunit.
Charles
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A
Department of Medicine, Brown University School of Medicine, Rhode Island 02908, USA.
Dawicki
D D
DD
Oldmixon
E
E
Kuhn
C
C
Cutaia
M
M
Rounds
S
S
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010042:Ouabain / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011651:Pulmonary Artery / Q000201:enzymology*; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016877:Oxidants; D015240:Phorbol 12,13-Dibutyrate; D010042:Ouabain; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium