Rogério Faustino Ribeiro Junior

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.

Low Mercury Concentration Produces Vasoconstriction, Decreases Nitric Oxide Bioavailability and Increases Oxidative Stress in Rat Conductance Artery

Mercury is an environmental pollutant that reduces nitric oxide (NO) bioavailability and increases oxidative stress, having a close link with cardiovascular diseases, as carotid atherosclerosis, myocardial infarction, coronary heart disease and hypertension. One of the main sites affected by oxidative stress, which develops atherosclerosis, is the aorta. Under acute exposure to low mercury concentrations reactive oxygen species (ROS) production were only reported for resistance vessels but if low concentrations of mercury also affect conductance arteries it is still unclear. We investigated the acute effects of 6 nM HgCl2 on endothelial function of aortic rings measuring the reactivity to phenylephrine in rings incubated, or not, with HgCl2 for 45 min, the protein expression for cyclooxygenase 2 (COX-2) and the AT1 receptor. HgCl2 increased Rmax and pD2 to phenylephrine without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage abolished the increased reactivity to phenylephrine. The increase of Rmax and pD2 produced by L-NAME was smaller in the presence of HgCl2. Enalapril, losartan, indomethacin, furegrelate, the selective COX-2 inhibitor NS 398, superoxide dismutase and the NADPH oxidase inhibitor apocynin reverted HgCl2 effects on the reactivity to phenylephrine, COX-2 protein expression was increased, and AT1 expression reduced. At low concentration, below the reference values, HgCl2 increased vasoconstrictor activity by reducing NO bioavailability due to increased ROS production by NADPH oxidase activity. Results suggest that this is due to local release of angiotensin II and prostanoid vasoconstrictors. Results also suggest that acute low concentration mercury exposure, occurring time to time could induce vascular injury due to endothelial oxidative stress and contributing to increase peripheral resistance, being a high risk factor for public health.

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 leads 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 disease.

Myocardial contractility is preserved early but reduced late after ovariectomy in young female rats

BackgroundOvarian sex hormones (OSHs) are implicated in cardiovascular function. It has been shown that OSHs play an important role in the long term regulation of cardiac sarcoplasmic reticulum (SR) function and contractility, although early effects of OSHs deprivation on myocardial contractility have not yet been determined. This study evaluated the early and late effects of OSHs deficiency on left ventricular contractility in rats after ovariectomy.MethodsYoung female Wistar rats were divided into 3 groups (n = 9-15): sham operated (Sham), ovariectomized (Ovx) and Ovx treated with estradiol (1 mg/kg, i.m., once a week) (Ovx+E2). After 7, 15, 30 and 60 days post Ovx, left ventricle papillary muscle was mounted for isometric tension recordings. The inotropic response to Ca2+ (0.62 to 3.75 mM) and isoproterenol (Iso 10-8 to 10-2 M) and contractility changes in response to rate changes (0.25 to 3 Hz) were assessed. Protein expression of SR Ca2+-ATPase (SERCA2a) and phospholamban (PLB) in the heart was also examined.ResultsThe positive inotropic response to Ca2+ and Iso at 7, 15, and 30 days after Ovx was preserved. However, at 60 days, the Ovx group had decreased myocardial contractility which was subsequently restored with E2 replacement. The reduction in SERCA2a and increase in PLB expression observed at 60 days after Ovx were restored with E2 replacement.ConclusionThis study demonstrated that myocardial contractility and expression of key Ca2+ handling proteins were preserved in the early phase and reduced at long-term during OSHs deprivation.

Exposure to low mercury concentration in vivo impairs myocardial contractile function

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl₂ (1st dose 4.6 μg/kg, subsequent dose 0.07 μg/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and β-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na+-K+ ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and α-1 isoform of NKA, whereas α-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function.

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats.

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K+ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K+ channels and Na+/K+)-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K+-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K+-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K+ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.

Tributyltin chloride increases phenylephrine-induced contraction and vascular stiffness in mesenteric resistance arteries from female rats.

Tributyltin chloride (TBT) is an organotin compound that reduces estrogen levels in female rats. We aimed to investigate the effects of TBT exposure on vascular tonus and vascular remodelling in the resistance arteries of female rats. Rats were treated daily with TBT (500 ng/kg) for 15 days. TBT did not change arterial blood pressure but did modify some morpho-physiological parameters of third-order mesenteric resistance arteries in the following ways: (1) decreased lumen and external diameters; (2) increased wall/lm ratio and wall thickness; (3) decreased distensibility and increased stiffness; (4) increased collagen deposition; and (5) increased pulse wave velocity. TBT exposure increased the phenylephrine-induced contractile response in mesenteric resistance arteries. However, vasodilatation responses induced by acetylcholine and sodium nitroprusside were not modified by TBT. It is suggested that TBT exposure reduces vascular nitric oxide (NO) production, because:(1) L-NAME incubation did not cause a leftward shift in the concentration-response curve for phenylephrine; (2) both eNOS protein expression; (3) in situ NO production were reduced. Incubation with L-NAME; and (4) SOD shifted the phenylephrine response curve to the left in TBT rats. Tiron, catalase, ML-171 and VAS2870 decreased vascular reactivity to phenylephrine only in TBT rats. Moreover, increased superoxide anion production was observed in the mesenteric resistance arteries of TBT rats accompanied by an increase in gp91phox, catalase, AT1 receptor and total ERK1/2 protein expression. In conclusion, these findings show that TBT induced alterations are most likely due to a reduction of NO production combined with increased O2(-) production derived from NADPH oxidase and ERK1/2 activation. These findings offer further evidence that TBT is an environmental risk factor for cardiovascular disease.

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K+) channels and Na+K+-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K+ channels and Na+K+-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K+ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K+ channel (Kv) participates more in males. Inhibition of the three types of Ca2+-activated K+ channels induced a greater reduction in Rmax in females than in males. The functional activity of the Na+K+-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K+-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na+K+-ATPase functional activity. L-NAME reduced K+-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na+K+-ATPase activity. These results suggest that the K+ channels involved in the gender-based vascular relaxation differences are the large conductance Ca2+-activated K+ channels (BKCa) in females and Kv in males and in the K+-induced relaxation and the Na+K+-ATPase vascular functional activity is greater in males.

Exercise modulates the aortic renin-angiotensin system independently of estrogen therapy in ovariectomized hypertensive rats.

HIGHLIGHTSSome components of the renin‐angiotensin system (RAS) are altered under conditions of estrogen deficiency and hypertension.Exercise training reverses the detrimental effects of ovariectomy on the aortic reactivity to ANG II and ANG‐(1–7) in hypertensive rats.Exercise training modulates RAS independently of estrogen therapy, protecting the hypertensive postmenopausal women against CVD. ABSTRACT The renin‐angiotensin‐system is an important component of cardiovascular control and is up‐regulated under various conditions, including hypertension and menopause. The aim of this study was to evaluate the effects of swimming training and estrogen therapy (ET) on angiotensin‐II (ANG II)‐induced vasoconstriction and angiotensin‐(1–7) [ANG‐(1–7)]‐induced vasorelaxation in aortic rings from ovariectomized spontaneously hypertensive rats. Animals were divided into Sham (SH), Ovariectomized (OVX), Ovariectomized treated with E2 (OE2), Ovariectomized plus swimming (OSW) and Ovariectomized treated with E2 plus swimming (OE2 + SW) groups. ET entailed the administration of 5 &mgr;g of 17&bgr;‐Estradiol three times per week. Swimming was undertaken for sixty minutes each day, five times per week. Both, training and ET were initiated seven days following ovariectomy. Forty‐eight hours after the last treatment or training session, the animals’ systolic blood pressures were measured, and blood samples were collected to measure plasma ANG II and ANG‐(1–7) levels via radioimmunoassay. In aortic rings, the vascular reactivity to ANG II and ANG‐(1–7) was assessed. Expression of ANG‐(1–7) in aortic wall was analyzed by immunohistochemistry. The results showed that both exercise and ET increased plasma ANG II levels despite attenuating systolic blood pressure. Ovariectomy increased constrictor responses to ANG II and decreased dilatory responses to ANG‐(1–7), which were reversed by swimming independently of ET. Moreover, it was observed an apparent increase in ANG‐(1–7) content in the aorta of the groups subjected to training and ET. Exercise training may play a cardioprotective role independently of ET and may be an alternative to ET in hypertensive postmenopausal women.

Low-dose ouabain administration increases Na+,K+-ATPase activity and reduces cardiac force development in rats

BACKGROUND Ouabain is a digitalis compound that inhibits the Na(+),K(+)-ATPase (NKA) activity inducing increment in cardiac force. However, this effect seems to be dose dependent. At low concentration, ouabain can induce an increase of NKA activity. METHODS We investigated the effects of ouabain administration (25 μg/kg/day) for 15 days on cardiac contractility and NKA activity. Blood pressure and left ventricular papillary muscle contraction from placebo and ouabain-treated rats for 15 (OUA15) days were evaluated. Isometric force, post-rest potentiation, positive inotropic intervention produced by isoproterenol, and tetanic tension were measured. The activity and protein expression levels of α1 and α2 isoforms of NKA, sodium calcium exchanger (NCX), sarcoplasmic reticulum calcium ATPase (SERCA2a) and phospholamban (PLB) were also measured. RESULTS Systolic and diastolic blood pressures increased after treatment with ouabain. However, isometric tension was reduced in the ouabain treated group. Post-rest potentiation, time parameters, inotropic interventions by isoproterenol and tetanic tension did not change. In the ouabain treated group, NKA activity was increased (Oua 406.16 ± 70.6 vs. CT 282.80 ± 80.5) while protein expression of the α1 isoform of NKA was reduced (Oua 0.97 ± 0.06 vs. CT 0.76 ± 0.05). No changes were observed in protein expression of α2 isoform of NKA, NCX, SERCA2a and PLB. Therefore, although 15-day ouabain treatment increases blood pressure (Oua: 116.4 ± 3 vs. CT: 99.9 ± 3), treatment also reduces isometric tension development (Oua: 0.34 ± 0.14 vs. CT: 0.56 ± 0.22). CONCLUSION We suggest that the effects induced by ouabain in the isolated cardiac muscle could be related at least in part, to changes in NKA activity.