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Vanadyl sulfate inhibits NO production via threonine phosphorylation of eNOS.



Exposure to excessive vanadium vanadium (vənā`dēəm), metallic chemical element; symbol V; at. no. 23; at. wt. 50.9415; m.p. about 1,890°C;; b.p. 3,380°C;; sp. gr. about 6 at 20°C;; valence +2, +3, +4, or +5. Vanadium is a soft, ductile, silver-grey metal.  occurs in some occupations and with consumption of some dietary regimens for weight reduction and body building. Because vanadium is vasoactive vasoactive /vaso·ac·tive/ (va?zo-) (vas?o-ak´tiv) exerting an effect upon the caliber of blood vessels.

va·so·ac·tive
adj.
, individuals exposed to excessive vanadium may develop adverse vascular effects. We have previously shown that vanadyl sulfate causes acute pulmonary vasoconstriction vasoconstriction /vaso·con·stric·tion/ (-kon-strik´shun) decrease in the caliber of blood vessels.vasoconstric´tive

va·so·con·stric·tion
n.
, which could be attributed in part to inhibition of nitric oxide production. In the present study we investigated whether NO inhibition was related to phosphorylation phosphorylation, chemical process in which a phosphate group is added to an organic molecule. In living cells phosphorylation is associated with respiration, which takes place in the cell's mitochondria, and photosynthesis, which takes place in the chloroplasts.  of endothelial nitric oxide synthase The nitric oxide synthase (NOS; EC 1.14.13.39) is an enzyme in the body that contributes to transmission from one neuron to another, to the immune system and to dilating blood vessels.  (eNOS). VOS An operating system used in Stratus computers. FTX is Stratus' Unix operating system. [O.sub.4] produced dose-dependent constriction of pulmonary arteries in isolated perfused lungs and pulmonary arterial rings and a right shift of the acetylcholine-dependent vasorelaxation curve. VOS[O.sub.4] inhibited constitutive as well as A23187-stimulated NO production. Constitutive NO inhibition was accompanied by increased [Thr.sup.495] (threonine threonine (thrē`ənēn), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  at codon codon: see nucleic acid.  495) phosphorylation of eNOS, which would inhibit eNOS activity. [Thr.sup.495] phosphorylation of eNOS and inhibition of NO were partially reversed by pretreatment pretreatment,
n the protocols required before beginning therapy, usually of a diagnostic nature; before treatment.

pretreatment estimate,
n See predetermination.
 with calphostin C, a protein kinase C Protein kinase C ('PKC', EC 2.7.11.13) is a family of protein kinases consisting of ~10 isozymes.[1] They are divided into three subfamilies: conventional (or classical), novel, and atypical based on their second messenger requirements.  (PKC PKC Protein Kinase C (biochemistry)
PKC Public Key Cryptography
PKC Public Key Certificate
PKC PaKua Chang (Chinese martial art)
PKC Paroxysmal Kinesigenic Choreoathetosis
) inhibitor. There were no changes in [Ser.sup.1177] (serine serine (sĕr`ēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  at codon 1177) or tyrosine phosphorylation of eNOS. These results indicate that VOS[O.sub.4] induced acute pulmonary vasoconstriction that was mediated in part by the inhibition of endothelial NO production via PKC-dependent phosphorylation of [Thr.sup.495] of eNOS. Exposure to excessive vanadium may contribute to pulmonary vascular diseases. Key words: boilermakers, protein kinase C, pulmonary hypertension, vanadium. Environ Health Perspect 112:201-206 (2004). doi:10.1289/ehp.6477 available via http://dx.doi.org/ [Online 22 October 2003]

**********

Vanadium was first discovered in 1971 as a trace element that is essential for normal growth. Since then, vanadium has been found to regulate the activity of various enzymes that induce pronounced changes in metabolic functions. Some of these enzymes include [Na.sup.+],[K.sup.+] ATPase, acid phosphatase, alkaline phosphatase, and protein tyrosine phosphatases. Its vanadyl (IV) derivative, vanadyl sulfate (VOS[O.sub.4]), has a potent effect on transportation and metabolism of glucose.

Humans may be exposed to excessive vanadium in several situations, for example, over-consumption of vanadium-rich foods (e.g., some wines and seafood) (Bu-Olayan and al-Yakoob 1998; Teissedre et al. 1998), ingestion of certain dietary regimens, and inhalation of vanadium-rich environmental pollutants in certain occupations (Levy et al. 1984; Woodin et al. 1999, 2000). These occupations include boilermakers and power plant workers, who are often exposed to high levels of vanadium-rich compounds at work. Vanadium exposures have been associated with acute upper and lower respiratory symptoms in these workers (Levy et al. 1984; Woodin et al. 1999, 2000).

Exposure to excessive vanadium may also produce adverse effects on the cardiovascular system. Rats fed vanadium-containing drinking water for 2 months developed pulmonary hypertension (Susic and Kentera 1986). VOS[O.sub.4] and vanadium-rich pollutant dust (residual oil fly ash) produced an acute increase in pulmonary artery pressure in buffer-perfused rabbit lungs (Huang et al. 2002) and vasoconstriction in isolated rat aortic rings (Cadene et al. 1997). In boilermakers, the vanadium concentration in particulate air was associated with increases in the heart rate variability Heart rate variability (HRV) is a measure of variations in the heart rate. It is usually calculated by analysing the time series of beat-to-beat intervals from ECG or arterial pressure tracings.  index, indicating alterations in cardiac autonomic functions (Magari et al. 2002). The mechanisms by which VOS[O.sub.4] induced cardiovascular adverse effects, however, are unclear.

We have shown previously that VOS[O.sub.4] induced acute pulmonary vasoconstriction in perfused rabbit lungs and that this effect was associated with inhibition of nitric oxide production (Huang et al. 2002). The inhibition occurred within 10 min after the administration of VOS[O.sub.4], indicating the mechanisms for the inhibition may be at the levels of nitric oxide synthase (NOS) activity. Because phosphorylation of endothelial NOS (eNOS) is an important posttranslational post·trans·la·tion·al  
adj.
Of or relating to a substance or process, such as the addition of sugar groups to form a glycoprotein, that occurs or is formed after translation of protein: a posttranslational modification. 
 regulatory mechanism for its activity, in this study we investigated the effects of VOS[O.sub.4] on the phosphoryation of threonine (Thr) residues of eNOS, which would inhibit its enzymatic activity. The hypothesis was tested in isolated perfused lungs, isolated pulmonary artery rings, and pulmonary artery endothelial cells.

Materials and Methods

Reagents and chemicals. Vanadyl sulfate (VOS[O.sub.4]), zinc sulfate, copper, zinc superoxide dismutase (Cu,ZnSOD), PAPANONOate, calphostin C, and A23187 (a calcium ionophore ionophore /ion·o·phore/ (i´on-ah-for?) any molecule, as of a drug, that increases the permeability of cell membranes to a specific ion.

i·on·o·phore
n.
) were obtained from Sigma Chemical Co. (St. Louis, MO). Human pulmonary artery endothelial cells and endothelial cell growth medium were purchased from Cell Applications, Inc. (San Diego, CA), and the monoclonal antibodies to phosphothreonine and phosphoserine were purchased from Alexis Corp. (San Diego, CA). Rabbit anti-phosphothreonin(495) (phospho-[Thr.sup.495]) eNOS, mouse anti-phosphotyrosine (PY99), rabbit anti-eNOS (NOS3, N-20), rabbit anti-p-Akt1(serine at codon 473), goat anti-human Akt, protein A agarose, and secondary antibodies (both horseradish peroxidase-conjugated and fluorescein-conjugated) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Molecular mass standards, polyacrylamide, and buffers were purchased from Bio-Rad (Richmond, CA). We obtained ECL (Emitter-Coupled Logic) A digital circuit composed of bipolar transistors in which the emitter ends are wired together. ECL gates switch faster than TTL gates, but consume more power. See TTL, I2L and bipolar.

1.
 Western blotting detection reagents from Amersham Pharmacia Biotechnology (Piscataway, NJ).

Isolated perfused rabbit lung. The isolated buffer-perfused lung (IPL (Initial Program Load) Same as boot.

1. IPL - Information Processing Language.
2. IPL - Internet Public Library.
3. IPL - Initial Program Load.
4. IPL - Initial Program Loader.
) model using male New Zealand white rabbits (Robinson's Farm, Clemens, NC) weighing 2.5-3.0 kg has been described previously (Huang et al. 2002). Briefly, the pulmonary artery and the left atrium were cannulated can·nu·late also can·u·late  
tr.v. can·nu·lat·ed, can·nu·lat·ing, can·nu·lates
To insert a cannula into (a bodily cavity, duct, or vessel), as for the drainage of fluid or the administration of medication.

adj.
 for monitoring the mean pulmonary artery pressure and the left atrial pressure, respectively. A reservoir was connected with the perfusion circuit downstream from the left atrium and was suspended freely from a force displacement transducer (Model FT100; Grass Instrument Company, Quincy, MA) for monitoring the weight gain of the lung. The reservoir was placed at the lowest portion of the lung to maintain a left atrial pressure of zero. The perfusion circuit also consisted of a roller pump, a bubble trap, and a heat exchanger connected with Tygon tubing. The volume of the system was approximately 250 mL. The trachea trachea (trā`kēə) or windpipe, principal tube that carries air to and from the lungs. It is about 4 1-2 in. (11.4 cm) long and about 3-4 in. (1.9 cm) in diameter in the adult.  was also cannulated for monitoring tracheal pressure. The lung was ventilated ven·ti·late  
tr.v. ven·ti·lat·ed, ven·ti·lat·ing, ven·ti·lates
1. To admit fresh air into (a mine, for example) to replace stale or noxious air.

2.
 with 21% [O.sub.2] + 5% C[O.sub.2] through the tracheostomy using an animal respirator delivering 30 breaths/min at 2-3 cm [H.sub.2]O positive end-expiratory pressure positive end-expiratory pressure
n. Abbr. PEEP
A technique used in respiratory therapy in which pressure is maintained in the airway so that the lungs empty less completely in expiration.
. The tidal volume was adjusted to achieve a peak tracheal pressure of 7-10 mmHg (~20 mL). All pressures and weight gain were transmitted to a 4-channel analog-to-digital converter and amplifier connected to a personal computer equipped with data acquisition software (DATAQ; DATAQ Instruments, Inc. Akron, OH). After the pulmonary circulation was washed free of blood with Krebs-Henseleit (KH)-3% albumin buffer (82.8 mM sodium chloride, 4.7 mM potassium chloride, 2.4 mM monobasic monobasic /mono·ba·sic/ (-ba´sik) having but one atom of replaceable hydrogen.

mon·o·ba·sic
adj.
1. Having only one hydrogen ion to donate to a base in an acid-base reaction.
 potassium phosphate, 25 mM sodium bicarbonate, 1.2 mM magnesium sulfate, 2.7 mM calcium chloride, 11.1 mM dextrose dextrose: see glucose. , and 3% w/v bovine serum albumin, fraction V at a pH of 7.3-7.4), a recirculated perfused lung system with a flow of 100 mL/min was established. The lung was then allowed to stabilize for 10-15 min. Lungs with visible leaks and/or high pulmonary artery pressure (> 20 mmHg) during this period were excluded.

Measurements of microvascular pressure and pulmonary vascular resistance. At the end of the experiments, we measured the microvascular pressure ([P.sub.mv]) using the double occlusion technique (Linehan et al. 1982). Pulmonary vascular resistance was partitioned into upstream (arterial; [R.sub.a]) and downstream (venous; [R.sub.v]) resistance using the following equations:

[R.sub.a] = ([P.sub.pa] - [P.sub.mv])/Flow

[R.sub.v] = ([P.sub.mv] - [P.sub.la])/Flow

where [P.sub.la] is left atrial pressure. In our study, the flow was a constant at 100 mL/min, and [P.sub.la] Was set at zero.

Isolated rat pulmonary artery ring. Segments of the right and left main pulmonary arteries of Sprague-Dawley rats (250-350 g) approximately 2-3 mm in length were removed carefully and placed in KH buffer. The artery segments were then suspended in the Radnoti 4-unit organ bath system (Glass Technology Inc., Monrovia, CA). Each reservoir held 20 mL of KH buffer and was bubbled constantly with a 21% [O.sub.2] + 5% C[O.sub.2] gas mixture. After a 10-15 min stabilization period, the baseline tension of the rings was adjusted to 1 g before all experiments. In acetylcholine experiments, all rings were preconstricted with 1 [micro]M phenylephrine phenylephrine /phen·yl·eph·rine/ (-ef´rin) an adrenergic used as the hydrochloride salt for its potent vasoconstrictor properties.

phen·yl·eph·rine
n.
. The degree of relaxation and constriction was expressed as a percentage of constriction induced by 1 [micro]M phenylephrine.

Cultured human pulmonary artery endothelial cells (HPAEC HPAEC High-Performance Anion-Exchange Chromatography ). HPAEC (Cell Applications, Inc. San Diego, CA) were cultured in the endothelial cell growth medium in 6-well plates and were used during passages 3-5. HPAEC were isolated from normal human pulmonary arteries and have been shown to respond to a variety of vasoactive substances (Ryan et al. 1976a; 1976b).

Measurements of nitrite/nitrate. To determine the production of NO, we measured nitrite/nitrate concentrations in serial perfusate perfusate /per·fu·sate/ (per-fu´zat) a liquid that has been subjected to perfusion.

perfusate

a liquid that has been subjected to perfusion.
 samples and in the culture medium of HPAEC by the Griess reaction using a colorimetric col·or·im·e·ter  
n.
1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards.

2.
 nitrite/nitrate assay kit (Nitric Oxic Assay Kit; R&D Systems, Minneapolis, MN).

Immunoprecipitation and Western blot. Confluent con·flu·ent
adj.
1. Flowing together; blended into one.

2. Merging or running together so as to form a mass, as sores in a rash.
 HPAEC cells were washed once with ice-cold phosphate-buffered saline (PBS PBS
 in full Public Broadcasting Service

Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural,
) and then lysed with RIPA RIPA. The bank of a river, or the place beyond which the waters do not in their natural course overflow.
     2. An extraordinary overflow does not change the banks of the river. Poth. Pand. lib. 50, h.t. See Banks of rivers; Riparian proprietors; Rivers.
 buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set.
 in PBS, pH 7.4) containing 0.1 mM VOS[O.sub.4] and protease inhibitors (0.5 mg/mL aprotinin aprotinin /apro·ti·nin/ (ap?ro-ti´nin) an inhibitor of proteolytic enzymes used to reduce perioperative blood loss in patients undergoing cardiopulmonary bypass during coronary artery bypass graft. , 0.5 mg/mL E-64, 0.5 mg/mL pepstatin, 0.5 mg/mL bestatin, 10 mg/mL chymostatin, 0.1 ng/mL leupeptin). Cells were scraped up and passed through a 21-gauge needle 3-5 times. The cell samples were then centrifuged at 3,000 x g for 10 min at 4[degrees]C and the supernatants were collected. For immunoprecipitation, the supernatant samples were pre-cleared with protein A-agarose for 30 min at 4[degrees]C and centrifuged at 10,000 x g for 1 min. A rabbit anti-human eNOS antibody (1 [micro]g) was added to the supernatant (250 [micro]g) and incubated for 2 hr at 4[degrees]C, followed by another 2-hr incubation with 20 [micro]L protein A-agarose at 4[degrees]C with end-to-end rotation. After centrifugation at 10,000 x g for 1 min, the pellets were collected, washed twice with 500 [micro]L ice-cold RIPA buffer and once with ice-cold PBS, resuspended in electrophoresis sample buffer, and boiled for 5 min. Supernatant samples were then separated by 7.5% SDS-PAGE SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
 and transferred to a polyvinylidene difluoride (PVDF PVDF polyvinylidene difluoride ) membrane. The blots were blocked with 3% gelatin in TBS for 2 hr at room temperature and probed with antibodies against phosphothreonine, phosphoserine, or phosphotyrosine (PY99) overnight at 4[degrees]C. This was followed by incubation with appropriate HRP-conjugated secondary antibodies. To determine the amount of eNOS, the blot was stripped and reprobed with a rabbit anti-human eNOS antibody and HRP-conjugated goat anti-rabbit IgG. Bands were detected using chemiluminescence chemiluminescence /chemi·lu·mi·nes·cence/ (kem?i-loo?mi-nes´ens) luminescence produced by direct transformation of chemical energy into light energy.  reagents and film.

Immunocytochemical staining of phosphorylated eNOS. HPAEC grown on slides were treated with VOS[O.sub.4] (50 [micro]M) for 10 min. The cells were fixed with 4% paraformaldehyde paraformaldehyde: see formaldehyde.  and blocked with 3% donkey serum. The fixed cells were probed with an antibody against phospho-[Thr.sup.495] eNOS overnight at 4[degrees]C followed by incubation with a fluorescein-conjugated secondary antibody for 30 min. Images were examined under a fluoresence microscope equipped with a fluorescein isothiocyanate filter. Photographs were taken using a digital camera system (Nikon Microphot-SA; Southern MicroInstruments, Marietta, GA) and imaging software (ACT-1, version 2.10, Nikon).

Statistical analysis. We present data as mean [+ or -] SE. Time-series data were analyzed using repeated measures analysis of variance (ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
). We evaluated differences among groups at selective time points using one-way ANOVA followed by the Fisher protected least-significant-difference post-hoc test. The statistical analysis was performed using Statview software (SAS Institute Inc., Cary, NC). We report actual p-values where statistical analyses were performed. A p-value of < 0.05 was considered statistically significant.

Results

VOS[O.sub.4]-induced pulmonary artery constriction. The baseline mean pulmonary artery pressure of the IPL was 13 [+ or -] 1 mmHg. Vanadyl sulfate at 0.5 [micro]M injected into the pulmonary artery produced a transient increase in pulmonary artery pressure, which peaked at 5 min and returned to the baseline by 40 min (Figure 1A). At 5 [micro]M, VOS[O.sub.4] produced an acute and persistent increase in pulmonary artery pressure, which plateaued at approximately 24 mmHg after 20 min. At 50 [micro]M, VOS[O.sub.4] produced such severe pulmonary hypertension and lung edema edema (ĭdē`mə), abnormal accumulation of fluid in the body tissues or in the body cavities causing swelling or distention of the affected parts.  that the IPL could not be sustained for 40 min. All subsequent experiments in IPL were thus performed with 5 [micro]M of VOS[O.sub.4].

[FIGURE 1 OMITTED]

The increase in pulmonary vascular resistance induced by VOS[O.sub.4] was primarily due to constriction of upstream pulmonary vessels (Figure 1B). VOS[O.sub.4] produced vasoconstriction in pulmonary artery rings (Figure 2A) and IPL, whereas zinc sulfate had little vasoactive effect (Figure 2B).

[FIGURE 2 OMITTED]

VOS[O.sub.4] shifted the acetylcholine-induced vasorelaxation curve to the right (Figure 3A) in pulmonary artery rings, which is consistent with a loss of vasodilatory activity of NO. Inhibition of NO production by an NOS inhibitor, L-NAME L-NAME G -nitro-L-Arginine-Methyl Ester
L-NAME N-Nitro-L-Arginine Methyl Ester
, increased pulmonary artery pressure in our IPL system (Huang et al. 1997). VOS[O.sub.4]-induced pulmonary hypertension was attenuated Attenuated
Alive but weakened; an attenuated microorganism can no longer produce disease.

Mentioned in: Tuberculin Skin Test


attenuated

having undergone a process of attenuation.
 by an NO donor, PAPANONOate (1 mM) and Cu,ZnSOD (Figure 3B and 3C).

[FIGURE 3 OMITTED]

VOS[O.sub.4] inhibited NO production. In control IPL, NO production measured as nitrite/nitrate accumulation in the perfusate increased gradually over time; the total increase was 3.4 [+ or -] 0.8 [micro]M during 40 min of perfusion (Figure 4A). VOS[O.sub.4] (5 [micro]M) inhibited nitrite/nitrate accumulation, which occurred as early as 10 min after the treatment. At 40 min, VOS[O.sub.4] inhibited nitrite/nitrate accumulation by approximately 80%. The average change in nitrite/nitrate concentration after 40 min of perfusion was decreased to 0.7 [+ or -] 0.6 [micro]M (p = 0.003 vs. control) (Figure 4A). In HPAEC, VOS[O.sub.4] inhibited constitutive and A23187-stimulated NO production (Figure 4B and 4C).

[FIGURE 4 OMITTED]

Effect of VOS04 on eNOS phosphorylation. In HPAEC, VOS[O.sub.4] induced an approximately 2-fold increase in Thr phosphorylation of eNOS (Figure 5A and 5B). Immunostaining with a phospho-[Thr.sup.495] eNOS antibody was also increased in VOS[O.sub.4]-treated HPAEC (Figure 5C). [Thr.sup.495] was located in the calcium-calmodulin binding domain of eNOS, and phosphorylation of this Thr residue has been shown to inhibit eNOS activity (Fulton et al. 2001; Govers and Rabelink 2001). These findings are consistent with the VOS[O.sub.4]-induced inhibition of NO production stimulated by A23187, which enhances calcium binding to calmodulin calmodulin /cal·mod·u·lin/ (kal-mod´u-lin) a calcium-binding protein present in all nucleated cells; it mediates a variety of cellular reponses to calcium.

cal·mod·u·lin
n.
. VOS[O.sub.4] did not alter phosphorylation on [Ser.sup.1177] tyrosine residues of eNOS or Akt1 phosphorylation (data not shown).

[FIGURE 5 OMITTED]

Effects of protein kinase C inhibitor on VOS[O.sub.4] effects. The VOS[O.sub.4]-induced pulmonary artery hypertension in perfused rabbit lungs was partially reversed by pretreatment with calphostin C (0.1 [micro]M), a protein kinase C (PKC) inhibitor (Figure 6A). The attenuation Loss of signal power in a transmission.
Attenuation

The reduction in level of a transmitted quantity as a function of a parameter, usually distance. It is applied mainly to acoustic or electromagnetic waves and is expressed as the ratio of power densities.
 of pulmonary hypertension was associated with a reversal of VOS[O.sub.4]-induced inhibition of NO production (Figure 6B). In HPAEC, 0.1 [micro]M calphostin C also attenuated VOS[O.sub.4]-induced inhibition of NO production (Figure 7A). This was associated with decreased [Thr.sup.495] phosphorylation of eNOS (Figure 7B).

[FIGURES 6-7 OMITTED]

Discussion

Vanadium compounds have been known to cause vasoconstriction in systemic arteries. Orthovanadate ([V.sup.+5]) constricts renal arteries (Benabe et al. 1984; Kumar and Corder 1980; Larsen and Thomsen 1980a, 1980b), splanchnic splanchnic /splanch·nic/ (splangk´nik) pertaining to the viscera.

splanch·nic
adj.
Of or relating to the viscera; visceral.



splanchnic

pertaining to the viscera.
 arteries (Larsen and Thomsen 1980a), and the aorta (Borchard et al. 1981), and VOS[O.sub.4] ([V.sup.+4]) constricts mesenteric arteries and the aorta (Cadene et al. 1997). In the present study, we showed in intact lungs and isolated pulmonary artery rings that VOS[O.sub.4] produced pulmonary artery constriction, which could be attributed in part to the loss of vasodilator vasodilator /vaso·di·la·tor/ (-di-la´ter)
1. causing dilatation of blood vessels.

2. a nerve or agent that does this.


va·so·di·la·tor
n.
 activity provided by endothelial NO. This was supported by the inhibition of nitrite/nitrate accumulation in the perfusate of IPL and HPAEC and by the rightward shift of the acetylcholine-induced vasorelaxation curve induced by VOS[O.sub.4].

VOS[O.sub.4]-induced NO inhibition in our study was associated with increased [Thr.sup.495] phosphorylation of eNOS. [Thr.sup.495] phosphorylation is one of the few negative posttranslational mechanisms that regulate eNOS activity (Fulton et al. 2001; Govers and Rabelink 2001). Many agonist stimuli, such as vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).  (VEGF VEGF vascular endothelial growth factor. ) (Michell et al. 2001), bradykinin bradykinin /brady·ki·nin/ (-ki´nin) a nonapeptide kinin formed from HMW kininogen by the action of kallikrein; it is a very powerful vasodilator and increases capillary permeability; in addition, it constricts smooth muscle and  (Fleming et al. 2001; Harris et al. 2001), and ischemic Ischemic
An inadequate supply of blood to a part of the body, caused by partial or total blockage of an artery.

Mentioned in: Antiangiogenic Therapy, Subarachnoid Hemorrhage, Ventricular Fibrillation


ischemic
 stress (Chen ZP et al. 1999), inhibit phosphorylation at the [Thr.sup.495] residue. Phosphorylation of [Thr.sup.495] by antagonists such as VOS[O.sub.4], as shown in our study, would thus interfere with the binding of calcium-calmodulin and inhibit eNOS activity. Several previous studies in systemic arteries have shown that the vanadium-induced contraction was related to entry of extracellular calcium (Benabe et al. 1984; Cadene et al. 1997). Extracellular calcium may activate protein phosphatases (e.g., PP1, PP2A PP2A Protein Phosphatase 2A ) and dephosphorylate [Thr.sup.495] (Michell et al. 2001).

The mechanism for [Thr.sup.495] phosphorylation was partially mediated by activation of PKC became the PKC inhibitor, calphostin C, attenuated VOS[O.sub.4]-induced Thr phosphorylation and restored NO production in HPAEC. These findings agree with those in bovine and porcine aortic endothelial cells, in which PKC activation phosphorylates [Thr.sup.495] of eNOS (Fleming et al. 2001; Michell et al. 2001).

There are other phosphorylative mechanisms that regulate eNOS activity post-translationally. Phosphorylation of [Ser.sup.1177] enhances eNOS activity, a mechanism shared by many NO agonists such as insulin (Montagnani et al. 2001; Schnyder et al. 2002), insulin-like growth factor-1 (Michell et al. 1999), VEGF (Brouet et al. 2001; Dimmeler et al. 2000; Fulton et al. 1999; Michell et al. 2001), estrogen (Hisamoto et al. 2001), and stress (Boo et al. 2002). In our study, VOS[O.sub.4] did not affect Ser phosphorylation of eNOS or Akt1. eNOS may also be phosphorylated at tyrosine residues, although the functional consequence of the enzyme is less clear. Tyrosine phosphorylation of eNOS produced by hydrogen peroxide and orthovanadate decreased enzyme activity (Garcia-Cardena et al. 1996). In contrast, VEGF-, estrogen- or stress-induced eNOS activation could be inhibited by tyrosine kinase inhibitors (Ayajiki et al. 1996; Chen Z et al. 1999; Corson et al. 1996; Papapetropoulos et al. 1997). We did not see tyrosine phosphorylation of eNOS in HPAEC exposed to 50 [micro]M of VOS[O.sub.4], but we could not exclude tyrosine residues of other proteins functionally linked to NOS activation.

Our findings that VOS[O.sub.4] produces pulmonary vasoconstriction by inhibiting NOS activity have several clinical implications. First, certain occupations, such as boilermakers, are exposed to fuel oil ash that contains a high concentration of vanadium (up to 30 [micro]g/[m.sup.3]) for many hours a day during a boiler overhaul (Hauser et al. 1995, 1998; Woodin et al. 1999, 2000). Inhalation of vanadium in this situation has been associated with not only airway inflammation and constriction (Hauser et al. 1995, 2001, 2002; Hessel et al. 1998; Woodin et al. 1998, 2000) but also cardiovascular effects measured as increased heart rate variability (Magari et al. 2002). Vanadium contained in vanadium-rich residual oil fly ash given intratracheally can gain access into the pulmonary circulation (Huang et al. 2002).

Second, certain weight-reduction and muscle-building regimens contain significant amounts of VOS[O.sub.4]. For example, BioLean Free contains 400 [micro]g VOS[O.sub.4] per serving (four tablets), Mass Appeal contains 5 mg VOS[O.sub.4] per serving, and Satiete contains 660 [micro]g of VOS[O.sub.4] per serving (all from Wellness International Network, Ltd., Piano, TX). With the recommended doses of several servings a day on the label, regular users can consume a significant amount of VOS[O.sub.4]. The pulmonary vasoconstrictor vasoconstrictor /vaso·con·stric·tor/ (-kon-strik´ter)
1. causing constriction of blood vessels.

2. a nerve or agent that does this.


va·so·con·stric·tor
n.
 property of VOS[O.sub.4] demonstrated in our study raised the possibility of increased risk for pulmonary hypertension in these individuals, especially in view of the long elimination half-life of vanadium (12 days) after ingestion (Ramanadham et al. 1991). Indeed, rats fed with vanadium for 2 months developed pulmonary hypertension and right ventricular hypertrophy right ventricular hypertrophy Cardiology An ↑ in myocardial mass which may be due to interventricular septal defects or ↑ blood flow–eg, hyperthyroidism  (Susic and Kentera 1986).

Vanadium is a unique metal that is known to avidly permeate cell membranes. Once inside the cells, vanadium chelates to many intracellular ligands, such as creatine phosphate, proteins, glutathione, and ascorbic acids (Nechay et al. 1986), and presumably pre·sum·a·ble  
adj.
That can be presumed or taken for granted; reasonable as a supposition: presumable causes of the disaster.
 is detoxified. When vanadium loads exceed the capacity of the chelators, however, free vanadyl ions ([V.sup.+4]) and the more potent vanadate van·a·date  
n.
Any of three anions, VO3, VO4, or V2O7, containing pentavalent vanadium.



[vanad(ium) + -ate2.]

Noun 1.
 ([V.sup.+5]) may be released at high local concentration and may attack important biomolecules This page aims to list articles on Wikipedia that describe particular biomolecules or types of biomolecules.

This list is not necessarily complete or up to date - if you see an article that should be here but isn't (or one that shouldn't be here but is), please update the page
, resulting in cellular dysfunction (Hirao 2000; Liochev and Fridovich 1987; Nechay 1984). Our results indicate that eNOS in endothelial cells is a potential target. Given the long elimination half-life of vanadium, the endothelial dysfunction with loss of NO-mediated vasodilator activity raised the possibility that individuals exposed to excessive vanadium in the environment may be at a greater risk for developing pulmonary hypertension and perhaps other vascular diseases. Our results indicate the need for further studies on the prevalence of cardiovascular diseases in at-risk workers and individuals consuming vanadium-rich dietary supplements on a regular basis.

REFERENCES

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Zhuowei Li, (1) Jacqueline D. Carter, (2) Lisa A. Dailey, (2) and Yuh-Chin T. Huang (2)

(1) Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina, Chapel Hill, North Carolina Chapel Hill is a town in North Carolina and the home of the University of North Carolina at Chapel Hill (UNC-CH), the oldest state-supported university in the United States. As of the 2000 census, it had a population of 48,715. As of 2004 its estimated population was 52,440. , USA; (2) National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency Environmental Protection Agency (EPA), independent agency of the U.S. government, with headquarters in Washington, D.C. It was established in 1970 to reduce and control air and water pollution, noise pollution, and radiation and to ensure the safe handling and , Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. , North Carolina, USA

Address correspondence to: Y-C. T. Huang, CB 7315, 104 Mason Farm Road, Chapel Hill, NC 27599, USA. Telephone: (919) 843-9504. Fax: (919) 966-6271. E-mail: huang.tony@epa.gov

We thank C. Marshall of Duke University Medical Center for procuring rat pulmonary artery rings and J. Soukup of the U.S. EPA for measuring nitrite/ nitrate in the samples.

The research described in this article has been reviewed by the Health Effects and Environmental Research Laboratory, U.S. EPA, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use.

The authors declare they have no competing financial interests.

Received 23 May 2003; accepted 22 October 2003.
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Title Annotation:Research
Author:Huang, Yuh-Chin T.
Publication:Environmental Health Perspectives
Date:Feb 1, 2004
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