Yong D. Li

Angiotensin IV receptor-mediated activation of lung endothelial NOS is associated with vasorelaxation.

The hexapeptide angiotensin (ANG) IV, a metabolic product of ANG II, has been reported to play a functional role in the regulation of blood flow in extrapulmonary tissues. Here, we demonstrate that ANG IV-specific (AT4) receptors are present in porcine pulmonary arterial endothelial cells (PAECs) and that the binding of ANG IV to AT4 receptors can be blocked by its antagonist divalinal ANG IV but not by the ANG II-, AT1-, and AT2-receptor blockers [Sar1,Ile8]ANG II, losartan, and PD-123177, respectively. ANG IV significantly increased endothelial cell constitutive nitric oxide synthase (ecNOS) activity ( P < 0.05) as well as cellular cGMP content ( P < 0.001). Western blot analysis revealed that ecNOS protein expression was comparable in control and ANG IV-stimulated cells. Divalinal ANG IV but not [Sar1,Ile8]ANG II, losartan, or PD-123177 inhibited the ANG II- and ANG IV-stimulated increases in ecNOS activity and cGMP content in PAECs. Incubation in the presence of N-nitro-l-arginine methyl ester (l-NAME) or methylene blue but not of indomethacin significantly diminished ANG IV-stimulated as well as basal levels of cGMP ( P < 0.001). Similarly, in situ studies with precontracted porcine pulmonary arterial rings showed that ANG IV caused an endothelium-dependent relaxation that was blocked byl-NAME or methylene blue. Collectively, these results demonstrate that ANG IV binds to AT4 receptors, activates ecNOS by posttranscriptional modulation, stimulates cGMP accumulation in PAECs, and causes pulmonary arterial vasodilation, suggesting that ANG IV plays a role in the regulation of blood flow in the pulmonary circulation.

Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells.

We recently reported that nitric oxide (NO) induces posttranscriptional modulation of lung endothelial cell NO synthase (ecNOS) that results in loss of activity. The loss of activity can be reversed by the redox regulatory proteins thioredoxin (Thx)/thioredoxin reductase (Thx-R). The present study was designed to examine whether diminished expression of endogenous Thx and Thx-R may account for regulation of ecNOS activity in NO-exposed cells and whether overexpression of Thx can prevent NO-induced reduction of ecNOS activity in cultured porcine pulmonary artery endothelial cells (PAEC). Exposure to 8.5 ppm NO gas for 24 h resulted in an 80% decrease of Thx and a 27% decrease of Thx-R mRNA expression. Similarly, NO exposure caused 30 and 50% reductions in Thx and Thx-R protein mass, respectively. This NO-induced decrease in the expression of Thx-R mRNA and protein was accompanied by a significant ( P < 0.05) decrease in the catalytic activity of Thx-R but not of glutaredoxin or the cellular levels of reduced glutathione and oxidized glutathione. Overexpression of Thx gene in PAEC was achieved by transient transfection of these cells with pcDNA 3.1 vector inserted in sense or antisense (native) orientation in a human Thx cDNA. Thx mRNA and protein contents in transfected cells were four- and threefold higher, respectively, than those in native PAEC. Exposure of native cells to 10 μM NO solution for 30 min resulted in a significant ( P < 0.01) loss of ecNOS activity, whereas ecNOS activity was comparable in Thx-overexpressed cells with or without NO exposure. These results demonstrate that NO exposure results in diminished expression of Thx and Thx-R in PAEC. Endogenous levels of Thx are critical to restoring the NO-induced loss of ecNOS activity because overexpression of Thx prevented the NO-induced loss of ecNOS catalytic activity. These results also demonstrate that NO modulation of ecNOS and Thx proteins is regulated by a physiologically relevant redox mechanism.We recently reported that nitric oxide (NO) induces posttranscriptional modulation of lung endothelial cell NO synthase (ecNOS) that results in loss of activity. The loss of activity can be reversed by the redox regulatory proteins thioredoxin (Thx)/thioredoxin reductase (Thx-R). The present study was designed to examine whether diminished expression of endogenous Thx and Thx-R may account for regulation of ecNOS activity in NO-exposed cells and whether overexpression of Thx can prevent NO-induced reduction of ecNOS activity in cultured porcine pulmonary artery endothelial cells (PAEC). Exposure to 8.5 ppm NO gas for 24 h resulted in an 80% decrease of Thx and a 27% decrease of Thx-R mRNA expression. Similarly, NO exposure caused 30 and 50% reductions in Thx and Thx-R protein mass, respectively. This NO-induced decrease in the expression of Thx-R mRNA and protein was accompanied by a significant (P < 0.05) decrease in the catalytic activity of Thx-R but not of glutaredoxin or the cellular levels of reduced glutathione and oxidized glutathione. Overexpression of Thx gene in PAEC was achieved by transient transfection of these cells with pcDNA 3.1 vector inserted in sense or antisense (native) orientation in a human Thx cDNA. Thx mRNA and protein contents in transfected cells were four- and threefold higher, respectively, than those in native PAEC. Exposure of native cells to 10 microM NO solution for 30 min resulted in a significant (P < 0.01) loss of ecNOS activity, whereas ecNOS activity was comparable in Thx-overexpressed cells with or without NO exposure. These results demonstrate that NO exposure results in diminished expression of Thx and Thx-R in PAEC. Endogenous levels of Thx are critical to restoring the NO-induced loss of ecNOS activity because overexpression of Thx prevented the NO-induced loss of ecNOS catalytic activity. These results also demonstrate that NO modulation of ecNOS and Thx proteins is regulated by a physiologically relevant redox mechanism.

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 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.

Overexpression of Plasma Membrane Annexin II In NO2-Exposed Pulmonary Artery Endothelial Cells

Because exposure to nitrogen dioxide (NO2) alters plasma membrane structure and function in pulmonary artery endothelial cells (PAEC), we examined whether NO2 exposure is associated with upregulation of plasma membrane-specific proteins in PAEC. Exposure to 5 ppm NO2 for 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of isolated plasma membranes from [35S]-methionine pulse-labeled PAEC exposed to NO2 for 24 h demonstrated 3- to 9-fold increases in the synthesis of several proteins with molecular masses of 36, 39, and 40 kDa compared with controls. N-terminal amino acid sequencing and immunodetection analysis identified the 36kDa plasma membrane protein as annexin II (lipocortin II). Northern blotting analysis demonstrated that the mRNA expression for annexin II in NO2-exposed cells was also increased. These results suggest that exposure to NO2 results in induction of plasma membrane annexin II, an important multifunctional calcium- and phospholipid-binding protein in PAEC.

Calreticulin Regulation of Lung Endothelial NOS Activity

Increased synthesis of a multifunctional calcium binding protein calreticulin has been reported under diverse physiologic and pathophysiologic conditions in various tissues in eluding stimulation of vascular endothelium by angiotensin IV (Ang-IV), a metabolic product of Ang-II. Ang-IV-mediated early and sustained activation of lung endothelial cell nitric oxide synthase (eNOS) is mediated through increased mobilization of intracellular calcium and by increased expression of calreticulin. Immunoprecipitation and confocal imaging studies revealed that eNOS and calreticulin are co-localized in Ang-IV-stimulated lung endothelial cells. Catalytic activity of purified eNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. The studies monitoring the effect of calreticulin on the rate of electron transfer from the reductase to the oxygenase domain of eNOS revealed that the calreticulin/eNOS interaction promotes electron transfer and mimics eNOS activation in the absence of exogenous calmodulin and enhances electron transfer and the catalytic activity of eNOS in the presence of calmodulin. Thus, calreticulin/eNOS proteimprotein interaction enhances the rate of electron transfer, a critical event in the regulation of the catalytic activity of eNOS.

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.