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

An NADPH oxidase complex composed of a membrane-bound flavocytochrome b(558 ) consisting Of two subunits (p22(phox) and gp91(phox)) and cytosolic activ ating factors (p471(phox) and p67(phox)) generates superoxide anions from o xygen in the respiratory burst of phagocytic cells. Inconsistent results ha ve been previously obtained concerning its additional occurrence in pulmona ry artery endothelial cells (PAEC), and this issue was addressed in the pre sent study. PAEC isolated from porcine pulmonary trunk contained mRNA for p 22(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 PAE C and in situ in endothelial cells in tissue sections of the pulmonary trun k. isolated PAEC generated reactive oxygen species (ROS; measured by lucige nin-induced chemiluminescence and conversion of dihydrorhodamine 123 into r hodamine 123) in response to stimulation with phorbol 12-myristate 13-aceta te. This stimulated ROS production was sensitive to the flavoprotein inhibi tor diphenyleneiodonium, and reduced when the supcroxide scavenger superoxi de dismutase was added. Chemiluminescence measurements of superoxide genera tion 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 identi fied in phagocytic cells.