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Abstract

Mercury, a heavy metal, is widespread and persistent in the environment and has been elucidated as a possible risk factor in cardiovascular diseases. Mercury has been reported to selectively impair the nitric oxide (NO) pathway in the vascular endothelium as a consequence of oxidative stress. Conversely, mercury per se causes endothelium-dependent vasorelaxation at lower concentration via the NO pathway. Little is known about the effects of mercury per se on other endothelial mediators. To elucidate possible mechanisms involved in this action, isometric tension was measured in aortic rings precontracted with phenylephrine (10 µM) from Wistar rats. Responses to increasing concentrations of inorganic mercuric chloride (10⁻¹²–10⁻⁵ M) were obtained in the presence and absence of endothelium. Inorganic mercury produced a biphasic response in endothelium-intact aortic rings and produced only vasoconstriction in endothelium-denuded aortic rings. To study the possible underlying mechanisms for the biphasic response of mercury, increasing concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) were used before and after N^G-nitro-l-arginine methyl ester (L-NAME (10⁻⁴ M)), glybenclamide (10⁻⁵ M), superoxide dismutase (10 U/ml) + catalase (100 U/ml), and nifedipine (10⁻⁴ M) treatment. Results suggest that mercury produces endothelium-dependent relaxation at low concentration mediated by endothelial-generated NO and endothelium-derived hyperpolarizing factor and endothelium-independent contraction resulting from the blockade of l-type Ca²⁺ channels by generation of free radicals.

Keywords

Rat aorta mercury endothelium NO EDHF oxidative stress

Introduction

Vascular endothelium plays an important role in vascular homeostasis. It maintains a delicate balance between endothelium-derived smooth muscle relaxing factors and contracting factors to regulate the vascular tone and blood flow to tissues/organs of the body.¹ Any pollutant when enters the body reaches various organs through the circulatory system. Therefore, blood vessels are the primary site of exposure to the toxic effects of pollutants.

Mercury is a well-known environmental pollutant, and the role of mercury toxicity as a possible risk factor in cardiovascular disease has been discussed and documented by several workers.^2
–4 One of the most commonly observed cardiovascular effects of mercury exposure is loss of endothelial function.^5

–8 Mercury exposure reportedly causes oxidative stress leading to endothelial dysfunction and vasoconstriction.^7,8 Previous study from our group have reported endothelial dysfunction in blood vessels from chronically inorganic mercury-exposed rats due to oxidative stress resulting in the selective loss of nitric oxide (NO)-mediated relaxation⁵ and persistence/upregulation of an endothelium-derived hyperpolarizing factor (EDHF) pathway. In endothelial dysfunction, there is either decrease in endothelial NO synthase (eNOS) activity and NO production or decreased bioavailability of NO due to oxidative stress. In vascular smooth muscle, NO stimulates production of cyclic guanosine monophosphate (cGMP), which promotes vasodilation by lowering intracellular calcium (Ca² ⁺) ion concentration and reducing the sensitivity of the contractile proteins to Ca² ⁺.⁹ The treatment of endothelial cells with Ca² ⁺ channel blocker, nifedipine increases the release of NO that is not due to an altered expression of eNOS messenger RNA (mRNA) and protein but because of an enhanced availability of NO.¹⁰ Contrary to the above, Golpon et al.⁶ have demonstrated that in the isolated aortic tissue, mercury induced an endothelial-dependent vasorelaxation and altered structure and function of vascular endothelial cells. Acute methyl mercury chloride exposure also modulates the vasorelaxant response by inducing NOS and increasing the NO production along with inducing oxidative stress.¹¹ From the above studies, it is conceivable to hypothesize that mercury exerts its effects on the NO-mediated relaxation, which in turn may contribute to the mercury-induced cardiovascular diseases in the populations exposed to mercury exposure.

Therefore, the aim of this study was to evaluate whether mercury, a redox inactive metal, per se affects the endothelial cells by involving NO, adenosine triphosphate-sensitive potassium channel (K⁺ _ATP channel), reactive oxygen species (ROS), and l-type Ca²⁺ channel pathways that may contribute to mercury-induced vascular disorders.

Methods

Wistar rats (n = 12), weighing 250–300 g, of either sex were used. The animals were maintained according to recommendations by National Accreditation Board of Testing and Calibration Laboratories (NABL), and the study was approved by the VP Chest Institute’s animal ethical committee. Rats were anesthetized with urethane (1.5 g/kg body weight intraperitoneally). The thoracic aorta was quickly removed, cleaned of adhering fat, and connective tissue. Ring segments (3–5 mm) were mounted between two stainless steel wires in 10 ml organ baths filled with modified Krebs solution, under passive tension of 2 g for 60 min. The Krebs solution had the following composition: sodium chloride (118 mM); potassium chloride (KCl; (4.7 mM); calcium chloride (2.5 mM); magnesium sulfate (1.2 mM); mono potassium phosphate (1.17 mM); sodium bicarbonate (20 mM); ethylenediaminetetraacetic acid (0.01 mM), and glucose.¹²

The rings were maintained at 37°C and gassed with 95% oxygen and 5% carbon dioxide mixture (pH = 7.4). The contractile response (isometric tension, in grams) was measured by a force transducer (model FT-03, Grass Technologies, Warwick, Rhode Island, USA) and coupled to a data acquisition system (AD Instruments, Dunedin, New Zealand). Vessels were initially exposed to 75 mM KCl to check their functional integrity, and the presence of endothelium was confirmed by the ability of acetylcholine (ACh; 10 µM) to relax segments precontracted with phenylephrine (PE) at a concentration that produces close to 50% of the contraction induced by 75 mM KCl. Endothelium was removed by rubbing the lumen with forceps. Response to ACh (10 µM) was observed to confirm denudation (no vasorelaxation), which was validated by histology.¹² For each set of experiments described below four rats were used and three tissues from each animal were studied. There was equal distribution of female and male rats in each set.

Protocols

Effects of mercury on aortic rings

To evaluate the effects of mercury on the aortic rings, experiments were performed using rings with intact endothelium as well as denuded endothelium. After equilibration, aortic rings were precontracted with PE (10⁻⁷ M) and once the plateau was attained, concentration-dependent response curves of mercuric chloride (10⁻¹²–10⁻⁵ M) were obtained.

Effects of mercury on endothelial mediators

To evaluate the effects of mercury on the endothelial mediators: NO and EDHF, two different experimental designs were used.

Endothelium-intact aortic rings were preincubated separately with the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME; 10⁻⁴ M) and K⁺ ATP inhibitor glybenclamide (10⁻⁵ M) for 30 min separately. Cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) were then applied during the sustained (tonic) phase of PE (10⁻⁷ M)-induced contraction. The effects of these inhibitors were studied by comparing the amount of relaxation or contraction induced by mercury in the absence or presence of these inhibitors.

Role of free radical scavengers on mercury-induced relaxation and contraction

To investigate the possible involvement of ROS in the vasorelaxing and vasoconstricting effects of mercury, endothelium-intact aortic rings were preincubated with the superoxide dismutase (SOD; 10 U/ml) + catalase (100 U/ml), and l-NAME (10⁻⁴ M) along with SOD (10 U/ml) + catalase (100 U/ml) for 30 min separately. Cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) were then applied during the sustained (tonic) phase of PE (10⁻⁷ M)-induced contraction. The effects of these scavengers and scavengers along with l-NAME were studied by comparing the amount of relaxation induced by mercury in the absence or presence of these scavengers and scavengers along with l-NAME.

Effect of l-type Ca²⁺ channel inhibitor on mercury-induced contraction

To investigate the possible involvement of l-type Ca²⁺ channel inhibitor in the vasoconstricting effect of mercury, endothelium-intact aortic rings were preincubated with the nifedipine (10⁻⁴ M) for 30 min. Cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) were then applied during the sustained (tonic) phase of PE (10⁻⁷ M)-induced contraction. The effects of these inhibitors were studied by comparing the amount of contraction induced by mercury in the absence or presence of this inhibitor.

Statistical analysis

The responses produced by mercuric chloride are expressed in grams. Data are presented as mean ± standard error of the mean. Statistical evaluations were performed using Student’s unpaired t test and the value of p < 0.05 was considered significant.

Results

Effect of mercury on aortic rings tension development

Inorganic mercury (mercuric chloride) produced a biphasic response—a significant vasorelaxation with concentrations 10⁻⁷–10⁻⁶ M and vasoconstriction with concentration 10⁻⁵ M in endothelium-intact aortic rings of normal rats precontracted by PE (10⁻⁷ M; Figure 1).

Figure 1.

Biphasic response of aortic rings precontracted with PE (10⁻⁷ M) to cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) in control rats. e⁺: endothelium intact (n = 12), e⁻: endothelium denuded (n = 12) n: number of tissues. Data represent mean ± SEM *p < 0.05: compared to baseline value; ¥p < 0.05: compared with the corresponding value before endothelium denudation. PE: phenylephrine.

Endothelial dependence of the vasorelaxant effects of mercury

Concentration–response curves for cumulative concentration of mercuric chloride (10⁻¹²–10⁻⁵ M) treatment (Figure 1) showed a biphasic response in endothelium-intact aortic rings. However, in endothelium-denuded aortic rings, mercury produced vasoconstriction with concentrations 10⁻⁷–10⁻⁵ M (Figure 1). These results suggest that the vasorelaxation produced by mercury is endothelium dependent and the vasoconstriction produced is by its effect upon smooth muscle activity. The endothelium-dependent vasorelaxation is a primary event and it masks the vasoconstrictor effect of mercury at lower concentration.

Effects of endothelial mediators in mercury-induced vasorelaxation

To investigate the role of NO or EDHF concentration–response curves for cumulative mercuric chloride (10⁻¹²–10⁻⁵ M) treatment in endothelium-intact aortic rings precontracted with PE (10⁻⁷ M) were performed before and after treatment with l-NAME (10⁻⁴ M) or glybenclamide (10⁻⁵ M). The results indicated that treatment with these inhibitors abolished the relaxation effect.

These results were very similar to those obtained with endothelium-denuded preparations, suggesting that vasorelaxant actions of mercury methyl chloride affected by removal of endothelium were mediated by NO and EDHF (Figures 2 and 3).

Figure 2.

Inhibition of vasorelaxant responses of aortic rings precontracted with PE (10⁻⁷ M) to cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) in control rats after incubation with l-NAME (n = 12). n: number of tissues. Data represent mean ± SEM; *p < 0.05: compared with baseline value; ¥p < 0.05: compared with the corresponding value before incubation with l-NAME. l-NAME: N^G-nitro-l-arginine methyl ester; PE: phenylephrine.

Figure 3.

Inhibition of vasorelaxant response of aortic rings precontracted with PE (10⁻⁷ M) to cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) in control rats before incubation with glybenclamide (n = 12) and after incubation with glybenclamide (n = 12). n: number of tissues. Data represent mean ± SEM. *p < 0.05: compared to baseline value; ¥p < 0.05: compared to corresponding value before incubation with glybenclamide. PE: phenylephrine.

Effects of free radicals in mercury-induced vasorelaxation

Additional experiments were performed to elucidate the mechanisms behind the dual effects of mercury on the aortic vasculature. Vascular responsiveness to mercury was examined after incubation of endothelium-intact aortic rings with free radical scavengers SOD and catalase. Mercuric chloride, at concentrations 10⁻¹¹–10⁻⁵ M, produced significant (p < 0.05) vasorelaxation (Figure 4) suggesting that mercury induces oxidative stress resulting in vasoconstriction. When aortic rings were incubated with free radical scavengers along with l-NAME, mercury did not produce any significant response (Figure 4).

Figure 4.

Responses of aortic rings precontracted with PE (10⁻⁷ M) to cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) in control rats before incubation with SOD and catalase (n = 12) and after incubation with SOD and catalase (n = 12) and SOD + catalase along l-NAME. n: number of tissues. Data represent mean ± SEM. *p < 0.05: compared with the baseline value; ¥p < 0.05: compared with the corresponding values before incubation with SOD and catalase. l-NAME: N^G-nitro-l-arginine methyl ester; PE: phenylephrine; SOD: superoxide dismutase.

Effect of Ca²⁺ channel inhibitor on mercury-induced contraction

Treatment with Ca²⁺ channel inhibitor, nefidipine abolished the vasoconstrictor response of mercury. There was a significant increase in the vasorelaxant response (p < 0.05) at concentrations (10⁻⁸–10⁻⁵ M) of mercuric chloride, though at 10 µM, there was a tendency for a reduction in the vasorelaxant response (Figure 5).

Figure 5.

Inhibition of vasoconstrictor responses of aortic rings precontracted with PE (10⁻⁷ M) to cumulative concentrations of mercuric chloride (10⁻¹²–10⁻⁵ M) in control rats after incubation with nifedipine (n = 12). n: number of tissues. Data represent mean ± SEM. *p < 0.05: compared with the baseline value; ¥p < 0.05: compared with the corresponding values before incubation with nifedipine. PE: phenylephrine.

Discussion

In the present study, inorganic mercury (mercuric chloride) produced a biphasic response—a vasorelaxation at lower concentrations and vasoconstriction at higher concentration (10 µM) in isolated endothelium-intact aortic rings. However, in aortic rings whose endothelium was removed, mercury produced only vasoconstriction. These results suggest that the vasorelaxation produced by mercury is endothelium-dependent and the vasoconstriction produced is by its effect upon smooth muscle activity. Our results are consistent with previous studies that mercury at concentrations of 0.5–10 µM produces vasoconstriction.⁷ Thus, we can say that endothelium-dependent vasorelaxation is a primary event and it camouflages the vasoconstrictor effect of mercury. A mercury level of 15 µg/l of blood is used as the biological exposure index,¹³ therefore we used concentrations of mercury between 10⁻¹² and 10⁻⁵ M for our experiments. The concentration of 10 µM of mercury approximately corresponds to 15 µg/l of mercury in blood. The fact that the endothelium is affected by mercury below the reference values highlights the importance and the need to better understand the mechanisms behind the dual effects of mercury on the aortic vasculature.

One of the most commonly observed vascular effects of mercury exposure is the loss of endothelial function.^5

–8 Endothelial dysfunction is attributed to reduction in NO bioavailability because of increased ROS production.^5,8,14 Previous studies from our group have demonstrated that chronic mercuric chloride exposure selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.⁵ Although an increase in NO was observed in our study, reductions in NO bioavailability due to the increased ROS production was responsible for the endothelial dysfunction.^7
–9 In contrast, interestingly, previous study by Golpon et al. has reported that mercury in vitro induces an endothelium-dependent vasorelaxation, which is totally blocked by the NOS inhibitor l-NAME indicating that mercury per se acts through the NO pathway.⁶ An increase in the ACh response due to an increase in plasma NO levels following methyl mercury chloride exposure, which overcomes the inhibitory effects of the free radicals has also been reported.¹¹

In the present study, the vasorelaxant response of mercury was abolished after incubation of aortic rings with l-NAME and glybenclamide suggesting that mercury acts through activation of NOS and K⁺ _ATP channel. This finding suggests that mercury stimulates NOS, which in presence of l-arginine forms NO. NO causes synthesis of cGMP, which increases extrusion of Ca²⁺ from the cytosol in vascular smooth muscle, causing vasorelaxation.¹⁵ The cGMP and NO also phosphorylate K⁺ _ATP channels to induce hyperpolarization, which results in vasorelaxation too.^16,17

In the presence of free radical scavengers SOD and catalase, we observed augmentation of the vasorelaxant response of mercury and the abolishment of the vasoconstrictor response. The vasorelaxation response occurring even with as low a concentration as 10⁻¹² M of mercury suggests that free radicals play an important role in this response. Numerous studies have shown that mercury induces oxidative stress with subsequent damage to several organs or systems^{4,8,9,17
–19} and also reduces NO production and increases the inducible NO synthase gene expression.^20,21 Although multiple processes may lead to endothelial damage, the generation of oxygen-derived free radicals and subsequent lipid peroxidation may be key components in the cascade of events.²² In the presence of mercury, the vascular endothelium releases NO that interacts with free radicals to form peroxynitrite, a potent vasoconstrictor and reduces the bioavailability of NO resulting in increased Ca²⁺ in the cytosol of the vascular smooth muscle and a decrease of vasorelaxation. SOD, and catalase scavenge the free radicals and make most of NO available to the vascular tissue. Hence, there is potentiation in the endothelium-dependent vasorelaxation response of mercury.²³ In the presence of free radical scavengers along with l-NAME mercury did not produce a significant vasorelaxant or vasoconstrictor response. l-NAME blocked the NO-mediated vasorelaxant response and SOD + catalase blocked the ROS-mediated vasoconstrictor response. These results suggest that mercury produces both NO and ROS, and it is their ratio that produces the response. At 10⁻¹²–10⁻⁶ M concentration of mercury, the NO produced may be more than ROS. This is tipped in favor of ROS at 10⁻⁵ M resulting in a switch to biphasic response. Previous studies from our group have also demonstrated that chronic mercury exposure increases the NO levels and reduction in NO bioavailability was due to the increased ROS production.⁵ Acute methyl mercury exposure studies demonstrate a significant increase in the NO-dependent vasodilator response in aorta of rats due to increased production of NO even when there is oxidative stress suggesting a state of redox imbalance.¹¹ It is also possible that oxidative stress may facilitate the contractile mechanisms especially by activating the Ca²⁺ channel.²⁴

When the aortic rings were treated with nifedipine, only the vasorelaxant response of mercury was observed suggesting that the vasoconstrictor response may be dependent on l-type Ca²⁺ channel. Activation of l-type Ca²⁺ channel increases the Ca²⁺ influx and initiates release of Ca²⁺ from sarcoplasmic reticulum to cause vasoconstriction. A previous study has reported that l-type Ca²⁺ channels are activated by ROS, and it has been shown that mercury increases the production of ROS.²⁵ That such a mechanism is possible is supported by the present study also. Nifedipine, besides being a l-type Ca²⁺ channel blocker, may prevent mercury-induced vasoconstriction by its antioxidative property also.²⁶ In a recent study, l-type Ca²⁺ channel agonists, nefidipine protected against mercury-induced cytotoxicity in endothelial cells, lacking l-type Ca²⁺ channels. The treatment of endothelial cells with Ca²⁺ channel blocker, nifedipine increased the release of NO that was not due to an altered expression of eNOS mRNA and protein but because of an enhanced availability of NO by an antioxidative protection mechanism.²⁶

The results reveal that mercury per se produces an endothelium-dependent vasorelaxation which is mediated through production of NO which phosphorylate K⁺ _ATP channels. This endothelium-mediated vasorelaxation is potentiated in presence of free radical scavengers and l-type Ca²⁺ channel blocker by making NO available to the vascular tissue. Therefore, we may conclude that mercury below the reference values also exerts its effects on the NO-mediated relaxation, which in turn may contribute to the mercury-induced cardiovascular diseases in the populations exposed to mercury.

Footnotes

Funding

This work was funded by the Indian Council of Medical Research, New Delhi, India.

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Vasorelaxant effects of mercury on rat thoracic aorta

Abstract

Keywords

Introduction

Methods

Protocols

Effects of mercury on aortic rings

Effects of mercury on endothelial mediators

Role of free radical scavengers on mercury-induced relaxation and contraction

Effect of l-type Ca2+ channel inhibitor on mercury-induced contraction

Statistical analysis

Results

Effect of mercury on aortic rings tension development

Endothelial dependence of the vasorelaxant effects of mercury

Effects of endothelial mediators in mercury-induced vasorelaxation

Effects of free radicals in mercury-induced vasorelaxation

Effect of Ca2+ channel inhibitor on mercury-induced contraction

Discussion

Footnotes

Funding

References

Effect of l-type Ca²⁺ channel inhibitor on mercury-induced contraction

Effect of Ca²⁺ channel inhibitor on mercury-induced contraction