Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells.

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.

Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release.

Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca(2+) concentration ([Ca(2+)](i)) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5'-O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca(2+) stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca(2+)](i) release through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.

Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells.

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O(2)-95% N(2)-5% CO(2)) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.

Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake.

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells.

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.

Nitric oxide-induced reduction of lung cell and whole lung thioredoxin expression is regulated by NF-kappaB.

We examined whether nitric oxide (NO)-induced inhibition of thioredoxin (Thx) expression is regulated by a mechanism mediated by a transcription factor, i.e., nuclear factor-kappaB (NF-kappaB), in cultured porcine pulmonary artery endothelial cells (PAEC) and in mouse lungs. Western blot analysis revealed that IkappaB-alpha content was reduced by 20 and 60% in PAEC exposed to 8.5 ppm NO for 2 and 24 h, respectively. NO exposure also caused significant reductions of cytosol fraction p65 and p52 content in PAEC. The nuclear fraction p65 and p52 contents were significantly reduced only in PAEC exposed to NO for 24 h. Exposure to NO resulted in a 50% reduction of p52 mRNA but not of the IkappaB-alpha subunit. DNA binding activity of the oligonucleotide encoding the NF-kappaB sequence in the Thx gene was significantly reduced in PAEC exposed to NO for 24 h. Exposure of mice to 10 ppm NO for 24 h resulted in a significant reduction of lung Thx and IkappaB-alpha mRNA and protein expression and in the oligonucleotide encoding Thx and NF-kappaB/DNA binding. These results 1) demonstrate that the effects of NO exposure on Thx expression in PAEC are comparable to those observed in intact lung and 2) suggest that reduced expression of the NF-kappaB subunit, leading to reduced NF-kappaB/DNA binding, is associated with the loss of Thx expression in PAEC and in intact mouse lungs.

Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS.

This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of Ca(2+) concentration and by increased expression and release of the Ca(2+) binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca(2+)-free medium and in the presence of the ANG II AT(1) and AT(2) receptor antagonists losartan and PD-123319 (1 microM each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca(2+) release in PAEC (P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation (P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular Ca(2+) mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.

Angiotensin II-stimulated nitric oxide release from porcine pulmonary endothelium is mediated by angiotensin IV.

In this study, a nitric oxide (NO) sensor was used to examine the ability of angiotensin II (AngII), AngIV, and bradykinin (Bk) to stimulate NO release from porcine pulmonary artery (PPAE) and porcine aortic endothelial (PAE) cells and to explore the mechanism of the AngII-stimulated NO release. Physiologic concentrations of AngII, but not Bk, caused release of NO from PPAE cells. In contrast, Bk, but not AngII, stimulated NO release from PAE cells. AngIII-stimulated NO release from PPAE cells required extracellular L-arginine and was inhibited by L-nitro-arginine methyl ester. AT1 and AT2 receptor inhibition had no affect on AngII-mediated NO release or activation of NO synthase (NOS). AngIV, an AngII metabolite with binding sites that are pharmacologically distinct from the classic AngII receptors, stimulated considerably greater NO release and greater endothelial-type constitutive NOS activity than the same amount of AngII. The AngIV receptor antagonist, divalinal AngIV, blocked both AngII- and AngIV-mediated NO release as well as NOS activation. The results demonstrate that AngIV and the AngIV receptor are responsible, at least in part, for AngII-stimulated NO release and the associated endothelium-dependent vasorelaxation. Furthermore, these results suggest that differences exist in both AngII- and Bk-mediated NO release between PPAE and PAE cells, which may reflect important differences in response to these hormones between vascular beds.

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 by L-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.

Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells.

Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS activity, and incubation of PAEC with 3 mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.

Hypoxia-specific upregulation of calpain activity and gene expression in pulmonary artery endothelial cells.

The effects of exposure to hypoxia on the catalytic activity and mRNA expression of calpain, a calcium-regulated neutral cysteine protease, were examined in porcine pulmonary artery endothelial cells (PAECs). Specificity of the response to hypoxia was determined by comparing the effects of hypoxic exposure with exposure to oxidants such as nitrogen dioxide (NO2) and nitric oxide (NO), as well as to the sulfhydryl reactive chemical acrolein. Exposure of cells to hypoxia (0% O2) for 1 and 12 h significantly increased catalytic activity (P < 0.01 for both 1 and 12 h vs. control cells), as well as mRNA expression (P < 0.01 for 1 h and P < 0.05 for 12 h vs. control cells) of calpain. With more prolonged exposure to 24 h of hypoxia, calpain activity remained significantly elevated, whereas calpain mRNA expression returned to the control level. Calpain activities in cells exposed to NO2 [5 parts/million (ppm)] or NO (7.5 ppm) for 1 h or to acrolein (5 microM) for 1 and 24 h were unchanged. However, calpain activities in cells exposed to NO2 or NO for 24 h were significantly (P < 0.05) reduced compared with control cells. The hypoxia-induced increases in calpain mRNA content were prevented by the transcriptional inhibitor actinomycin D and by calpain inhibitor I. In addition, hypoxia increased the degradation of nuclear factor-kappaB (NF-kappaB) inhibitor IkappaB and enhanced the translocation of the p50 subunit of NF-kappaB to the nuclear membrane. Pretreatment with the calpain-specific inhibitor E-64d prevented hypoxia-induced mRNA expression and degradation of IkappaBalpha, as well as translocation of p50 subunit to the nuclear membrane. These results demonstrate for the first time that hypoxia upregulates calpain activity and mRNA expression in PAECs and that the upregulation is specific to hypoxia. Upregulation appears to involve activation of the transcription factor NF-kappaB.

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 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.

Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells.

We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.

Hypoxia inhibits the induction of argininosuccinate synthetase by endotoxin in lung endothelial cells.

Pulmonary artery endothelial cells (PAEC) possess a two-step pathway for synthesizing L-arginine from L-citrulline. The first and rate-limiting step is catalyzed by argininosuccinate synthetase (AS). We have previously shown that hypoxia inhibits synthesis of L-arginine from L-citrulline in PAEC. In this study, we examined the effect of hypoxia on the induction of AS in PAEC. Porcine PAEC were incubated with or without endotoxin under normoxia (air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2) for 24 h, and then AS activity and AS mRNA content were determined. Incubation with endotoxin resulted in increases in AS activity and mRNA, and the latter was blocked by actinomycin D. Exposure to hypoxia for 24 h decreased AS activity and mRNA content and stability, and it also abolished the increases in AS activity and mRNA induced by endotoxin. These results indicate that hypoxia inhibits endotoxin-mediated induction of AS. This inhibition might reduce the availability of intracellular L-arginine and thereby limit immunostimulant-induced nitric oxide production by lung endothelial cells.

Proinflammatory cytokines downregulate gene expression and activity of constitutive nitric oxide synthase in porcine pulmonary artery endothelial cells.

We evaluated the effects of cytokines on the catalytic activity and expression of porcine pulmonary artery endothelial cell (PAEC) constitutive (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS). Exposure of PAEC to the combination of IFN-gamma, TNF-alpha, and IL-1 beta did not alter iNOS activity in cytosolic and membrane fractions but significantly (p < 0.01) reduced eNOS activity in the membrane fraction, but not in the cytosolic fraction, after a 24-h exposure. The cytokine-induced loss of membrane fraction eNOS activity was associated with significant reductions of eNOS mRNA and protein content (p < 0.01 for both). Treatment with the protein synthesis inhibitor, cycloheximide, but not the transcriptional inhibitor actinomycin D prevented cytokine-induced reduction of eNOS mRNA expression. These results suggest that cytokine-induced loss of catalytic activity of eNOS is associated with a reduction in eNOS mRNA and protein mass and that cytokines alter eNOS mRNA stability. Inhibition of protein synthesis prevented reduction of eNOS mRNA by cytokines, suggesting that the mechanism by which cytokines alter eNOS mRNA stability involves protein synthesis.

Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells.

The lack of sequence information and clones of porcine pulmonary artery endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) cDNA limits comparative analysis between porcine and human PAEC. Therefore, we cloned, characterized and expressed the ecNOS cDNA from porcine PAEC. Two oligonucleotide primers were designed based on the published human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS using 5' and 3' rapid amplification of cDNA ends reverse transcriptase polymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molecular mass of 134 kDa. A mammalian expression vector (pcDNA3) containing this cDNA was transfected into COS-7 cells, and ecNOS activity was detected by monitoring the formation of [3H]-citrulline from [3H]-L-arginine. Expression of ecNOS activity was predominantly associated (> 90%) with the total membrane fraction of these transfected cells. The deduced amino acid sequence of porcine ecNOS cDNA, containing binding sites for NADPH, flavin adenine dinucleotide and bound flavin mononucleotide, shows 94% identity to human ecNOS. The molecular weight of porcine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, similar to human ecNOS mRNA. This suggests that porcine ecNOS is similar to human ecNOS in deduced amino acid sequence and structure.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

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.

Role of membrane potential in hypoxic inhibition of L-arginine uptake by lung endothelial cells.

System y+ accounts for the majority of L-arginine transport by pulmonary artery endothelial cells (PAEC). Given that membrane potential is a driving force for transport via system y+, we examined the hypothesis that hypoxia inhibits this transport by decreasing membrane potential. Porcine PAEC or plasma membrane vesicles derived from these cells were exposed to normoxia (room air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2). After exposure, L-[3H]arginine transport and/or accumulation of the lipophilic cation [3H]tetraphenylphosphonium, a quantitative sensor of changes in cell membrane potential, were measured. Hypoxia caused reversible time-dependent decrease in L-arginine transport and membrane potential in PAEC and in plasma membrane vesicles. Comparable decreases in membrane potential and L-arginine transport by PAEC were also observed after depolarization induced by KCl or ouabain. Hyperpolarization, induced by valinomycin, increased membrane potential and L-arginine transport in PAEC and plasma membrane vesicles. Valinomycin also prevented the hypoxia-mediated decreases in membrane potential and L-arginine transport in PAEC. These results indicate that hypoxia-induced plasma membrane depolarization is responsible for reduced L-arginine transport by system y+ in hypoxic porcine PAEC.

Substrate inhibition of nitric oxide synthase in pulmonary artery endothelial cells in culture.

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0-100 microM arginine increased NO production, detected as nitrite in the culture medium, in a dose-dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 microM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that the Km of arginine as a substrate for NOS is 3-5 microM, the Vmax occurred at 100 microM arginine, and substrate inhibition occurred at >100 microM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, and D-arginine did not change NOS kinetics. These results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.