Effect of hypoxic exposure on Na+/H+ antiport activity, isoform expression, and localization in endothelial cells.
Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+ antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+ exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.
Cutaia
M V
MV
Pulmonary Disease Division, Department of Medicine, Veterans Affairs Medical Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 02908-9019, USA.
Parks
N
N
Centracchio
J
J
Rounds
S
S
Yip
K P
KP
Sun
A M
AM
D000332:Aerobiosis; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011651:Pulmonary Artery; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic
D012333:RNA, Messenger; C081517:SLC9A2 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1