Endothelial dysfunction of rat coronary arteries after exposure to low concentrations of mercury is dependent on reactive oxygen species

Abstract

Background and purpose: Exposure to mercury is known to increase cardiovascular risk but the underlying mechanisms are not well explored. We analysed whether chronic exposure to low mercury doses affects endothelial modulation of the coronary circulation.

Experimental approach: Left coronary arteries and hearts from Wistar rats treated with either HgCl(2) (first dose 4.6 µg·kg(-1) , subsequent doses 0.07 µg·kg(-1) day(-1) , 30 days) or vehicle were used. Endothelial cells from pig coronary arteries incubated with HgCl(2) were also used.

Key results: Mercury treatment increased 5-HT-induced vasoconstriction but reduced acetylcholine-induced vasodilatation. It also reduced nitric oxide (NO) production and the effects of NO synthase inhibition with L-NAME (100 µmol·L(-1) ) on 5-HT and acetylcholine responses. Superoxide anion production and mRNA levels of NOX-1 and NOX-4 were all increased. The superoxide anion scavenger tiron (1 mmol·L(-1)) reduced 5-HT responses and increased acetylcholine responses only in vessels from mercury-treated rats. In isolated hearts from mercury-treated rats, coronary perfusion and diastolic pressure were unchanged, but developed isovolumetric systolic pressure was reduced. In these hearts, L-NAME increased coronary perfusion pressure and diastolic pressure while it further reduced developed systolic pressure.

Conclusions and implications: Chronic exposure to low doses of mercury promotes endothelial dysfunction of coronary arteries, as shown by decreased NO bioavailability induced by increased oxidative stress. These effects on coronary function increase resistance to flow, which under overload conditions might cause ventricular contraction and relaxation impairment. These findings provide further evidence that mercury, even at low doses, could be an environmental risk factor for cardiovascular disease.

Figure 1

Concentration–response curves to 5-HT, acetylcholine (ACh) and diethylamine (DEA)-NONOate (NO) in left coronary arteries from control and HgCl₂-treated rats. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control. Number of animals used is indicated in parentheses.

Figure 2

Effect of chronic treatment with mercury chloride (HgCl₂) on the modulation of vascular response by NO in coronary arteries. (A) Effect of L-NAME (100 µmol·L⁻¹) on the vasoconstrictor responses to 5-HT in left coronary arteries from untreated and HgCl₂-treated rats. (B) Effect of L-NAME on the vasodilator responses to ACh in untreated and HgCl₂-treated rats. The insert graphs show differences in area under the concentration–response curve (dAUC) in the presence and absence of L-NAME. (C) Vascular production of NO in septal coronary arteries from control and HgCl₂-treated rats. (D) Effect of apocynin (300 µmol·L⁻¹) and tiron (1 mmol·L⁻¹) on nitrite production in endothelial cells exposed to 5 µg·mL⁻¹ HgCl₂; the effect of 0.05 and 0.5 µg·mL⁻¹ HgCl₂ is also shown. (E) Representative blot and densitometric analysis showing eNOS protein expression normalized with β-actin expression in endothelial cells exposed to HgCl₂*P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control situation; #P < 0.05 versus HgCl₂. Number of animals used is indicated in parentheses. Ctrl: control.

Figure 3

Effects of chronic treatment with HgCl₂ on the participation of reactive oxygen species (ROS) in vascular responses in coronary arteries. Effect of tiron (1 mmol·L⁻¹) on the vasoconstrictor responses to 5-HT (A) and the vasodilator responses to ACh (B) in left coronary arteries from untreated and HgCl₂-treated rats. *P < 0.05, *P < 0.01 versus the corresponding control situation. Number of animals used is indicated in parentheses.

Figure 4

Effects of HgCl₂ on vascular superoxide anion production. (A) Images and quantification of superoxide anion production measured by fluorescence microscopy in untreated (Ctrl) and HgCl₂-treated endothelial cells from porcine coronary arteries. (B) Images and quantification of production of superoxide anion measured by fluorescence microscopy in endothelial cells exposed to HgCl₂ (5 µg·mL⁻¹) in the absence or the presence of tempol (1 mmol·L⁻¹), apocynin (0.3 mmol·L⁻¹), tiron (1 mmol·L⁻¹) or L-NAME (100 µmol·L⁻¹). (C) Vascular production of superoxide anion, measured by lucigenin chemiluminescence, in septal coronary arteries from control and HgCl₂-treated rats. Quantitative RT-PCR assessment of (D) NOX-1 and NOX-4 and (E) SOD-1 and SOD-2 mRNA levels in coronary arteries from control and HgCl₂-treated rats; results are expressed as the relative expression of mRNA compared with control. *P < 0.05, **P < 0.01 versus Ctrl, #P < 0.05 versus HgCl₂. Number of animals used or independent experiment in the case of cell cultures is indicated in parentheses.

Figure 5

Effect of chronic treatment with HgCl₂ on the participation of prostanoids and K_Ca channels in vascular responses in coronary arteries. Effect of indomethacin (10 µmol·L⁻¹) (A) and tetraethylammonium (TEA, 1 mol·L⁻¹) (B) on the vasoconstrictor responses to 5-HT in left coronary arteries from untreated and HgCl₂-treated rats. The inset graph shows differences in area under the concentration–response curve (dAUC) in the presence and absence of TEA. *P < 0.05 versus the corresponding control situation. Number of animals used is indicated in parentheses.

Figure 6

(A) Developed left ventricle isovolumetric systolic pressure (LVISP), (B) coronary perfusion pressure (CPP) and (C) left ventricle isovolumetric diastolic pressure (LVIDP) from control (Ctrl) and HgCl₂-treated rats before and after perfusion with L-NAME (100 µmol·L⁻¹) in isolated hearts. *P < 0.05 versus Control, #P < 0.05 versus HgCl₂, $P < 0.05 versus control + L-NAME. Number of animals used is indicated in parentheses.

Figure 7

Morphometric analysis of coronary vessels of control and HgCl₂-treated rats. (A) Representative microphotographs (original magnification × 40) of cross sections of intramyocardial vessels from control (Ctrl; on left) and HgCl₂-treated rats (on right) stained with Masson's trichrome. (B) Total vessel area. (C) Vascular luminal area. (D) Vascular media area. *P < 0.05. Number of animals used is indicated in parentheses.