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Involvement of endothelium and endothelin-1 in lead-induced smooth muscle cell dysfunction in rats

2006; Elsevier BV; Volume: 69; Issue: 4 Linguagem: Inglês

10.1038/sj.ki.5000103

1523-1755

Laura Molero, Carolina Carrasco, María Marqués, Nosratola D. Vaziri, Petra J. Mateos‐Cáceres, Santiago Casado, Carlos Macaya, A. Suárez Barrientos, Antonio López‐Farré,

Cardiovascular, Neuropeptides, and Oxidative Stress Research

Lead exposure induces dysfunction of the cyclic guanosine monophosphate-dependent vasodilator system through downregulation of soluble guanylate cyclase (sGC) expression. The endothelium not only releases vasodilators but also vasoconstrictors such as endothelin-1 (ET-1). Our aim was to explore the role of the vascular endothelium and ET-1 as possible mediators of lead-induced downregulation of sGC. Isolated aortic segments from Wistar Kyoto rats were incubated in the presence or absence of lead (1 parts per million) for 24 h. Endothelium was mechanically removed in some of the aorta segments. As reported previously, lead exposure induced downregulation of sGC protein expression in the intact aortic segments. However, lead exposure failed to significantly modify sGC-β1 subunit expression in the endothelium-denuded aortic segments. Incubation with a selective ETA-type receptor inhibitor, BQ-123 (10−6 mol/l), restored sGC protein expression in lead-exposed intact aortic segments. As it has also been previously observed, incubation in lead-containing medium resulted in the upregulation of cyclooxygenase-2 (COX-2) in the intact aortic segments. Denudation of endothelium partially abrogated this effect of lead. Incubation with BQ-123 prevented the lead-induced upregulation COX-2 in the intact aortic segments. However, neither ET-1 content nor ETA-type receptor expression were modified by lead exposure of the aortic segments. As conclusion, the endothelium through the activation of ETA-type receptors mediates the downregulation of sGC expression by lead in the vascular wall. Lead exposure induces dysfunction of the cyclic guanosine monophosphate-dependent vasodilator system through downregulation of soluble guanylate cyclase (sGC) expression. The endothelium not only releases vasodilators but also vasoconstrictors such as endothelin-1 (ET-1). Our aim was to explore the role of the vascular endothelium and ET-1 as possible mediators of lead-induced downregulation of sGC. Isolated aortic segments from Wistar Kyoto rats were incubated in the presence or absence of lead (1 parts per million) for 24 h. Endothelium was mechanically removed in some of the aorta segments. As reported previously, lead exposure induced downregulation of sGC protein expression in the intact aortic segments. However, lead exposure failed to significantly modify sGC-β1 subunit expression in the endothelium-denuded aortic segments. Incubation with a selective ETA-type receptor inhibitor, BQ-123 (10−6 mol/l), restored sGC protein expression in lead-exposed intact aortic segments. As it has also been previously observed, incubation in lead-containing medium resulted in the upregulation of cyclooxygenase-2 (COX-2) in the intact aortic segments. Denudation of endothelium partially abrogated this effect of lead. Incubation with BQ-123 prevented the lead-induced upregulation COX-2 in the intact aortic segments. However, neither ET-1 content nor ETA-type receptor expression were modified by lead exposure of the aortic segments. As conclusion, the endothelium through the activation of ETA-type receptors mediates the downregulation of sGC expression by lead in the vascular wall. Continuous exposure to low levels of lead is a well-known cause of arterial hypertension in humans and in experimental animals.1.Sharp D.S. Osterloh J. Becker C.E. et al.Blood pressure and blood lead concentration in bus drivers.Environ Health Perspect. 1988; 78: 131-137Crossref PubMed Scopus (76) Google Scholar, 2.Sharp D.S. Becker C.E. Smith A.H. Chronic low-level lead exposure: its role in the pathogenesis of hypertension.Med Toxicol. 1987; 2: 210-232Crossref PubMed Scopus (108) Google Scholar However, the mechanisms involved in lead-induced hypertension are not fully understood. Several alterations in the nitric oxide (NO)-dependent vasodilator system have been implicated in the pathogenesis of lead-induced hypertension.3.Vaziri N.D. Ding Y. Ni Z. Nitric oxide synthase expression in the course of lead-induced hypertension.Hypertension. 1999; 34: 558-562Crossref PubMed Scopus (84) Google Scholar Recently, we have described that lead not only affects the endothelial function but also the smooth muscle cells function.4.Marques M. Millás I. Jiménez A. et al.Alteration of the soluble guanylate cyclase system in the vascular wall of lead-induced hypertension in rats.J Am Soc Nephrol. 2001; 12: 2594-2600PubMed Google Scholar In fact, lead exposure was shown to reduce the expression of the soluble guanylate cyclase (sGC) in the vascular wall. This effect was independent of lead-induced hypertension since in vitro incubation of isolated rat aortic segments with lead also promoted downregulation of sGC expression.5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar sGC is the enzyme that is involved in the NO-induced vasodilatation by catalyzing the formation of cyclic guanosine monophosphate (cGMP) from guanine 5′-triphosphate in the smooth muscle cells.6.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. 1977; 3: 23-35PubMed Google Scholar sGC is a heterodimer composed of large (α1) and small (β1) subunits.7.Gerzar R. Bohme E. Hoffman R. Shultz G. Soluble guanylate cyclase purified from bovine lung contains heme copper.FEBS Lett. 1981; 132: 71-74Abstract Full Text PDF PubMed Scopus (262) Google Scholar It is of note that the presence of both subunits of sGC is required for NO to exert its vasodilatory action.8.Buechler W.A. Nekane M. Murad F. Expression of soluble guanylate cyclase activity requires both enzyme subunits.Biochem Biophys Res Commun. 1991; 174: 351-357Crossref PubMed Scopus (140) Google Scholar Although the intracellular signal regulators of sGC expression are not yet completely elucidated, different studies have demonstrated that cyclic adenosine monophosphate-stimulating agents, such as prostaglandins, downregulates sGC expression.9.Parapetropoulos A. Marczin N. Mora G. et al.Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA and protein levels by cAMP-elevating agents.Hypertension. 1995; 26: 696-704Crossref PubMed Scopus (83) Google Scholar In this regard, lead has been shown to not only reduce the expression of sGC but also induce the expression of the cyclooxygenase-2 (COX-2). Moreover, the blockade of COX-2 activity has been shown to prevent the effect of lead on the sGC expression.5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar These observations point to the involvement of COX-2 as the mediator of the lead-induced downregulation of sGC. Vascular endothelium plays a pivotal role in the maintenance of vascular tone by the release of several vasoactive substances. The presence and the absence of endothelium have been demonstrated to modify the level of different proteins in the vascular wall.10.Montón M. López-Farré A. Mosquera J.R. et al.Endogenous angiotensin II produced by endothelium regulates interleukin-1β-stimulated nitric oxide generation in rat isolated vessels.Hypertension. 1997; 30: 1191-1197Crossref PubMed Scopus (18) Google Scholar, 11.Werba J.P. Martinez V. Abulafia D.P. et al.Marked neointimal lipoprotein lipase increase in distinct models of proclivity to atherosclerosis: a feature independent of endothelial layer integrity.Atherosclerosis. 2001; 156: 91-101Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar The endothelium not only releases vasodilators such as NO but also vasoconstrictor factors such as endothelin-1 (ET-1).12.Yanagisawa M. Kurihara H. Kimura S. et al.A novel potent vasoconstrictor peptide produced by vascular endothelial cells.Nature. 1998; 332: 411-415Crossref Scopus (10247) Google Scholar ET-1 exerts its vasoconstrictor effects via activation of ETA-type receptors located on the vascular smooth muscle layer.13.Russell F.D. Skepper J.N. Davenport A.P. Detection of endothelin receptors in human coronary artery vascular smooth muscle cells but not endothelial cells by using electron microscope autoradiography.Cardiovasc Pharmacol. 1997; 29: 820-826Crossref PubMed Scopus (40) Google Scholar Several authors have shown that ET-1 is not only a potent vasoconstrictor but also possess proinflammatory properties. Interestingly, similar to lead, ET-1 induces COX-2 expression in glomerular mesangial cells, myocardium and circulating leukocytes.14.Sugimoto T. Haneda M. Sawano H. et al.Endothelin-1 induces cyclooxygenase-2 expression via nuclear factor of activated T-cell transcription factor in glomerular mesangial cells.J Am Soc Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar, 15.Molero L. Farré J. García-Mendez A. et al.Endothelin-1 induced proinflammatory markers in the myocardium and leukocytes of guinea-pigs: role of glycoprotein IIB/IIIA receptors.Cardiovasc Res. 2003; 57: 109-118Crossref PubMed Scopus (19) Google Scholar However, it is not clear if the endothelium and particularly ET-1 may be involved in the effect of lead on sGC and COX-2 expressions in the vascular wall. Therefore, the aim of the present study was to determine the role of the endothelium and ET-1 in the genesis of lead-induced alteration of sGC expression in the blood vessel wall. As we have reported previously,5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholarin vitro incubation of intact aortic segments with lead significantly decreased sGC-β1 subunit expression (Figure 1). However, lead exposure of de-endothelialized aortic rings failed to significantly modify sGC-β1 subunit expression (Figure 1). The absence of endothelium, per se, did not change the sGC-β1 subunit expression when the aortic rings were not exposed to lead (Figure 1). No changes in β-actin expression were observed in either intact or de-endothelialized aortic rings after incubation in either control or lead-containing media, thus supporting the specificity of the above-mentioned changes in sGC-β1 subunit expression (Figure 1). Since the presence of endothelium seems to be pivotal for lead-induced downregulation of sGC-β1 subunit expression, we sought to determine whether ET-1 (one of the main vasoactive mediators released by the endothelium) may be involved. Addition of a specific ETA-type receptor antagonist, BQ-123 (10−6 mol/l), to the intact aortic rings partially reversed the lead-induced downregulation of sGC-β1 subunit (Figure 2). BQ-123 (10−6 mol/l), by itself, failed to modify sGC-β1 subunit expression in the aortic segments incubated in the lead-free medium (Figures 1 and 2). As we have demonstrated previously,5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar the exposure of intact aortic segments to lead stimulated COX-2 expression (Figure 3). However, in de-endothelized aortic rings, lead exposure failed to modify COX-2 expression (Figure 3). There was no modification in the level of COX-2 expression in the control de-endothelialized aortic rings compared with the control intact aortic ings (Figure 3). BQ-123 (10−6 mol/l) partially prevented the upregulation of COX-2 by lead in intact aortic segments (Figure 4). BQ-123 (10−6 mol/l) failed to modify COX-2 expression in aortic segments incubated in lead-free medium (Figures 3 and 4).Figure 4Representative Western blot showing the expression of COX-2 protein in endothelized aortic rings incubated with and without 1 p.p.m. lead in the presence of the selective ETA receptor inhibitor BQ-123 (10−6 mol/l). The bar graph represents the densitometric values in arbitrary units. Results are presented as mean±s.e.m. of five different aortic segments. *P<0.05, versus control.View Large Image Figure ViewerDownload (PPT) Despite the fact that BQ-123 prevented both downregulation of sGC and upregulation of COX-2 isoform induced by lead in the intact aortic segments, the content of ET-1 in the vascular wall was not modified by lead exposure (Figure 5). Exposure of intact aortic segments to lead did not modify the level of expression of the ETA-type receptor (Figure 6). De-endothelization of the aortic segments, per se, did not change the expression of ETA-type receptor as compared to that observed in the intact aortic segments (Figure 6). Moreover, exposure of de-endothelized aortic segments to lead tended to slightly reduced ETA-type receptor, although the difference did not reach statistical significance (Figure 6). The main finding of this study was the demonstration of involvement of the endothelium and ET-1 as mediator of the lead-induced downregulation of sGC in the vascular wall. As described in a recent review,16.Vaziri N.D. Sica D. Lead-induced hypertension: role of oxidative stress.Curr Hypertens Rep. 2004; 6: 314-320Crossref PubMed Scopus (71) Google Scholar numerous mechanisms have been implicated in the pathogenesis of lead-induced hypertension. Chief among them is oxidative stress and altered NO pathway, which has been the subject of several investigations during the past decade.3.Vaziri N.D. Ding Y. Ni Z. Nitric oxide synthase expression in the course of lead-induced hypertension.Hypertension. 1999; 34: 558-562Crossref PubMed Scopus (84) Google Scholar, 4.Marques M. Millás I. Jiménez A. et al.Alteration of the soluble guanylate cyclase system in the vascular wall of lead-induced hypertension in rats.J Am Soc Nephrol. 2001; 12: 2594-2600PubMed Google Scholar, 5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar, 17.Vaziri N.D. Ding Y. Ni Z. Gonick H.C. Altered nitric oxide metabolism and increased oxygen free radical activity in lead-induced hypertension: effect of lazaroid therapy.Kidney Int. 1997; 52: 1042-1046Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 18.Gonick H.C. Ding Y. Bondy S.C. et al.Lead-induced hypertension: interplay of nitric oxide and reactive oxygen species.Hypertension. 1997; 30: 1487-1492Crossref PubMed Scopus (150) Google Scholar, 19.Vaziri N.D. Liang K. Ding Y. Increased NO inactivation and sequestration in lead-induced hypertension: Effect of vitamin E.Kidney Int. 1999; 56: 1492-1498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 20.Vaziri N.D. Ding Y. Ni Z. Compensatory upregulation of nitric oxide synthase isoforms in lead-induced hypertension; reversal by a superoxide dismutase-mimetic drug.J Pharmacol Exp Therap. 2001; 298: 679-685PubMed Google Scholar, 21.Khalil-Manesh F. Gonick H.C. Elmar W.J. Lead-induced hypertension: possible role of endothelial factors.Am J Hypertens. 1993; 6: 723-729PubMed Google Scholar In this regard, it has been demonstrated that lead exposure reduces the availability of the endothelial-derived NO, mainly as a result of NO inactivation by reactive oxygen species.17.Vaziri N.D. Ding Y. Ni Z. Gonick H.C. Altered nitric oxide metabolism and increased oxygen free radical activity in lead-induced hypertension: effect of lazaroid therapy.Kidney Int. 1997; 52: 1042-1046Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 19.Vaziri N.D. Liang K. Ding Y. Increased NO inactivation and sequestration in lead-induced hypertension: Effect of vitamin E.Kidney Int. 1999; 56: 1492-1498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 20.Vaziri N.D. Ding Y. Ni Z. Compensatory upregulation of nitric oxide synthase isoforms in lead-induced hypertension; reversal by a superoxide dismutase-mimetic drug.J Pharmacol Exp Therap. 2001; 298: 679-685PubMed Google Scholar In addition, we have recently demonstrated that independently of the effect of lead on the NO-generating system, lead exposure downregulates sGC, the main receptor of NO in the vascular wall.4.Marques M. Millás I. Jiménez A. et al.Alteration of the soluble guanylate cyclase system in the vascular wall of lead-induced hypertension in rats.J Am Soc Nephrol. 2001; 12: 2594-2600PubMed Google Scholar, 5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar Khalil-Manesh et al.21.Khalil-Manesh F. Gonick H.C. Elmar W.J. Lead-induced hypertension: possible role of endothelial factors.Am J Hypertens. 1993; 6: 723-729PubMed Google Scholar were among the first to show that lead-induced hypertension in rats is associated with significant reductions of plasma concentration and urinary excretion of cGMP. In a subsequent study,22.Khalil-Manesh F. Gonick H.C. Elmar W.J. et al.Effect of chelation treatment with dimercaptosuccinic acid (DMSA) on lead-related blood pressure changes.Environ Res. 1994; 65: 86-99Crossref PubMed Scopus (43) Google Scholar they showed a significant rise in cGMP levels in rats with lead-induced hypertension treated with the chelating agent, dimercaptosuccinic acid, which possesses potent antioxidant properties. Based on these observations, they theorized that lead-induced hypertension may be due to the inactivation of endothelium-derived relaxing factor. In a later study, Gonick et al.18.Gonick H.C. Ding Y. Bondy S.C. et al.Lead-induced hypertension: interplay of nitric oxide and reactive oxygen species.Hypertension. 1997; 30: 1487-1492Crossref PubMed Scopus (150) Google Scholar documented the occurrence of oxidative stress and compensatory upregulation of NO synthases in rats with lead-induced hypertension. It is of note that while mean values for plasma concentration and urinary excretion of cGMP were less than the corresponding values found in their control animals, the differences did not reach statistical significance. Moreover, plasma and urine levels of total NO3− and NO2− (NOx) measured during the established phase of hypertension were comparable among rats with lead-induced hypertension and the control animals. In a separate study,17.Vaziri N.D. Ding Y. Ni Z. Gonick H.C. Altered nitric oxide metabolism and increased oxygen free radical activity in lead-induced hypertension: effect of lazaroid therapy.Kidney Int. 1997; 52: 1042-1046Abstract Full Text PDF PubMed Scopus (122) Google Scholar these investigators monitored urinary excretion of NOx in rats with lead-induced hypertension exposed to lead for 12 weeks. They found a dramatic decline in urinary NOx excretion followed by a gradual rise to the control levels during the maintenance phase of hypertension. The restoration of plasma and urinary NOx in this model occurred despite persistent NO inactivation by reactive oxygen species as evidenced by overabundance of nitrotyrosine in various tissues in these animals.19.Vaziri N.D. Liang K. Ding Y. Increased NO inactivation and sequestration in lead-induced hypertension: Effect of vitamin E.Kidney Int. 1999; 56: 1492-1498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar It should be noted that the normality of plasma concentration or urinary excretion of the end products of NO metabolism (i.e. total NO3−+NO2−) is not necessarily indicative of the normality of biologically active NO, especially when oxidative stress is present. This is because nearly all of the NO produced in the body is eventually converted to NO3 and NO2 irrespective of having served its biological action or being intercepted and inactivated by reactive oxygen species. For instance, the primary and secondary by-products of NO interaction with reactive oxygen species are either rapidly converted to NO3− and NO2− via isomerization (ONOO− → NO3) and degradation (ONOOH → OH+NO2) or at a later stage via turnover of the nitrated molecules. Thus, caution should be exercised in the interpretation of the plasma and urinary NO2+NO3 in the assessment of the bioavailability of NO. Similarly, measurements of plasma and urinary cGMP do not exclusively represent the biological action of NO. This is because cGMP level is a function of its production, which is regulated not only by NO but also by natriuretic peptides as well as its degradation by phosphodiesterases. These factors can vary independently to affect cGMP levels in the intact animals. However, some of these variabilities are reduced by the in vitro experiments using isolated tissues such as those employed here. Modification in the sGC expression has been found in the vascular wall of aging rats, experimental model of myocardial infarction and hypertension as well as during pulmonary maturation.23.López-Farré A. Rodríguez-Feo J.A. García Colis E. et al.Reduction of the soluble cyclic GMP vasorelaxing system in the vascular wall of stroke prone spontaneously hypertensive rats. Effect of the α1-receptor blocked doxazosin.J Hypertens. 2002; 20: 463-470Crossref PubMed Scopus (36) Google Scholar, 24.Moreno L. Gonzalez-Luis G. Cogolludo A. et al.Soluble guanylyl cyclase during postnatal porcine pulmonary maturation.Am J Physiol Lung Cell Mol Physiol. 2005; 288: L125-L130Crossref PubMed Scopus (24) Google Scholar, 25.Bauersachs J. Bouloumie A. Mulsch A. et al.Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression and in superoxide anion production.Cardiovasc Res. 1998; 37: 772-779Crossref PubMed Scopus (146) Google Scholar, 26.Bauersachs J. Bouloumie A. Fraccarollo D. et al.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. 1999; 100: 292-298Crossref PubMed Scopus (316) Google Scholar Indeed, several factors have been shown to modulate sGC expression including cyclic adenosine monophosphate, nerve growth factor and interleukin-1β.9.Parapetropoulos A. Marczin N. Mora G. et al.Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA and protein levels by cAMP-elevating agents.Hypertension. 1995; 26: 696-704Crossref PubMed Scopus (83) Google Scholar, 27.Liu H. Force T. Bloch K.D. Nerve growth factor decreases soluble guanylate cyclase in rat PC12 cells.J Biol Chem. 1997; 272: 6038-6043Crossref PubMed Scopus (48) Google Scholar In a recent study, lead exposure was shown to raise production of reactive oxygen species in both cultured endothelial and smooth muscle cells, suggesting that alteration of both endothelial and vascular smooth muscle cells may contribute to the deleterious effect of lead on the vascular wall.28.Ni Z. Hou S. Barton C.H. Vaziri N.D. Lead exposure raises superoxide and hydrogen peroxide in human endothelial and vascular smooth muscle cells.Kidney Int. 2004; 6: 2329-2336Abstract Full Text Full Text PDF Scopus (80) Google Scholar However, to our knowledge, it is not known whether downregulation of sGC by lead is an endothelium-mediated effect and/or it is related to a direct effect of lead on the smooth muscle layer. The present study revealed that the lead-induced downregulation of sGC-β1 subunit is significantly reduced in the absence of endothelium, suggesting the major involvement of endothelium in this phenomenon. ET-1 is an endogenous vasoconstrictor released by the endothelium. ET-1 exerts its effects on the smooth muscle cells by the activation of ETA-type receptors. Several reports have implicated the role of ET-1 in endothelial dysfunction.29.Nohria A. Garrett L. Johnson W. et al.Endothelin-1 and vascular tone in subjects with atherogenic risk factors.Hypertension. 2003; 42: 43-48Crossref PubMed Scopus (48) Google Scholar, 30.Amiri F. Virdis A. Neves M.F. et al.Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction.Circulation. 2004; 110: 2233-2240Crossref PubMed Scopus (260) Google Scholar Therefore, the question raised was whether ET-1 may be involved in the observed endothelial-dependent effect of lead on sGC expression. The lead-induced downregulation of sGC-β1 subunit expression in the intact aorta segments was substantially mitigated by the specific ETA-type receptor antagonist, BQ-123. This finding points to the involvement of ET-1 in the endothelial-dependent effect of lead on sGC expression. In a previous work, we demonstrated that lead exposure stimulated the expression of the COX-2 isoform in the vascular wall.5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar Moreover, upregulation of COX-2 isoform by lead was associated with lead-induced downregulation of sGC-β1 subunit.5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar Previous in vitro studies have suggested that ET-1, not only is a potent vasoconstrictor but it also has proinflammatory properties. Indeed, ET-1 induces leukocyte adhesion to cultured endothelial cells and stimulates release of cytokines from isolated leukocytes.31.López-Farré A. Riesco A. Espinosa G. et al.Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart.Circulation. 1993; 88: 1166-1171Crossref PubMed Scopus (196) Google Scholar, 32.McCarron R.M. Wang L. Stanimirovic D.B. Spatz M. Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells.Neurosci Lett. 1993; 156: 31-34Crossref PubMed Scopus (141) Google Scholar, 33.McMillen M.A. Huribal M. Cunningham M.E. et al.Endothelin-1 increases intracellular calcium in human monocytes and causes production of interleukin-6.Crit Care Med. 1995; 23: 34-40Crossref PubMed Scopus (58) Google Scholar Moreover, in vivo infusion of ET-1 stimulates the expression of COX-2 in the myocardium and circulating leukocytes obtained from guinea-pigs.15.Molero L. Farré J. García-Mendez A. et al.Endothelin-1 induced proinflammatory markers in the myocardium and leukocytes of guinea-pigs: role of glycoprotein IIB/IIIA receptors.Cardiovasc Res. 2003; 57: 109-118Crossref PubMed Scopus (19) Google Scholar Therefore, we sought to determine if the endothelium and ET-1 are also involved in the stimulation of COX-2 expression by lead. The denudation of endothelium and the blockade of ETA-type receptor by BQ-123 partially prevented the upregulation of COX-2 isoform by lead in the vascular wall. These findings point to the role of endothelium in lead-induced upregulation of COX-2 in vascular tissue by a mechanism, which may involve ET-1. The next step was to determine if the tissue content of ET-1 is modified by lead exposure in intact aortic rings. We found no differences in the content of ET-1 between control and lead-exposed intact aortic rings. This finding suggested that the ET-1-mediated downregulation of sGC-β1 subunit by lead did not depend on quantitative changes of ET-1 production by the endothelium. This observation is concordant with an earlier in vivo study, which reported that plasma ET-1 level was unaffected by the administration of lead to rats.21.Khalil-Manesh F. Gonick H.C. Elmar W.J. Lead-induced hypertension: possible role of endothelial factors.Am J Hypertens. 1993; 6: 723-729PubMed Google Scholar Since the ETA-type receptor antagonist, BQ-123, prevented both lead-induced downregulation of sGC-β1 subunit and upregulation of COX-2, it is logical to assume that lead must exert these actions by mechanisms that act at the level of ETA receptor. In this regard, Daswood et al.34.Dashwood M.R. Noertersheuser P. Kirchengast M. Munter K. Altered endothelin-1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252.Cardiovasc Res. 1999; 43: 445-456Crossref PubMed Scopus (35) Google Scholar have previously demonstrated that balloon angioplasty of pig coronary arteries, a process that induces de-endothelialization, increased ETA-type receptor expression. However, in our study, the expression of ETA receptor was not modified by either de-endothelization or lead exposure. Therefore, the BQ-123-inhibitable effect of lead on the expression of sGC-β1 subunit and COX-2 should occur downstream of ETA-type receptor. Further studies are required to address this issue. In conclusion, through the activation of the ETA-type receptors, the endothelium plays a major role in the lead-induced downregulation of sGC-β1 subunit and upregulation of COX-2 in the rat vascular wall. These phenomena may be involved, at least in part, in the vascular dysfunctionality associated with lead-induced hypertension. Studies were approved by the Institutional Ethics Committee for animals and were performed in accordance with the international convention on animal experimentation. The experiments were carried out on male Wistar rats with average body weight of 300 g. The animals were anesthetized with pentobarbital (30 mg/kg intramuscularly) and exanguinated. The remaining blood was removed by perfusing the aorta with 100 ml of isotonic saline. The descending thoracic aorta was removed and cut into 5-mm-long segments and individually suspended in culture solution (RPMI (Roswell Park Memorial Institute) medium containing 5% fetal calf serum, at 37°C). In a subset of tissues, the endothelium was mechanically eliminated by gentle rubbing. Using hematoxylin and eosin staining, we verified the complete endothelial denudation. As described previously,5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar aortic segments were cut into three portions and were then preincubated in RPMI medium containing 5% red phenol-free fetal calf serum, 5 mmol/l glutamine, 2 × 10−5 U/l penicillin and 3 × 10−5 μg/l streptomycin for 1 h. Afterwards, the medium was removed and replaced with fresh RPMI medium containing 5% fetal calf serum. The aortic segments were then incubated in the absence and in the presence of lead acetate (1 parts per million (p.p.m.)) for 24 h. The dose of lead acetate chosen was based on a previous study in which we observed that 1 p.p.m. lead induced a maximal reduction in sGC-β1 subunit abundance.5.Courtois E. Marques M. Barrientos A. et al.Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxigenase-2.J Am Soc Nephrol. 2003; 14: 1464-1470Crossref PubMed Scopus (98) Google Scholar In a subset of tissues, ETA receptor inhibitor, BQ-123 (10−6 mol/l), was added to the culture solution. After the conclusion of the incubation period, the aortic segments were immediately frozen in liquid nitrogen and processed for molecular biological determinations. The expression of sGC-β1 subunit, COX-2, ET-1 and ETA-type receptor were analyzed by Western blot as described elsewhere.23.López-Farré A. Rodríguez-Feo J.A. García Colis E. et al.Reduction of the soluble cyclic GMP vasorelaxing system in the vascular wall of stroke prone spontaneously hypertensive rats. Effect of the α1-receptor blocked doxazosin.J Hypertens. 2002; 20: 463-470Crossref PubMed Scopus (36) Google Scholar, 24.Moreno L. Gonzalez-Luis G. Cogolludo A. et al.Soluble guanylyl cyclase during postnatal porcine pulmonary maturation.Am J Physiol Lung Cell Mol Physiol. 2005; 288: L125-L130Crossref PubMed Scopus (24) Google Scholar In brief, the aortic rings were pulverized and solubilized in Laemmli buffer containing 2-mercaptoethanol.35.Laemmli N.K. Change of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206999) Google Scholar The proteins obtained were separated in denaturing sodium dodecyl sulfate/10% polyacrylamide gels. Equal amount of proteins (20 μg/lane) estimated by bicinchonic acid reagent (Pierce, Rockford, IL, USA) were loaded. The proteins were then blotted into nitrocellulose (Immobilion-P, Millipore Ibérica, S.A. Cerdanyola del Vallès, Barcelona). The blots were blocked overnight at 4°C with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween (20 mmol/l Tris-HCl, 137 mmol/l NaCl, 0.1% Tween-20). Western blot analysis was developed with antibodies against sGC-β1 subunit, COX-2 isoform, ET-1 and ETA-type receptor. The blots were incubated with the first monoclonal rabbit immunoglobulin G antibody sGC-β1 (1:2500) (Alexis Biochem., Lausen, Switzerland), a monoclonal rabbit immunoglobulin G antibody COX-2 (1:2500) (Alexis Biochem), a monoclonal murine immunoglobulin G antibody ET-1 (1:500) (EMD Biosciences, San Diego, CA, USA) and a polyclonal sheep immunoglobulin G antibody (15 μg/ml) (Alexis Biochemical), respectively, for 1 h at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase-conjugated immunoglobulin antibody) at a dilution of 1:1500 for another hour. Specific sGC-β1 subunit, COX-2, ET-1 and ETA-type receptor proteins were detected by enhanced chemoluminiscence (Amersham Corp, Freiburg, Germany) and evaluated by densitometry (Quantity One; Biorad, Hercules, CA, USA). Prestained protein markers (Sigma) were used for molecular mass determinations. To compare sGC-β1 subunit, COX-2, ET-1 and ETA-type receptor protein expression with the expression of another constitutive protein, we analyzed the expression of β-actin by Western blot, using a β-actin monoclonal antibody (Sigma Aldrich, St Louis, MO, USA). For this purpose, a parallel gel with identical samples was run and, after blotting onto nitrocellulose, Western blot analysis was then performed with the β-actin monoclonal antibody (1:2000). Results are expressed as mean±s.e.m. To determine statistical significance, the Mann–Whitney test was performed. A P-value <0.05 was considered statistically significant. This work was supported by a grant from Fondo de Investigación Sanitaria de la Seguridad Social (FISS PI020347) and RED Heracles (FISS G03/045). We thank Ruth Fernández for technical support and Begoña Larrea for secretarial assistance.