NO2-INDUCED EXPRESSION OF SPECIFIC PROTEIN-KINASE-C ISOFORMS AND GENERATION OF PHOSPHATIDYLCHOLINE-DERIVED DIACYLGLYCEROL IN CULTURED PULMONARY-ARTERY ENDOTHELIAL-CELLS

The present study examines whether nitrogen dioxide (NO2)-induced acti vation of protein kinase C (PKC) is associated with increased expressi on of specific PKC isoforms and/or with enhanced generation of phospha tidylcholine(PC)derived diacylglycerol (DAG) in pulmonary artery endot helial cells (PAEC), Western blot analysis revealed that exposure to 5 ppm NO2 resulted in increased expression of PKC alpha and epsilon iso forms in both cytosol and membrane fractions in a time-dependent fashi on compared with controls, A time-dependent elevated expression of PKC isoform beta was observed in the cytosol fraction only of NO2-exposed cells, PKC isoform gamma was not detectable in either the cytosolic o r membrane fractions from control or NO2-exposed cells. Scatchard anal ysis of [H-3]phorbol 12,13-dibutyrate (PDBu) binding showed that expos ure to NO2 for 24 h increased the maximal number of binding sites (B-m ax) from 15.2+/-2.3 pmol/mg (control) to 42.3+/-5.3 pmol/mg (p < 0.01, n = 4) (NO2-exposed), Exposure to NO2 significantly increased PC spec ific-phospholipase C and phospholipase D activities in the plasma memb rane of PAEC (p < 0.05 and p < 0.001, respectively), When [H-3]myristi c acid-labeled cells were exposed to NO2, significantly increased radi oactivity was associated with cellular DAG, These results show for the first time that exposure of PAEC to NO2 results in elevated expressio n 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.