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

Chronic lead exposure increases blood pressure and myocardial contractility in rats.

We investigated the cardiovascular effects of lead exposure, emphasising its direct action on myocardial contractility. Male Wistar rats were sorted randomly into two groups: control (Ct) and treatment with 100 ppm of lead (Pb) in the drinking water. Blood pressure (BP) was measured weekly. At the end of the treatment period, the animals were anaesthetised and haemodynamic parameters and contractility of the left ventricular papillary muscles were recorded. Blood and tissue samples were properly stored for further biochemical investigations. Statistical analyses were considered to be significant at p<0.05. The lead concentrations in the blood reached approximately 13 µg/dL, while the bone was the site of the highest deposition of this metal. BP in the Pb-treated group was higher from the first week of lead exposure and remained at the same level over the next four weeks. Haemodynamic evaluations revealed increases in systolic (Ct: 96 ± 3.79 vs. Pb: 116 ± 1.37 mmHg) and diastolic blood pressure (Ct: 60 ± 2.93 vs. Pb: 70 ± 3.38 mmHg), left ventricular systolic pressure (Ct: 104 ± 5.85 vs. Pb: 120 ± 2.51 mmHg) and heart rate (Ct: 307 ± 10 vs. Pb: 348 ± 16 bpm). Lead treatment did not alter the force and time derivatives of the force of left ventricular papillary muscles that were contracting isometrically. However, our results are suggestive of changes in the kinetics of calcium (Ca++) in cardiomyocytes increased transarcolemmal Ca++ influx, low Ca++ uptake by the sarcoplasmic reticulum and high extrusion by the sarcolemma. Altogether, these results show that despite the increased Ca++ influx that was induced by lead exposure, the myocytes had regulatory mechanisms that prevented increases in force, as evidenced in vivo by the increased systolic ventricular pressure

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

Protein expression levels of the ventricular α-1 Na⁺, K⁺-ATPase subunit.

<p>Bands that are representative of the Western blots of the expression of α-1 Na⁺, K⁺-ATPase subunit and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) are presented at the top of the figure. Results are expressed as the ratio between the area and density of α-1 Na⁺, K⁺-ATPase subunit and GAPDH in the control (Ct) and lead treated (Pb) groups. Number of animals = 7 for each group. <i>p</i>>0.05, Ct vs Pb, Student t<i>-</i>test.</p

Concentration-response curve for increasing extracellular [Ca⁺⁺] in isolated left ventricular papillary muscles from control and lead-treated rats.

<p>Symbols represent mean ± SEM. Open circles- controls (N = 9), filled squares- lead treated group (N = 10). *<i>p</i><0.05, Ct-group <i>vs.</i> Pb-group, two-way repeated measures ANOVA, followed by Bonferroni’s test.</p

Peak and plateau of the isometric force in isolated left ventricular papillary after tetanic stimulation in the absence and presence of verapamil (Ver) of control (Ct), lead treated (Pb), control+verapamil (Ct Ver), lead treatred+verapamil (Pb Ver).

<p>Each column represents the means ± SEM. Number of animals = 6 for each group. *<i>p</i><0.05, Pb <i>vs.</i> control, one-way ANOVA, followed by Tukey’s test.</p

Effects of lead exposure on haemodynamic arterial and ventricular parameters in anaesthetised rats.

<p>Systolic Arterial Pressure (SAP); Diastolic Arterial Pressure (DAP); Mean Arterial Pressure (MAP); Left Ventricular Systolic Pressure (LVSP); End Left Ventricular Diastolic Pressure (LVDP); Right Ventricular Systolic Pressure (RVSP); End Right Ventricular Diastolic Pressure (RVDP); Left Ventricular Positive (dP/dt+LV) and Negative (dP/dt – LV) Derivates; Right Ventricular Positive (dP/dt+RV) and Negative (dP/dt – RV) Derivates; and Heart Rate (HR). Means ± SEM. Animal number range = 9–12.</p><p>*<i>p</i><0.05, as assessed using Unpaired Student’s t-test.</p

Protein expression levels of the ventricular calcium pump SR (SERCA 2), phospholamban (PLB), phospho-Tre¹⁷-PLB, phospho-Ser¹⁶-PLB, as well as the ratios phospho-Tre¹⁷-PLB/PLB and phospho-Ser¹⁶-PLB/PLB from controls (Ct) and lead treated (Pb) groups.

<p>Bands are representative of the Western blots of the expression of SERCA-2, PLB, phospho-Tre¹⁷-PLB, phospho-Ser¹⁶-PLB and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) are presented at the top of the figure. The results are expressed as the ratio between the area and density of SERCA-2 or PLB and GAPDH in the Ct- and Pb-groups. Number of animals = 7 for each group.*<i>p</i><0.05, Ct vs Pb, Student t<i>-</i>test.</p

Tissue lead concentrations after 30 days of lead treatment (100 ppm).

<p>Means ± SEM. Number of animals = 7 each group.</p><p>*<i>p</i><0.05, as assessed using Student’s t<i>-</i>test.</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