NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells

Abstract

The present study examines whether nitrogen dioxide (NO₂)-induced activation of protein kinase C (PKC) is associated with increased expression of specific PKC isoforms and/or with enhanced generation of phosphatidylcholine(PC)-derived diacylglycerol (DAG) in pulmonary artery endothelial cells (PAEC). Western blot analysis revealed that exposure to 5 ppm NO₂ resulted in increased expression of PKC α and ε isoforms in both cytosol and membrane fractions in a time-dependent fashion compared with controls. A time-dependent elevated expression of PKC isoform β was observed in the cytosol fraction only of NO₂-exposed cells. PKC isoform λ was not detectable in either the cytosolic or membrane fractions from control or NO₂-exposed cells. Scatchard analysis of [³H]phorbol 12,13-dibutyrate (PDBu) binding showed that exposure to NO₂ for 24 h increased the maximal number of binding sites (B _max) from 15.2±2.3 pmollmg (control) to 42.3±5.3 pmol/mg (p < 0.01, n = 4) (NO₂-exposed). Exposure to NO₂ significantly increased PC specific-phospholipase C and phospholipase D activities in the plasma membrane of PAEC (p < 0.05 and p < 0.001, respectively). When [³H]myristic acid-labeled cells were exposed to NO₂, significantly increased radioactivity was associated with cellular DAG. These results show for the first time that exposure of PAEC to NO₂ results in elevated expression of specific PKC isoforms and in enhanced generation of cellular DAG, and the latter appears to arise largely from the hydrolysis of plasma membrane PC.