Acute Lead Exposure Increases Arterial Pressure: Role of the Renin-Angiotensin System

Background: Chronic lead exposure causes hypertension and cardiovascular disease. Our purpose was to evaluate the effects of acute exposure to lead on arterial pressure and elucidate the early mechanisms involved in the development of lead-induced hypertension. Methodology/Principal Findings: Wistar rats were treated with lead acetate (i.v. bolus dose of 320 μg/Kg), and systolic arterial pressure, diastolic arterial pressure and heart rate were measured during 120 min. An increase in arterial pressure was found, and potential roles of the renin-angiotensin system, Na+,K+-ATPase and the autonomic reflexes in this change in the increase of arterial pressure found were evaluated. In anesthetized rats, lead exposure: 1) produced blood lead levels of 37±1.7 μg/dL, which is below the reference blood concentration (60 μg/dL); 2) increased systolic arterial pressure (Ct: 109±3 mmHg vs Pb: 120±4 mmHg); 3) increased ACE activity (27% compared to Ct) and Na+,K+-ATPase activity (125% compared to Ct); and 4) did not change the protein expression of the α1-subunit of Na+,K+-ATPase, AT1 and AT2. Pre-treatment with an AT1 receptor blocker (losartan, 10 mg/Kg) or an ACE inhibitor (enalapril, 5 mg/Kg) blocked the lead-induced increase of arterial pressure. However, a ganglionic blockade (hexamethonium, 20 mg/Kg) did not prevent lead's hypertensive effect. Conclusion: Acute exposure to lead below the reference blood concentration increases systolic arterial pressure by increasing angiotensin II levels due to ACE activation. These findings offer further evidence that acute exposure to lead can trigger early mechanisms of hypertension development and might be an environmental risk factor for cardiovascular diseaseThis study was supported by grants from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)/FAPES (Fundação de Amparo à Pesquisa do Espírito Santo)/FUNCITEC (Fundação de Ciência e Tecnologia)(39767531/07), Brazil and from MCINN (Ministerio de Ciencia e Innovación) (SAF 2009- 07201) and ISCIII (Instituto de Salud Carlos III) (Red RECAVA- Red Temática de Investigación en Enfermedades Cardiovasculares del Instituto de Salud Carlos III, RD06/0014/0011), Spai

Low-level lead exposure increases systolic arterial pressure and endothelium-derived vasodilator factors in rat aortas.

Chronic lead exposure induces hypertension and alters endothelial function. However, treatment with low lead concentrations was not yet explored. We analyzed the effects of 7 day exposure to low lead concentrations on endothelium-dependent responses. Wistar rats were treated with lead (1st dose 4 µg/100 g, subsequent dose 0.05 µg/100 g, i.m. to cover daily loss) or vehicle; blood levels attained at the end of treatment were 9.98 µg/dL. Lead treatment had the following effects: increase in systolic blood pressure (SBP); reduction of contractile response to phenylephrine (1 nM-100 µM) of aortic rings; unaffected relaxation induced by acetylcholine (0.1 nM-300 µM) or sodium nitroprusside (0.01 nM-0.3 µM). Endothelium removal, N(G)-nitro-L-arginine methyl ester (100 µM) and tetraethylammonium (2 mM) increased the response to phenylephrine in treated rats more than in untreated rats. Aminoguanidine (50 µM) increased but losartan (10 µM) and enalapril (10 µM) reduced the response to phenylephrine in treated rats. Lead treatment also increased aortic Na(+)/K(+)-ATPase functional activity, plasma angiotensin-converting enzyme (ACE) activity, protein expression of the Na(+)/K(+)-ATPase alpha-1 subunit, phosphorylated endothelial nitric oxide synthase (p-eNOS), and inducible nitric oxide synthase (iNOS). Our results suggest that on initial stages of lead exposure, increased SBP is caused by the increase in plasma ACE activity. This effect is accompanied by increased p-eNOS, iNOS protein expression and Na(+)/K(+)-ATPase functional activity. These factors might be a compensatory mechanism to the increase in SBP

The effects of endothelium removal (E⁻) (A, B) and <i>N</i>^G-nitro-L-arginine methyl ester (L-NAME, 100 µM) (D, E) on the concentration-response curve for phenylephrine treatment in aortic rings from untreated (CT) and lead-treated rats (Pb⁺²).

<p>The inset shows differences in area under the concentration-response curves (dAUC) in endothelium–denuded and intact segments (C) and in the presence and absence of L-NAME (F). Densitometry analyses of western blots for endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) protein expression in aortas from untreated (CT) and lead-treated rats (Pb⁺²) (G). Representative blots are also shown. *P<0.05 by Student's <i>t-</i>test. Number of animals used is indicated in parentheses.</p

Potassium-induced relaxation in aortic rings from untreated (CT) and lead-treated (Pb⁺²) rats previously incubated in a K⁺-free medium and contracted with phenylephrine before and after incubation with 100 µM ouabain (A).

<p>Densitometry analyses of the western blots for the alpha-1 subunit (B) and alpha-2 subunit (C) in aortas from untreated (CT) and lead-treated rats (Pb⁺²). Representative blots are also shown. *P<0.05 (CT <i>vs.</i> Pb⁺²) by Student's <i>t-</i>test or two-way ANOVA followed by a Bonferroni test. ^#P<0.05 (CT OUA <i>vs.</i> Pb⁺² OUA) by two-way ANOVA followed by a Bonferroni test. Number of animals used is indicated in parentheses.</p

Changes in arterial pressure.

<p>Changes in systolic arterial pressure-SAP (A and C) and diastolic arterial pressure-DAP (B and D) before (Ct) and after Losartan (Los) or Enalapril (Enal) administration and following lead exposure (Los+Pb; Enal+Pb). A and B show the Losartan protocol; C and D show the Enalapril protocol. *p<0.05 compared with untreated controls. The number of animals used is indicated in parentheses.</p

Effect of losartan (10 µM) (A, B) and enalapril (10 µM) (C, D) on the concentration-response curves to phenylephrine in endothelium-intact aortic segments from untreated (CT) and lead-treated rats (Pb⁺²).

<p>Densitometry analyses of the western blots for receptors AT₁ (E) and AT₂ (F) in aortas from untreated (CT) and lead-treated rats (Pb⁺²). Representative blots are also shown.*P<0.05 by Student's <i>t-</i>test. Number of animals used is indicated in parentheses.</p

Angiotensin converting enzyme (ACE) activity (nmol His-Leu/min) in plasma; correlation between systolic arterial blood pressure and ACE activity (nmol His-Leu/min) in plasma of control and lead-treated rats (r = 0.787, P<0.05).

<p>*P<0.05 by Student's <i>t-</i>test. Number of animals used is indicated in parentheses.</p

Effects of lead on protein expression and ACE activity.

<p>Effects of lead exposure on the protein expression of the (A) AT1 and (B) AT2 receptors and on (C) ACE activity. *p<0.05 compared with untreated controls. The number of animals used is indicated in parentheses.</p

Hemodynamic parameters upon acute lead exposure.

<p>SAP- systolic arterial pressure, DAP- diastolic arterial pressure, HR- heart rate, Ct- Control. The results are expressed as the mean ± SEM.</p><p>*p<0.05 compared with controls (time 0); n = 10.</p

Effects of aminoguanidine, TEA, losartan and enalapril on the vascular responses to phenylephrine (R_max and pD₂) in aortas from untreated and lead-treated rats.

<p>Results are expressed as mean ± SEM of the number of animals shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017117#pone-0017117-g003" target="_blank">Figs. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017117#pone-0017117-g005" target="_blank">5</a> ; R_max, maximal effect (expressed as a percentage of the maximal response induced by 75 mM KCl); pD₂, −log one-half R_max; AG; aminoguanidine, TEA; tetraethylammonium, losartan, enalapril. P<0.05 <i>vs.</i> untreated control rats (^#) and lead-treated control rats (*).</p