Chronic exposure to low levels of lead results in arterial hypertension in humans and in experimental animals (1 , 2 3 4 , 5
Nitric oxide (NO) is a gas generated by the endothelium through the conversion of L-arginine into L-citrulline by the activity of the endothelial NO synthase (eNOS). The NO generated by the eNOS activity induces vasodilatation by stimulating the soluble guanylate cyclase (sGC) in the adjacent smooth muscle cells (6 7
sGC is an heterodimer composed of a large (α1 ) and a small (β1 ) subunit (8 1 and β1 subunits seem to contain a catalytic domain, the expression of enzymatic activity requires the presence of both subunits (9
In this regard, modifications in the sGC activity and expression have been found in the vascular wall of aging rats and experimental models of myocardial infarction, respectively (10 , 11 in vitro studies have demonstrated that guanylate cyclases can be inactivated by heavy metals such as cobalt, nickel, and manganese by binding within the porphyrin ring of heme, substituting the iron molecule, and thus locking heme in a deoxy state (12
Impaired endothelium-dependent vasodilatation has a relevant role in the development of several cardiovascular diseases, including hypertension in rats and humans (13 , 14 15 – 17
An increased production of reactive oxygen species (ROS) has been observed in lead-treated animals that contributed to the associated arterial hypertension (18 , 19 20 18 , 20
Materials and Methods
Animal Procedures
The study protocols were approved by the Institutional Ethics Committee for animals and were performed according the international conventions on animal experimentation. Experiments were carried out with the use of 20 male Wistar rats with age-matched pair-fed controls. Beginning at 3 mo of age, the experimental group (10 rats) was given 5 ppm lead acetate alone or in combination with physiologic doses of vitamin C (3 mmol/L) in the drinking water during 30 d. A parallel control group was given vitamin C alone (3 mmol/L).
Indirect pressure readings by the tail-cuff method were performed on each rat while it was conscious, by use of a Narco electrosphyngomanometer coupled to a computer system (Power Lab 400, AD Instruments, Casterhill, New South Wales, Australia). The rats were prewarmed for 10 to 15 min and placed into a restrainer for BP measurement. Five consecutive recordings (∼1 min apart) were performed.
At the end of the intoxication period, the animals were anesthetized with pentobarbital (30 mg/kg im), and the descending thoracic aorta was removed and cut into three portions. One of the aortic segments was immediately frozen in liquid nitrogen for the molecular biology determinations. The remaining vessel was processed to test the endothelium-dependent response to acetylcholine (Ach) and the endothelium-independent response to sodium nitroprusside (SNP). Blood samples were collected to determine lead content in whole blood by use of an atomic absorption spectrophotometer (Perkin-Elmer, model 5000 with a graphite furnace).
Preparation for Isometric Tension Measurements
Aortic segments were cut into portions of 2 mm in length and suspended in Kreb’s-Henseleit solution (in mmol/L: NaCl 115, KCl 4.6, KH2 PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, glucose 11.1, and calcium sodium ethylenediaminetetraacetate 0.02 [pH 7.4]). The organ bath contained 5 ml of the solution gassed with 95% O2 /5% CO2 . Aortic segments were connected to isometric force displacement transducers coupled to a computer system (Power Lab 400, AD Instruments, Casterhill, New South Wales, Australia). The segments were allowed to rest to the previously determined optimal resting force of 2 g, as determined by repeated exposure to 20 mmol/L KCl. The endothelium-dependent relaxation to Ach and the endothelium-independent relaxation to SNP were tested on arteries precontracted with 10−5 mol/L phenylephrine, as reported elsewhere (21 −5 mol/L) to block any effect mediated by the activation of cyclooxygenase.
Determination of eNOS and sGC β1 Subunit Expression by Western Blot Analysis
The expression of eNOS and sGC β1 subunit protein was analyzed by Western blot, as described elsewhere (22 23 1 subunit. Blots were incubated with the first antibody (1:2500) for 1 h at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase-conjugated anti-rabbit Ig antibody) at a dilution of 1:1500 for another hour. Specific eNOS and sGC β subunit proteins were detected by enhanced chemoluminiscense (Amersham Corp., Buckinghamshire, UK) and evaluated by densitometry (Molecular Dynamics, Buckinghamshire, UK). Prestained protein markers (Sigma) were used for molecular mass determinations. As reported elsewhere (24 , 25 1 subunit expression with the expression of another protein, we analyzed the expression of β-actin by Western blot, using a β-actin monoclonal antibody (Sigma Aldrich, St. Louis, MO). For this purpose, a parallel gel with identical samples was run, and, after blotting onto nitrocellulose, the Western blot analysis was performed with the β-actin monoclonal antibody (1:2000).
Determination of Vascular sGC Activity
Aortic segments obtained from controls and lead-treated rats were cut into five portions and individually suspended into culture solution (RPMI medium, 5% fetal calf serum, at 37°C) that contained the phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (2 mmol/L) to prevent breakdown of cGMP. SNP was added to each vessel in concentrations 0, 10−8 , 10−6 , 10−4 , and 10−2 mol/L during 2 min. Afterward, the vessels were immediately stored in liquid nitrogen. The frozen segments were pulverized and resuspended in an ice-cold solution that contained 200 mmol/L perchloric acid. After they were mixed, the homogenates were centrifuged (12,000 × g , 15 min, 4°C), and the supernatants were recovered and washed out five times with 5-vol diethyl ether. The remaining aqueous phase was dried under a stream of N2 .
cGMP was measured as described elsewhere (21 , 26
Determination of sGC by Reverse Transcriptase-PCR
A frozen aortic segment of each animal was homogenized. Briefly, the homogenate was resuspended and vortexed in 800 μl lysis buffer (4 mol/L guanidinium tyocianide, 0.75 mol/L sodium citrate, 10% serhosyl, and β-mercaptoethanol). After incubation for 30 min at 4°C, the sample was extracted twice by vortexing with an equal volume of phenol-chloroform-isoamylalcohol (24:1, vol/vol). RNA resuspended in H2 O diethyl pyrocarbonate was stored at −80°C until use. RNA samples were precipitated and resuspended in (mmol/L): Tris 10, NaCl 10, and ethylenediaminetetraacetate (pH 8.0) and were incubated with 3 U of RNAse-free DNAse per 35 μg total RNA for 30 min at 37°C to digest traces of genomic DNA. Total RNA (50 to 500 ng) was combined with 50 U Moloney murine leukemia virus reverse transcriptase (Promega) in (mmol/L): MgCl2 5, KCl 50, Tris-HCl 10, deoxyribonucleoside triphosphates 1, 10 U RNAse inhibitor, and 2.5 μmol/L random hexamers to prime the cDNA formation in a reaction volume of 20 μl for 15 min at 42°C. Samples were then heated for 5 min at 99°C to destroy reverse transcriptase activity before the PCR reaction.
Oligonucleotides complementary to sGC α1 and β1 subunit were purchased from Life Technologies BRL (Buckinghamshire, UK). We used the following sequences: α1 subunit (forward, base position 1071, 5′-GAA ATC TTC AAG GGT TAT G-3′ and reverse, base position 1896, 5′-CAC AAA GCC AGG ACA GTC-3′), β1 subunit (forward, base position 1450, 5′-GGT TTG CCA GAA CCT TGT ATC CAC C-3′ and reverse base position 1733, 5′-GAG TTT TCT GGG GAC ATG AGA CAC C-3′), and β-actin (forward, base position 35, 5′-GAT CAT GTT TGA GAC CTT CAA CAC C-3′ and reverse, base position 1017, 5′-AGT TTC ATG GAT GCC ACA GGA TTC C-3′).
cDNA was amplified (25 through 31 cycles) at 92°C for 1 min, 58°C for 1.5 min, and 72°C for 3 min (melting, annealing, and extension temperatures, respectively). For the PCR reaction, primers were used at 1 μmol/L for the sGC α1 and β1 subunits and 0.2 μmol/L for β-actin. MgCl2 concentration was 2 mmol/L, and 2.5 U polymerase (Biotools) were used per reaction. After the amplification, 10 μl of the reverse transcriptase-PCR reaction mixture were electrophoresed on 0.9% agarose gels, stained with etidium bromide, visualized on an ultraviolet transilluminator, and photographed. A molecular weight standard that consisted of 100-bp increments between 100 and 2600 bp (BioRad) was used to confirm the predicted PCR product size.
Statistical Analyses
Results are expressed as mean ± SEM. Each of the above mentioned studies was performed in at least a minimum of eight rats. Comparisons were performed by ANOVA or paired and unpaired t test when appropriate. Bonferroni’s correction for multiple comparisons was used to determine the level of significance of P . P < 0.05 was considered significant.
Results
Vasorelaxing Response to Ach and eNOS Expression
There was a sustained elevation of mean arterial BP (MAP) after 30 d of lead exposure (MAP: control 115 ± 5 mmHg, 30 d lead: 133 ± 7 mmHg, n = 10, P < 0.05). No significant differences were observed in MAP in shorter periods of lead exposure. Only in the 21-d lead exposure period, a tendency to increase MAP was detected, although it did not reach statistical significance. Therefore, the time of 30 d of lead exposure was chosen for the following experiments.
Treatment with vitamin C reduced the hypertensive effect of lead (in mmHg; lead + vitamin C: 130 ± 5, P < 0.05 with respect to lead group). Blood lead levels were not modified by cotreatment with vitamin C (lead: 42.5 ± 2.3 μg/dL, lead + vitamin C: 40.0 ± 3.1 μg/dL, P = NS).
Ach produced a dose-related relaxation in phenylephrine-precontracted aortic rings from control rats (Figure 1 ). Relaxation to Ach was significantly reduced in aortic segments from lead-poisoned rats (Figure 1 ). The 50% maximum effect dose (EC50 ) for control vascular segments was 5 × 10−8 ± 0.2 mol/L, and the EC50 for those from lead-exposed rats was 4.7 × 10−7 ± 0.4 mol/L (P < 0.05). As shown in Figure 1 , a greater maximum response to Ach was observed in the aortic rings obtained from control than from lead-exposed rats (Emax control group 100 ± 1%, Emax lead-treated rats 89 ± 2%, P < 0.05).
Figure 1.:
Graph showing vascular relaxation by acetylcholine in control (▪), 30-d lead-treated (•), and 30-d lead + vitamin C-treated (○) rats. Results are expressed as percentage of relaxation with respect to contraction by phenylephrine (10−5 mol/L). Results are expressed as mean ± SEM of segments from ten different rats for each experimental group. *P < 0.05 with respect to control segments.
Coadministration of lead with vitamin C restored vascular relaxation to Ach toward control levels (EC50 lead + vitamin C: 4.5 × 10−8 ± 0.3 mol/L; P = NS with respect to control). Vitamin C administration did not modify the relaxing response to Ach in control rats with respect to untreated control rats (EC50 control + vitamin C: 4.8 × 10−8 ± 0.3 mol/L; P = NS with respect to control).
Aortic rings isolated from 30 d lead-treated rats demonstrated an increased eNOS protein expression with respect to that of control rats (Figure 2, A and B ). Treatment with vitamin C prevented the increased eNOS protein expression in lead-exposed rats and resulted in a level of expression similar to that found in control segments (Figure 2, A and B ). In contrast, vitamin C did not modify NOS expression in control segments (Figure 2, A and B ).
Both nNOS and iNOS protein expression was almost undetectable in aortic segments from control Wystar Kyoto rats (Figure 3 ). Treatment with 5 ppm lead during 30 d alone or in combination with vitamin C failed to stimulate neither iNOS nor nNOS expression in the vascular wall (Figure 3 ). The specificity of the monoclonal antibodies used to detect nNOS and iNOS isoforms has been demonstrated elsewhere (27 , 28
Figure 2.:
(A) Representative Western blot showing endothelial nitric oxide synthase (eNOS) protein expression in the vascular wall of control, control treated for 30 d with vitamin C, 30-d lead-treated, and 30-d lead + vitamin C-treated rats. The expression of the constitutive protein β-actin is shown at the bottom of the figure. (B) Bar graph showing the densitometric analysis of eNOS expression normalized by the β-actin values. Results are represented as mean ± SEM of aortic segments obtained from ten different rats for each experimental group. *P < 0.05 with respect to control segments.
Figure 3.:
(Top) Representative Western blot showing inducible NOS (iNOS) protein expression in the vascular wall of control and 30-d lead-treated rats. The homogenate obtained from Escherichia coli lipopolysaccharide (LPS)-treated aortic rat segment was used as positive control. (Bottom) Representative Western blot showing neuronal NOS (nNOS) protein expression in the vascular wall of control and 30 d lead-treated rats. The homogenate obtained from rat cerebellum was used as a positive control.
Vasorelaxing Response to SNP and sGC Expression in Lead-Treated Rats
The endothelium-independent agent SNP produced a dose-related relaxation of precontracted aortic segments from both control and lead-treated rats (Figure 4 ). However, the relaxing response to SNP was significantly reduced in aortic segments from 30 d lead-treated rats (Figure 4 ). The EC50 for SNP-induced vasorelaxation was higher in 30 d lead-treated rats than in control rats (in mol/L, EC50 lead-treated: 5.8 × 10−8 ± 0.5 mol/L, EC50 control rats: 5.3 × 10−9 ± 0.6 mol/L, P < 0.05). Moreover, the maximum vasodilating response to SNP was greater in aortic segments obtained from controls than in those from 30 d lead-treated rats: (Emax control, 100 ± 0.8%; lead, 95 ± 0.7%; P < 0.05) (Figure 4 ).
Figure 4.:
Graph showing vascular relaxation by sodium nitroprusside (SNP) in control (▪), 30-d lead-treated (•), and 30-d lead + vitamin C-treated (○) rats. Results are expressed as percentage of relaxation with respect to contraction by phenilephrine (10−5 mol/L). Results are represented as mean ± SEM of segments from ten different rats for each experimental group. *P < 0.05 with respect to control segments.
Both basal and SNP-stimulated sGC activity were significantly decreased in lead-treated rats (Figure 5 ). The addition of the NO-donor SNP caused a dose-dependent increase in the accumulation of cGMP in the vascular wall of control rats (Figure 5 ). The curve of accumulation of cGMP was significantly reduced in 30 d lead-treated rats with respect to that of control rats (Figure 5 ). Therefore, the maximum cGMP concentration obtained with the maximum tested concentration of SNP (10−2 mol/L) was greater in controls than in 30 d lead-treated animals (Figure 5 ). However, no significant differences were found when cGMP levels were calculated as the increment between basal and 10−2 mol/L SNP-stimulated cGMP formation (control, 58.0 ± 10.0; lead-treated rats, 64.0 ± 8.8 pmol/mg; P = NS) The vascular wall from lead-treated rats showed a marked reduction of both α1 or β1 sGC subunits (Figure 6 ). No differences were found in the β-actin amplification used as internal standard of PCR. The Western blot analysis of the expression of sGC β1 subunit confirmed the PCR findings. The expression of sGC β1 subunit was markedly reduced in the vascular wall of lead-treated rats (Figure 7, A and B ).
Figure 5.:
Cyclic GMP (cGMP) accumulation in the vascular wall of control (▪) and lead-treated rats (•) before and after the addition of increasing concentration of the NO donor, SNP. All the measurements were performed in the presence of 3-isobutyl-1-methylxantine (2 mmol/L). Results are represented as mean ± SEM of six different experiments for each experimental group. *P < 0.05 with respect to control.
Figure 6.:
Reverse transcriptase-PCR (RT-PCR) analysis showing the expression of α1 - and β1 -soluble guanylate cyclase (sGC) subunits in the vascular wall of control and 30-d lead-treated rats. RT-PCR for β-actin was used as the control.
Figure 7.:
(A) Representative Western blot showing β1 -sGC subunit expression in the vascular wall of control, controls treated for 30 d with vitamin C, 30-d lead-treated, and 30-d lead + vitamin C-treated rats. The expression of β1 -sGC subunit in an homogenate of rat platelets (PLT) was used as control. (B) Densitometric analysis of the Western blot. The β1 -sGC subunit protein was normalized by the β-actin expression. Results are expressed as mean ± SEM of aortic segments obtained from seven different animals for each experimental group. *P < 0.05 with respect to control.
Effect of Vitamin C Administration on the Endothelium-Independent Vasorelaxation
As shown in Figure 4 , cotreatment of lead and vitamin C normalized the vasorelaxation response to SNP that was observed in lead-treated rats (EC50 lead + vitamin C, 6.2 × 10−9 ± 0.4 mol/L; Emax lead + vitamin C, 100 ± 0.6%; P < 0.05 with respect to the lead-treated group and P = NS with respect to control). Vitamin C administration had no effect on the relaxing response to SNP in aortic segments from control rats.
The Western blot analysis demonstrated that treatment with lead plus vitamin C protected the expression of the sGC β1 subunit (Figure 7, A and B ). No differences were found in the level of sGC β1 subunit expression in aortic segments from vitamin C-treated control rats compared with that of control untreated animals (Figure 7, A and B ).
Discussion
In this study, we have shown the existence of both an impaired endothelium-dependent and -independent vasodilating response in lead-induced hypertensive rats. This fact was accompanied by a marked increase in eNOS protein expression and downregulation of sGC. These effects were prevented when the antioxidant vitamin C was simultaneously administered with lead, which suggests the involvement of ROS in the regulation of the NO/cGMP relaxing system in the vascular wall of lead-treated rats.
Lead-induced hypertension is the element of chronic lead-intoxication syndrome that has received more attention in the literature. Different considerations have been raised to explain the pathogenesis of lead-induced hypertension. During the past decade, several studies have focused on the ability of endothelium to generate NO in this model of hypertension. These studies demonstrated the existence of an altered NO synthesis, probably because of a diminished eNOS activity and/or increased NO catabolism by oxygen free radicals. In this regard, Vaziri et al. (18 et al. (29
In our experiments, we observed an impaired endothelium-dependent vasorelaxing response in lead-treated rats. Paradoxically, the expression of eNOS protein, the NOS isoform involved in the endothelium-dependent vasodilatory response (30 31 , 32
In vitro studies have demonstrated that NOS activity is inhibited by lead (33 et al. (34
In this study we could not find modifications in either nNOS or iNOS protein expression in low-level lead-exposed rats. Expression of iNOS protein has, however, been observed in rats treated with higher doses of lead (100 ppm) than those used in our experimental model (34
As mentioned above, lead-induced hypertension has been suggested to be closely related to enhanced activity of ROS (18 , 29
Initially, eNOS protein was defined as a constitutive enzyme. However, it was later demonstrated that eNOS protein expression could be up- and downregulated by different factors, including chronic exercise, cytokines, and the growth state of endothelial cells (24 , 25 , 35 , 36 et al. (37 2 O2 caused an increase in eNOS protein expression in cultured endothelial cells through transcriptional and posttranscriptional mechanisms. In this sense, treatment with tempol, a superoxide dismutase-mimetic agent, or with the nonspecific antioxidant desmethyltirilazad reversed the upregulation of eNOS protein expression in lead-cultured endothelial cells (38
Independently of the ability of the endothelium to produce NO, this work demonstrates for the first time an impaired vasodilating response to exogenous NO in lead-treated rats. This fact was associated with the existence of a downregulation of the expression of both α1 and β1 sGC subunits in the vascular wall of lead-treated rats. The sGC is the main receptor for NO that mediates the NO-dependent vasodilation (7
The fact that the vasorelaxing response to the exogenous NO-donor SNP was impaired in lead-treated rats suggests either that sGC is not functional or that cGMP is rapidly degraded. In this regard, the reduction in basal and SNP-stimulated cGMP in the vascular wall of lead-exposed rats could be attributable to an increased cGMP degradation by phosphodiesterase. However, cGMP measurements were performed in the presence of the general phosphodiesterase inhibitor IBMX, discarding this possibility.
Previous in vitro studies have demonstrated that heavy metals inhibit guanylate cyclase activity (12 1 and α1 subunits of sGC mRNA levels were decreased in lead-hypertensive rats, as was observed by reverse transcriptase-PCR analysis. A conventional quantitative method, i.e., Western blot analysis, confirmed the reduction in the sGC β1 subunit in the vascular wall of lead-treated rats. Because the presence of both α1 and β1 subunits is required for sGC activity, and because at least β1 protein levels were found to be decreased in lead-treated rats, these findings suggest that the impairment in the endothelium-independent response to NO was due to a loss of sGC. The antioxidant therapy with vitamin C inhibited the downregulation of sGC protein expression elicited by lead administration and restored the NO-dependent vascular relaxation.
Several studies have suggested that vitamin C administration improves the endothelial-dependent relaxation in different cardiovascular diseases characterized by the existence of endothelial dysfunction (39 , 40
In summary, lead-induced hypertension provokes an impaired endothelium-dependent and -independent vasorelaxing response. Moreover, independent of the ability of endothelium to produce NO, the level of expression of sGC, the intracellular receptor of NO in vascular smooth muscle cells, was reduced, leading to a decreased effectiveness of NO to relax the vascular wall. Treatment with vitamin C restored both the endothelium-dependent and -independent relaxing response and prevented lead-induced downregulation of sGC, suggesting a new role for ROS in the pathogenesis of lead-induced hypertension.
This work was supported by the Fondo de Investigación Sanitaria de la Seguridad Social (Grant FISS 00/0145). A.J., J.A.R.-F., and S.V. are fellows from Fundación Conchita Rábago de Jiménez Díaz. We thank Begon[Combining Tilde]a Larrea for secretarial assistance.
1. Sharp DS, Osterloh J, Becker CE, Bernard B, Smith AH, Fisher JM, Syme SL, Holman BL, Johnson T: Blood pressure and blood lead concentration in bus drivers. Environ Health Perspect 78: 131–137, 1988
2. Sharp DS, Becker CE, Smith AH: Chronic low-level lead exposure. Its role in the pathogenesis of hypertension. Med Toxicol 2: 210–232, 1987
3. Perry HJ, Erlanger MW, Perry EF: Increase in the blood pressure of rats chronically fed low levels of lead. Environ Health Perspect 78: 107–111, 1988
4. United States Department of Labor, OSHA: Occupational exposure to lead. Final standard. Federal Register 43: 52952–53014, 54353–54616, 1978
5. Fowler BA, Kimmel CA, Woods JS, MacConnell EG, Grant LD: Chronic low level lead toxicity in the rat. Toxicol Appl Pharmacol 56: 59–77, 1980
6. Busse R, Mulsch A, Fleming I, Hecker M: Mechanisms of nitric oxide release from the vascular endothelium. Circulation 87: 18–25, 1993
7. Katsuki S, Arnold W, Mittal C, Murad F: Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerine and nitric oxide in various tissue preparations and comparison to the effects of sodium azyde and hychoxylamine. J Cyclic Nucleotide Res 3: 23–35, 1977
8. Gerzer R, Bohme E, Hoffman R, Shultz G: Soluble guanylate cyclase purified from bovine lung contains heme and copper. FEBS Lett 132: 71–74, 1981
9. Buechler WA, Nakane M, Murad F: Expression of soluble guanylate cyclase activity requires both enzyme subunits. Biochem Biophys Res Commun 174: 351–357, 1991
10. Bauersachs J, Bouloumie A, Mülsch A, Wiener G, Fleming I, Busse R: Vasodilator dysfunction in aged spontaneously hypertensive rats: Changes in NO synthase III and soluble guanylil cyclase expression, and in superoxide anion production. Cardiovasc Res 37: 772–779, 1998
11. Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Ertl G: Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: Role of enhanced vascular superoxide anion production. Circulation 100: 292–298, 1999
12. Taylor CT, Lisco SJ, Awtrey CS, Colgan SP: Hypoxia inhibits cyclic nucleotide-stimulated epithelial ion transport: Role for nucleotide cyclases as oxygen sensors. J Pharmacol Exp Ther 284: 568–575, 1998
13. Nava E, López-Farré A, Moreno C, Casado S, Moreau P, Cosentino F, Lüscher TF: Alterations to the nitric oxide pathway in spontaneously hypertensive rats. J Hypertens 16: 609–615, 1998
14. Vanhoutte PM, Boulanger CM: Endothelium-dependent responses in hypertension. Hypertens Res 18: 87–98, 1995
15. Piccinini F, Favalli L, Chiari MC: Experimental investigations on the contraction induced by lead in arterial smooth muscle. Toxicology 8: 43–51, 1977
16. Khalil-Manesh F, Gonick HC, Weiler EWJ, Prins B, Weber MA, Purdy RE, Ren Q: Effect of chelation treatment with dimercaptosuccinic acid (DMSA) on lead-related blood pressure changes. Environ Res 65: 86–89, 1994
17. Khalil-Manesh F, Gonick HC, Weiler EWJ, Prins B, Weber MA, Purdy RE: Lead-induced hypertension. Possible role of endothelial factors. Am J Hypertens 6: 723–729, 1993
18. Vaziri ND, Ding Y, Ni Z, Gonick HC: Altered nitric oxide metabolism and increased oxygen free radical activity in lead-induced hypertension: Effect of lazaroid therapy. Kidney Int 52: 1042–1046, 1997
19. Gonick HC, Ding Y, Bondy SC, Ni Z, Vaziri ND: Lead-induced hypertension: Interplay of nitric oxide and reactive oxygen species. Hypertension 30: 1487–1492, 1997
20. Vaziri ND, Liang K, Ding Y: Increased NO inactivation and sequestration in lead-induced hypertension. Effect of vitamin E. Kidney Int 56: 1492–1498, 1999
21. Gallego MJ, García-Villalón, López-Farré A, García JL, Garrón MP, Gómez B, Casado S, Hernando L, Carmelo C: Mechanisms of the endothelial toxicity of cyclosporine A (CyA). Circ Res 74: 477–484, 1994
22. Alonso J, Sanchez de Miguel L, Montón M, Casado S, López-Farré A: Endothelial cytosolic proteins bind to the 3′-untranslated region of endothelial nitric oxide synthase mRNA: Regulation by tumor necrosis factor alpha. Mol Cell Biol 17: 5719–5726, 1997
23. Laemmli NK: Change of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970
24. López-Farré A, Sánchez de Miguel L, Caramelo C, Gómez García J, García R, Mosquera JR, de Frutos T, Millás I, Rivas F, Echegarreta G, Casado S: Role of nitric oxide in the autocrine of growth and apoptosis of endothelial cells. Am J Physiol 272: H760–H768, 1997
25. Arriero MM, Rodriguez-Feo JA, Celdrán A, Sanchez de Miguel L, Gonzalez-Fernandez F, Fortes J, Reyero A, Frieyro O, de la Pinta JC, Franco A, Pastor C, Casado S, López-Farré A: Expression of endothelial nitric oxide synthase in human peritoneal tissue: Regulation by Escherichia coli lipopolysaccharide. J Am Soc Nephrol 11: 1848–1856, 2000
26. López-Farré A, Riesco A, Digiuni E, Mosquera JR, Caramelo C, Sánchez de Miguel L, Millás I, de Frutos I, Cernadas MR, Montón M, Alonso J, Casado S: Aspirin-stimulated nitric oxide production by neutrophils after acute myocardial ischemia in rabbits. Circulation 94: 83–87, 1996
27. García Durán M, de Frutos T, Diaz-Recasens J, García Galvez G, Jimenez A, Montón M, Farré J, Sanchez de Miguel L, Gonzalez-Fernandez F, Arriero MM, Rico L, García R, Casado S, López-Farré A: Estrogen stimulates neuronal nitric oxide synthase protein expression in human neutrophil. Circ Res 85: 1020–1026, 1999
28. Gonzalez-Fernandez F, López-Farré A, Rodriguez-Feo JA, Farré J, Guerra J, Millás I, García-Durán M, Rico L, Mata P, Sanchez de Miguel L, Casado S: Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery. Role of platelets. Circ Res 83: 1080–1087, 1998
29. Ding Y, Gonick HC, Vaziri ND: Lead promotes hydroxyl radical generation and lipid peroxidation in cultured aortic endothelial. Am J Hypertens 13: 552–555, 2000
30. Moncada S, Higgs A: The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002–2012, 1993
31. Cernadas MR, Sánchez de Miguel L, García Durán M, Ganzález-Fernández F, Millás I, Montón M, Rodrigo J, Rico L, Fernández P, de Frutos T, Rodriguez Feo JA, Guerra J, Caramelo C, Casado S, López-Farré A: Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ Res 83: 279–286, 1998
32. La Cras TD, Xue C, Rengasawy A, Johns RA: Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol 270: L164–L170, 1996
33. Mittal CK, Harell WB, Mehta CS: Interaction of heavy metal toxicant with brain constitutive nitric oxide synthase. Mol Cell Biochem 149–150: 269S–265S, 1995
34. Vaziri ND, Ding Y, Ni Z: Nitric oxide synthase expression in the course of lead-induced hypertension. Hypertension 34: 558–562, 1999
35. de Frutos T, Sanchez de Miguel L, Farré J, Romero J, Marcos-Alberca P, Nun[Combining Tilde]ez A, Rico L, López-Farré A: Expression of an endothelial-type nitric oxide synthase isoform in human neutrophils: Modification by tumor necrosis factor-alpha and during myocardial infarction. J Am Coll Cardiol 37: 800–807, 2001
36. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH: Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res 74: 349–353, 1994
37. Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG: Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res 86: 347–354, 2000
38. Vaziri N, Ding Y: Effect of lead on nitric oxide synthase expression in coronary endothelial cells. Hypertension 37: 223–226, 2001
39. Motoyama T, Kawano H, Kugiyama K, Hirashima O, Ohgushi M, Yoshimura M, Ogawa H, Yasue H: Endothelium-dependent vasodilatation in the brachial artery is impaired in smokers: Effect of vitamin C. Am J Physiol 273: H1644–H1650, 1997
40. Hornig B Arakawa N, Kohler C, Drexler H: Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure. Circulation 97: 63–368, 1998
