Nitrogen dioxide-induced expression of a 78 kDa protein in pulmonary artery endothelial cells

Exposure to nitrogen dioxide (NO2) activates signal transduction in cultured pulmonary artery endothelial cells (PAEC). We examined whether NO2-induced activation of signal transduction results in increased expression of proteins in PAEC. Exposure to 5 ppm NO2 for 4, 12, and 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of [35S]-methionine-labeled PAEC exposed to NO2 for 24 h, but not 4 and 12 h, demonstrated increased synthesis of several proteins including a two- to five-fold increase of some proteins with molecular masses of 47, 64, 78, and 105 kDa compared to controls. N-terminal amino acid sequencing and immunodetection analysis identified the 78 kDa protein as 78 kDa glucose-regulated protein (GRP-78). Induction of GRP-78 by NO2 exposure was regulated at the transcriptional level, and the induction required de novo protein synthesis. Exposure to NO2 for 24 h also significantly (p < .05) decreased glycosylation of proteins in PAEC. Exposure of cell monolayers to tunicamycin, an inhibitor of protein glycosylation, mimicked the effect of NO2 exposure on expression of GRP-78. Increased expression of GRP-78 was also detected when cell monolayers were exposed to the calcium ionophore A 23187, to 2-deoxyglucose, or to glucose-free medium, which are also known to cause perturbations in protein glycosylation. These results demonstrate that exposure to NO2 increases expression of a number of proteins including GRP-78 in PAEC. Increased expression of GRP-78 in NO2-exposed cells appears to be associated with inhibition of glycosylation or through coordinated alterations in metabolic events that lead to inhibition of protein glycosylation.