Hypoxia inhibits L-arginine synthesis from L-citrulline in porcine pulmonary artery endothelial cells

1995 ◽  
Vol 269 (5) ◽  
pp. L581-L587 ◽  
Author(s):  
Y. Su ◽  
E. R. Block
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Biosynthetic Pathway ◽  
Argininosuccinate Synthetase ◽  
Argininosuccinate Lyase ◽  
Arginine Biosynthesis ◽  
Intact Cells ◽  
Porcine Pulmonary Artery

Both non-arginine-depleted and arginine-depleted pulmonary artery endothelial cells (PAEC) actively convert citrulline into arginine. Exposure to hypoxia for 4-24 h inhibited arginine synthesis from citrulline in intact cells and in cell homogenates. The conversion of L-citrulline to L-argininosuccinate by argininosuccinate synthetase (AS) was inhibited by exposure to hypoxia for 4, 12, or 24 h. The conversion of argininosuccinate to arginine by argininosuccinate lyase was inhibited by exposure to hypoxia for 24 h but not for 4-12 h. The decrease of L-arginine biosynthesis during hypoxia coincided with the increase of intracellular glutamine content and was abrogated by preventing an increase in intracellular glutamine. In addition, AS activity was inversely related to glutamine content in the medium. These results indicate that hypoxia inhibited the L-arginine biosynthetic pathway via decreased activity of AS. The latter is related to increased glutamine content. Hypoxic inhibition of arginine synthesis from citrulline did not result in a decrease of arginine content, suggesting that PAEC are able to maintain intracellular arginine for up to 24 h despite reduction in the L-arginine biosynthetic pathway.

Hypoxia induces the synthesis of tropomyosin in cultured porcine pulmonary artery endothelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. L271-L281 ◽  
Author(s):  
U. J. Rao ◽  
N. D. Denslow ◽  
E. R. Block
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Pulmonary Artery ◽  
Monolayer Culture ◽  
Cross Reactivity ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Masses ◽  
35 Kda Protein ◽  
Porcine Pulmonary Artery

The present study examined the effect of hypoxia on protein synthesis by porcine pulmonary artery endothelial cells (PAEC). Hypoxia decreased protein synthesis in PAEC, but two-dimensional gel electrophoresis of [35S]methionine-labeled PAEC proteins demonstrated the increased synthesis of a set of proteins having molecular masses (M(r)) of 35, 36.5, 45, 116, and 205 kDa. The synthesis of the 35-, 36.5-, and 45-kDa proteins was increased in preconfluent and postconfluent cells. The 35- and 45-kDa proteins were not induced by hyperthermia, whereas the 36.5-kDa protein was induced slightly by hyperthermia. Induction of the 36.5- and 45-kDa proteins required a minimum of 8 h of hypoxia, whereas induction of the 35-kDa protein required only 4 h of exposure to hypoxia. The upregulated synthesis of the 35-, 36.5-, and 45-kDa proteins was reversible with return to normoxia. Actinomycin D, an inhibitor of transcription, did not block the hypoxic induction of the 35- and 36.5-kDa proteins but did block induction of the 45-kDa protein. The partial amino acid sequence of the 35-kDa protein obtained from cyanogen bromide cleavage of the molecule was Asp-Ala-Ile-Lys-Lys-Lys-Met-Gln-Met-Leu-Lys-Leu-Asp-Lys-Glu. This partial sequence of the 35-kDa protein identically matches the sequence of tropomyosin. Amino acid composition data and the isoelectric point (4.8) were also typical of tropomyosin. Finally, specific cross-reactivity was detected between the 35-kDa protein and a monoclonal antibody to chicken gizzard tropomyosin on immunoblot. Thus hypoxia induces the synthesis of tropomyosin, a major microfilament-associated protein, in porcine PAEC in monolayer culture.

Effect of hypoxia on phospholipid metabolism in porcine pulmonary artery endothelial cells

1992 ◽  
Vol 262 (5) ◽  
pp. L606-L613 ◽  
Author(s):  
G. B. Bhat ◽  
E. R. Block
Keyword(s):  
Endothelial Cells ◽  
Fatty Acid ◽  
Pulmonary Artery ◽  
Fatty Acid Fraction ◽  
Phospholipid Metabolism ◽  
Fatty Acid Esters ◽  
Hypoxic Exposure ◽  
Synthetase Activity ◽  
Diacylglycerol Lipase ◽  
Porcine Pulmonary Artery

The effect of exposure of porcine pulmonary artery endothelial cells to hypoxic (0% O2) and normoxic (20% O2) conditions for 24 and 48 h on phospholipid metabolism was studied. Sonicates prepared from endothelial cells that were exposed to 24 h of hypoxia showed significant increases in phospholipase A1 (91%), phospholipase C (75%), and diacylglycerol lipase (57%) activities. Hypoxic exposure of cells for 48 h caused an increase in diacylglycerol lipase activity (54%) only. Hypoxia also caused significant decreases in ATP levels and ATP-dependent arachidonyl coenzyme A (CoA) synthetase activity. Phospholipase A2, lysophosphatidylcholine acyltransferase, and diacylglycerol acyltransferase activities were not influenced by 24 or 48 h of hypoxia. When endothelial cells were prelabeled with [3H]arachidonic acid and then exposed to hypoxia, increased counts were recovered from the free fatty acid fraction of medium and from the cell fatty acid esters, lysophospholipids, diacylglycerols, and triacylglycerols. There was a concomitant decreased recovery of counts from cell phospholipids. These results indicate that hypoxic exposure of endothelial cells altered phospholipid metabolism by activating deacylation pathways and inhibiting reacylation via ATP-dependent arachidonyl CoA synthetase.

Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury

1997 ◽  
Vol 273 (4) ◽  
pp. L856-L865 ◽  
Author(s):  
Bruce R. Pitt ◽  
Margaret Schwarz ◽  
Elizabeth S. Woo ◽  
Emily Yee ◽  
Karla Wasserloos ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Metal Binding ◽  
High Performance ◽  
Binding Capacity ◽  
Peroxyl Radicals ◽  
Intact Cells ◽  
Tert Butyl Hydroperoxide ◽  
Liquid Chromatography Separation

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Further studies of L-arginine biosynthesis in germinating pea seeds. Evidence for the presence of argininosuccinate synthetase in cotyledon extracts

1969 ◽  
Vol 47 (4) ◽  
pp. 467-475 ◽  
Author(s):  
P. D. Shargool ◽  
E. A. Cossins
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Magnesium Ions ◽  
Argininosuccinate Synthetase ◽  
Argininosuccinate Lyase ◽  
Specific Enzyme ◽  
Arginine Biosynthesis ◽  
Specific Enzyme Activity ◽  
Pea Seeds

The synthesis and metabolism of arginine in germinating peas was examined by supplying micromolar quantities of L-citruiline-carbamyl-14C, DL-arginine-carbamyl-14C, and DL-arginine-5-14C to imbibing seeds. Citrulline was readily incorporated into arginine, but the labelled arginine solutions were not extensively metabolized.Extracts of 1-day-old cotyledons were found to catalyze the synthesis of arginine from citrulline in a reaction having absolute requirements for ATP, L-aspartate, and magnesium ions. The extracts were fractionated by (NH4)2SO4 precipitation followed by gel filtration on columns of Sephadex G-50 and G-200. These treatments increased the specific enzyme activity by approximately 36 times. After such treatments the preparations still contained appreciable amounts of argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) activity. The rate of arginine synthesis was altered by increasing the concentrations of L-citrulline, L-aspartate, and ATP. The latter compounds were found to be inhibitory at concentrations of 1 μmole/ml and 4 μmoles/ml, respectively. Arginine synthesis was markedly affected by pH and by additions of arginine and argininosuccinate. It is concluded that germinating pea cotyledons contain appreciable levels of argirrinosuccmate synthetase (L-citrulline:L-aspartate ligase (AMP), EC 6.3.4.5), and furthermore, that this enzyme has importance in arginine biosynthesis during germination.

scholarly journals Oxidized glutathione decreases luminal Ca2+ content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+ store

1995 ◽  
Vol 312 (2) ◽  
pp. 485-489 ◽  
Author(s):  
P N Henschke ◽  
S J Elliott
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Vascular Tissue ◽  
Oxidant Stress ◽  
Inhibitory Effect ◽  
Oxidized Glutathione ◽  
Intact Cells

The model oxidant, t-butyl hydroperoxide (t-buOOH), inhibits Ins(1,4,5)P3-dependent Ca2+ signalling in calf pulmonary artery endothelial cells. Metabolism of t-buOOH within the cytosol is coupled to the oxidation of glutathione. In this study, we investigated whether oxidized glutathione (GSSG) is the intracellular moiety responsible for mediating the effects of t-buOOH on Ca2+ signalling. The increase in cytosolic [Ca2+] stimulated by application of 2,5-di-t-butylhydroquinone (BHQ) was used to estimate the luminal Ca2+ content of the Ins(1,4,5)P3-sensitive store in intact cells. Luminal Ca2+ content was unaffected by t-buOOH (0.4 mM, 0-3 h) unless intracellular GSSG content was concomitantly elevated. The effect was specific for increased GSSG and was not replicated by depletion of GSH. These results suggest that cytosolic GSSG, produced endogenously within the endothelial cell, decreases the luminal Ca2+ content of Ins(1,4,5)P3-sensitive Ca2+ stores. Depletion of internal Ca2+ stores by GSSG may represent a key mechanism by which some forms of oxidant stress inhibit signal transduction in vascular tissue. At the plasma membrane, t-buOOH is known to inhibit the capacitative Ca2+ influx pathway. Increased intracellular GSSG potentiated the inhibitory effect of t-buOOH on Ca2+ influx, thereby providing the first evidence that activity of the capacitative Ca2+ influx channel is sensitive to thiol reagents formed endogenously within the cell.

Nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1133-L1141 ◽  
Author(s):  
M. P. Gupta ◽  
V. Evanoff ◽  
C. M. Hart
Keyword(s):  
Nitric Oxide ◽  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Cell Injury ◽  
Vascular Endothelial Cell ◽  
H2o2 Treatment ◽  
No Donors ◽  
Synthase Inhibitor ◽  
Porcine Pulmonary Artery

To examine the role of nitric oxide (.NO) in vascular endothelial cell injury, cultured porcine pulmonary artery endothelial cells (PAEC) were treated with H2O2 (100-500 microM) for 30 min in the presence or absence of the .NO donors (+/-)S-nitroso-N-acetylpenicillamine (SNAP) or diethylamine nitric oxide (DEANO). H2O2 caused dose-dependent PAEC cytotoxicity detected 2 h after H2O2 treatment as the release of lactate dehydrogenase. SNAP (100 microM) and DEANO (100 microM) attenuated H2O2-induced cytotoxicity if present during H2O2 treatment. In contrast, restricting treatment with .NO donors to periods before (30 min) or after (2 h) incubation with H2O2 did not prevent PAEC injury. Furthermore, the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (1 mM) sensitized PAEC to H2O2-induced injury. SNAP also attenuated H2O2-induced PAEC lipid peroxidation even if restricted to periods before or after exposure to H2O2. Thus, although .NO effectively attenuated H2O2-mediated PAEC lipid peroxidation and cytotoxicity, these effects were clearly dissociated, suggesting that the antiperoxidative effects of .NO are not sufficient to account for its cytoprotective properties.

Hypoxia stimulates endothelial cell angiotensin-converting enzyme antigen synthesis

1989 ◽  
Vol 256 (6) ◽  
pp. C1231-C1238 ◽  
Author(s):  
S. J. King ◽  
F. M. Booyse ◽  
P. H. Lin ◽  
M. Traylor ◽  
A. J. Narkates ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Angiotensin Converting Enzyme ◽  
Cell Number ◽  
Converting Enzyme ◽  
Hypoxic Conditions ◽  
Inactive Form ◽  
Immunoglobin G ◽  
Porcine Pulmonary Artery

Previous studies from our laboratory indicate that exposure of the rat to chronic normobaric hypoxia reduces stores of active angiotensin-converting enzyme (ACE) in the lung. This study assesses directly the effects of hypoxia on ACE synthesis in cultured porcine pulmonary artery endothelial cells. Confluent cultures were exposed to hypoxia [2.5% O2 at 1 atmosphere (atm)] in a triple gas incubator; controls were cultured in normoxic conditions. After 24-, 48-, and 72-h exposure to hypoxic or normoxic conditions, followed by incubation with [35S]methionine for an additional 24 h under the same conditions, newly synthesized radiolabeled ACE was quantitated. Radiolabeled ACE was isolated by an immunobead procedure using either anti-ACE (porcine lung) immunoglobin G (IgG) or nonimmune IgG. A single radiolabeled peak (150 kDa) with the same electrophoretic mobility as purified porcine lung ACE was observed. There was a significant time-dependent increase in endothelial cell ACE antigen synthesis without a concomitant change in either cell number or total trichloroacetic (TCA)-precipitable protein in hypoxic cells compared with normoxic controls. In contrast, ACE activity, assessed by conversion of 125I-labeled angiotensin I to 125I-labeled angiotensin II was unchanged in cultures exposed to hypoxia (2.5% O2). This suggests that an inactive form of ACE is synthesized by cultured pulmonary artery endothelial cells under hypoxic conditions.

Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells

1995 ◽  
Vol 269 (5) ◽  
pp. L574-L580 ◽  
Author(s):  
E. R. Block ◽  
H. Herrera ◽  
M. Couch
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Monolayer Culture ◽  
Kinetic Studies ◽  
No Production ◽  
Michaelis Constant ◽  
Arginine Transport ◽  
Sodium Dependent ◽  
Porcine Pulmonary Artery

Under physiological conditions, L-arginine transport by porcine pulmonary artery endothelial cells (PAEC) is mediated by system y+, a sodium-independent transport system that accounts for 60 +/- 5% of L-arginine transport, and system Bo,+, a sodium-dependent system that accounts for 40 +/- 5% of transport. Because NO production is dependent on intracellular L-arginine content and intracellular L-arginine content depends on transport of extracellular L-arginine, we examined the effect of hypoxia on L-arginine transport and intracellular L-arginine content in PAEC. Exposure of passage 3-7 PAEC in monolayer culture to 0% O2 for 4 h decreased L-arginine transport via system y+ from 120 +/- 10 to 81 +/- 23 (in pmol.mg protein-1.30 s-1) (P < 0.001), whereas 20-h exposures decreased transport from 122 +/- 17 to 84 +/- 18 (P < 0.001) in system y+ and from 104 +/- 19 to 90 +/- 26 (P < 0.05) in system Bo,+. Exposure to 5% O2 for 3-5 wk decreased L-arginine transport via system y+ from 128 +/- 15 to 73 +/- 13 (P < 0.001) and via system Bo,+, from 105 +/- 25 to 65 +/- 13 (P < 0.001). Kinetic studies revealed that hypoxia decreased the maximal transport velocity but not the apparent Michaelis constant for both system y+ and system Bo,+, and the decreases in transport were not reversible after return to normoxia for up to 24 h. Long-term exposure, i.e., 3-5 wk, to 5% O2 also resulted in decreases in intracellular L-arginine content (0.75 +/- 0.10 vs. 0.49 +/- 0.09 nmol/10(6) cells, P < 0.05) which did not reverse after return to normoxia for 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

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