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.

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)

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.

Protein Synthesis During Oxygen Deprivation in Cotton

1996 ◽  
Vol 23 (3) ◽  
pp. 341 ◽  
Author(s):  
AA Millar ◽  
ES Dennis
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Oxygen Deprivation ◽  
Two Dimensional ◽  
Root Tips ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Masses ◽  
Adh Activity

The alteration of protein synthesis induced by oxygen deprivation has been examined in the root tips of cotton (Gossypium hirsutum cv. Siokra), a plant that is intolerant to anoxia. Using [35S]methionine labelling and two-dimensional gel electrophoresis it was demonstrated that 14 major polypeptides are being selectively synthesised during oxygen deprivation. These polypeptides have been designated the cotton anaerobic polypeptides (ANPs), and have estimated molecular masses that correspond to molecular masses of ANPs from other plant species. However, compared to maize, several of the major molecular weight classes are absent, suggesting that the response to oxygen deprivation in cotton is simpler than that of maize. Alcohol dehydrogenase (ADH) activity is induced by oxygen deprivation. Using western analysis we have determined that this increase in activity is correlated with the accumulation of the ADH polypeptide and that three of the major cotton ANPs are ADH, including the most intensely labelled ANP, demonstrating that the synthesis of ADH constitutes a major part of the response in cotton.

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.

Monocrotaline pyrrole alters DNA, RNA and protein synthesis in pulmonary artery endothelial cells

1992 ◽  
Vol 262 (6) ◽  
pp. L740-L747 ◽  
Author(s):  
C. M. Hoorn ◽  
R. A. Roth
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Pulmonary Artery ◽  
Dna Content ◽  
Cell Number ◽  
Synthetic Activity ◽  
Cellular Activity ◽  
Rna And Protein Synthesis ◽  
High Concentrations ◽  
Monocrotaline Pyrrole

Administration of monocrotaline pyrrole (MCTP) to animals results in pulmonary vascular injury. Pulmonary vascular endothelium is a likely target for this pneumotoxicant. Cultured porcine pulmonary artery endothelial cells (PECs) treated with MCTP remain viable but are unable to divide and exhibit an altered morphology. Such responses raise a question about the extent to which affected cells carry out normal functions such as RNA and protein synthesis. Accordingly, the cellular activity of MCTP-treated PECs was examined in this study. PECs were treated with a single administration of MCTP or vehicle, and determinations of cell number, protein, and DNA content were made at times up to 7 days posttreatment. DNA, RNA, and protein synthesis were quantified by incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine, respectively. Increases in cell number that occurred with time in the control cells were reduced in MCTP-treated cells. At 7 days posttreatment, both protein and DNA content increased above control levels. Synthesis of DNA, RNA, and protein continued in all treatment groups throughout the posttreatment period, but cells treated with high concentrations of MCTP showed less synthetic activity than controls during the initial 48 h posttreatment. By 7 days, MCTP-treated cells were producing significantly more DNA, RNA, and protein. These results indicate that cells treated with MCTP continue to synthesize DNA, resulting in an increased DNA content. In addition, treated cells continue to synthesize RNA and translate RNA into protein. Thus, cellular activity is maintained but altered substantially by MCTP exposure.

Characterization of L-glutamine transport by pulmonary artery endothelial cells

1991 ◽  
Vol 260 (4) ◽  
pp. L241-L246 ◽  
Author(s):  
K. Herskowitz ◽  
B. P. Bode ◽  
E. R. Block ◽  
W. W. Souba
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Hormonal Regulation ◽  
Kinetic Studies ◽  
High Affinity ◽  
Sodium Dependent ◽  
Porcine Pulmonary Artery ◽  
External Ph ◽  
Glutamine Uptake

This study characterized the transport of L-glutamine by porcine pulmonary artery endothelial cells (PAECs). Uptake of 50 microM glutamine was determined and found to be linear for at least 45 min. The sodium-dependent velocity represented greater than 95% of the total uptake at all time points. Kinetic studies of glutamine uptake at concentrations between 0.005 and 10 mM showed a single saturable high-affinity carrier with a Michaelis constant of 100 +/- 6 microM and a maximal transport velocity of 1.0 +/- 0.08 nmol.mg protein-1.30s-1. Glutamine uptake by PAECs was markedly inhibited in the presence of L-cysteine, L-threonine, or L-alanine; lesser degrees of inhibition occurred when histidine and arginine were added. 2-Methylaminoisobutyric acid and 2-aminobicyclo [2,2,1]heptanedicarboxylic acid had little effect on glutamine uptake. Lithium did not substitute for sodium, strongly suggesting that L-glutamine was not transported by system N. Furthermore, transport of glutamine was not affected by hormones or by changes in external pH. Based on the intolerance of this high-affinity carrier to N-methylated substrate, its insensitivity to pH and hormonal regulation, and the failure of lithium to substitute for sodium, as well as its inhibition by alanine and cysteine, we conclude that in porcine pulmonary artery endothelial cells L-glutamine is predominantly taken up through system ASC.

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