Nitrogen dioxide (NO,), an environmental oxidant, is known to activate phospholipase A, and modulate the plasma membrane structure of porcine pulmonary artery endothelial cells. We evaluated the effects of exposure to NO,, purified phospholipase B (which acts as phospholipase A, and A,), or phospholipase A, on U51-angiotensin I1 (Ang 11) receptor binding, internalization, or both in pulmonary endothelial cells. Exposure to 5 ppm NO, for 48 hr at 37°C or 0.075 U each of phospholipase B or A, in phosphate-buffered saline (PBS) for 30 min at 24°C resulted in an increase in total Ang I1 binding (i.e., cell surface bound and internalized) by 45%
( p < 0.05), 50% ( p < 0.05), and 85% ( p < 0.001), respectively, compared to controls. An Ang I1 receptor antagonist, [Sar ' Ile'] Ang 11, competitively displaced Ang I1 binding to control, NO,-, phospholipase B-, and phospholipase A,-exposed cells. Dissociation of bound Ang I1 in the presence of PBS was <1% of total bound Ang I1 in control, NO,-, and phospholipase B-exposed cells and was 50% of total bound Ang I1 in phospholipase A,-exposed cells. In the presence of isotonic acetic acid/NaCl, in excess of 90% of cell surface-bound Ang I1 was dissociated from control, NO,-, and phospholipase B-exposed cells, and there was < 2% of Ang I1 detectable when acid-treated cells were subjected to NaOH solubilization. In cells exposed to phospholipase A,, acetic acid treatment did not release cell-bound Ang 11, and the remaining Ang I1 was recovered in the NaOH solubilized fraction. These results demonstrate that NO, exposure results in increased cell surface receptor binding and not internalization of Ang I1 and that phospholipase B-but not phospholipase A,-induced modulation of the membrane structure mimicked the effect of NO, on Ang I1 binding in pulmonary endothelial cells.