Oxidant and angiotensin II‐induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

Abstract

We recently reported that nitrogen dioxide (NO2), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO2 resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO2 exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO2 for 1-24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p less than 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO2-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar1 Ile8] Ang II. Exposure of 5 ppm NO2 for 1-24 hr provoked rapid increases in [3H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO2 increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.

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

“…Effect of NO2 exposure on expression of spectjic PKC isoforms Because exposure to NO2 increases total PKC activity [7], we examined whether NO2 selectively increases the expression of specific PKC isoforms. We used PKC isoform-specific antibodies directed against isoforms a, B, and y (classical PKC isoforms, both calcium and DAG dependent) and isoform E (calcium independent, DAG dependent) for immunodetection.…”

“…We have previously reported that exposure to NO2 results in PKC activation [7] and increased biosynthesis and translocation of PS to the plasma membrane of PAEC [8,19]. The results of the present study demonstrate for the first time that: (i) PKC isoforms a, 8, and E, but not y, are expressed in cultured porcine PAEC; (ii) exposure to NO2 specifically increases the expression of PKC isoforms ~1, 8, and E in the cytosolic fraction and isoforms a and E in the membrane fraction; and iii) NOT-induced activation of plasma membrane PLC and PLD resulted in elevated production of DAG that appears to arise largely from the hydrolysis of membrane PC in PAEC.…”

“…However, a recent report indicates that platelet-activating factor (PAF, l-radyl-2-acetyl-sn-glycerol-3-phosphocholine), an oxidative product of membrane PC, increases the mRNA transcripts and activates the a and I3 isoforms in human endothelial cells [24]. A number of oxidants including NO2 are known to cause peroxidation of membrane lipids, resulting in structural alterations and modulation of cell functions [6,7,11]. As such, NOz-induced lipid peroxidation products may be respnsible, at least in part, for increased expression of PKC proteins.…”

“…Endothelial cells were obtained from the main pulmonary artery of 6&7-month-old pigs as described by Pate1 et al [7] and propagated in monolayer cultures in loo-mm culture dishes. Confluent monolayers of unlabeled or [3H]myristic acid-labeled endothelial cells in 10 ml of fresh maintenance medium were exposed to a continuous flow of 5 ppm NOs in air containing 5% COs for 448 h as described earlier [6].…”

“…Immediately after NOs exposure, cells were homogenized and cytosol and membrane fractions were isolated by differential centrifugation as previously described [7]. Cytosol and membrane fraction PKC were partially purified from both NOz-exposed and control cells according to the procedure described by Pate1 et al [7].…”