NADPH oxidase subunits and superoxide production in porcine pulmonary artery endothelial cells

Abstract

An NADPH oxidase complex composed of a membrane-bound flavocytochrome b₅₅₈ consisting of two subunits (p22^phox and gp91^phox) and cytosolic activating factors (p47^phox and p67^phox) generates superoxide anions from oxygen in the respiratory burst of phagocytic cells. Inconsistent results have been previously obtained concerning its additional occurrence in pulmonary artery endothelial cells (PAEC), and this issue was addressed in the present study. PAEC isolated from porcine pulmonary trunk contained mRNA for p22^phox and gp91^phox as demonstrated by reverse transcription-polymerase chain reaction. Immunohistochemistry demonstrated cytochrome subunits, p22^phox, gp91^phox, p47^phox, and p67^phox, both in vitro in isolated PAEC and in situ in endothelial cells in tissue sections of the pulmonary trunk. Isolated PAEC generated reactive oxygen species (ROS; measured by lucigenin-induced chemiluminescence and conversion of dihydrorhodamine 123 into rhodamine 123) in response to stimulation with phorbol 12-myristate 13-acetate. This stimulated ROS production was sensitive to the flavoprotein inhibitor diphenyleneiodonium, and reduced when the superoxide scavenger superoxide dismutase was added. Chemiluminescence measurements of superoxide generation by stimulated PAEC accounted for approximately 1% of that generated by stimulated peritoneal macrophages. The data demonstrate a low-output NADPH oxidase system in porcine PAEC sharing several components with that identified in phagocytic cells.