Effects of mercury on the arterial blood pressure of anesthetized rats

The available data suggests that hypotension caused by Hg2+ administration may be produced by a reduction of cardiac contractility or by cholinergic mechanisms. The hemodynamic effects of an intravenous injection of HgCl2 (5 mg/kg) were studied in anesthetized rats (N = 12) by monitoring left and right ventricular (LV and RV) systolic and diastolic pressures for 120 min. After HgCl2 administration the LV systolic pressure decreased only after 40 min (99 ± 3.3 to 85 ± 8.8 mmHg at 80 min). However, RV systolic pressure increased, initially slowly but faster after 30 min (25 ± 1.8 to 42 ± 1.6 mmHg at 80 min). Both right and left diastolic pressures increased after HgCl2 treatment, suggesting the development of diastolic ventricular dysfunction. Since HgCl2 could be increasing pulmonary vascular resistance, isolated lungs (N = 10) were perfused for 80 min with Krebs solution (continuous flow of 10 ml/min) containing or not 5 μM HgCl2. A continuous increase in pulmonary vascular resistance was observed, suggesting the direct effect of Hg2+ on the pulmonary vessels (12 ± 0.4 to 29 ± 3.2 mmHg at 30 min). To examine the interactions of Hg2+ and changes in cholinergic activity we analyzed the effects of acetylcholine (Ach) on mean arterial blood pressure (ABP) in anesthetized rats (N = 9) before and after Hg2+ treatment (5 mg/kg). Using the same amount and route used to study the hemodynamic effects we also examined the effects of Hg2+ administration on heart and plasma cholinesterase activity (N = 10). The in vivo hypotensive response to Ach (0.035 to 10.5 μg) was reduced after Hg2+ treatment. Cholinesterase activity (μM h-1 mg protein-1) increased in heart and plasma (32 and 65%, respectively) after Hg2+ treatment. In conclusion, the reduction in ABP produced by Hg2+ is not dependent on a putative increase in cholinergic activity. HgCl2 mainly affects cardiac function. The increased pulmonary vascular resistance and cardiac failure due to diastolic dysfunction of both ventricles are factors that might contribute to the reduction of cardiac output and the fall in arterial pressure. Correspondence


Introduction
Acute intravenous (iv) administration of Hg 2+ produces important hemodynamic changes.The main effect is a progressive decrease of arterial blood pressure (ABP) associated with a reduction of heart rate (HR) (1,2).The mechanisms underlying this hemodynamic alteration are not known.Based on present knowledge, the effects of Hg 2+ reducing ABP may result from a reduction in cardiac mechanical activity (3,4), as supported by several findings.Inorganic mercury can produce profound cardiotoxicity (1,(3)(4)(5)(6)(7).Acute HgCl 2 poisoning in Langendorff-perfused rat hearts reduces left ventricular isovolumic systolic pressure and HR and delays atrio-ventricular conduction (2,3).In isolated papillary muscles 1 µM HgCl 2 increases force and rate of force development but above this concentration force is reduced (4,7).These cardiac effects result from a calcium overload developed as a consequence of the reduction of the sarcolemmal Na + ,K + -ATPase (8)(9)(10)(11)(12)(13) and of the sarcoplasmic reticulum (SR) Ca 2+ -ATPase activities (6,9,14,15).Simultaneous effects of Hg 2+ on the Ca 2+ channels of the SR also occur, increasing Ca 2+ release (16,17).Studies from our laboratory using tetanic contractions of isolated papillary muscles also suggested that a reduction in force might also result from the toxic effects of HgCl 2 on the contractile proteins (4).The participation of cholinergic mechanisms was also suggested by results showing that atropine pretreatment blocks the hypotensive effect (2).However, a vasodilatory effect is unlikely since previous reports have suggested that Hg 2+ induces vasoconstriction (18).
The present study was performed to examine the hemodynamic changes produced by acute administration of HgCl 2 .The study was conducted on anesthetized rats by measuring pressure development in both ventricles.We also studied the metals effects on the isolated perfused vascular bed from the rat lung.To determine whether Hg 2+ affects cholinergic mechanisms we analyzed the effects of acetylcholine (Ach) on ABP before and after Hg 2+ treatment.In addition, we measured the effects of Hg 2+ on heart and plasma pseudocholinesterase activity.

General methods
Studies were performed on 42 Wistar rats (150-330 g) of both sexes.Care and use of laboratory animals were in accordance with established NIH guidelines.All rats had free access to water and were fed rat chow ad libitum.

Experimental protocols
Hemodynamic effects of HgCl 2 .Rats were anesthetized with urethane (1.2 g/kg, ip) and the carotid artery and jugular and femoral veins were cannulated.The carotid artery and jugular vein cannulas were advanced into the left and right ventricular chambers, respectively, and connected to pressure transducers (Gold P23XL) to measure ventricular pressure (RG-300, FUNBEC).The electrocardiogram was also recorded (ME 100, FUNBEC) with electrodes placed according to the human standard using the D1 lead.The following parameters were analyzed: left ventricular (LV) and right ventricular (RV) systolic and end-diastolic pressures (LVSP, LVDP, RVSP and RVDP, respectively) and HR.
The following protocol was used: acute effects were achieved using an in bolus dose of HgCl 2 injected intravenously (5 mg/kg).This dose was selected to provide acute toxic concentrations above 10 µg/ml in blood, considering that the amount injected would be diluted in 40 ml of extracellular fluid per 100 g of body weight.According to Bakir et al. (19), 5 µg/ml of mercury in the blood causes 28% of deaths in humans.All ani-mals (N = 7) were followed for 120 min and LV and RV pressures and ECG were recorded before (control condition -C) and at 1, 3,5,10,15,20,30,40,50,60,70,80,90,100,110 and 120 min after HgCl 2 administration.Similar protocols were repeated in control animals (N = 5) without HgCl 2 injection.
Lung perfusion.To assess the effects of Hg 2+ on lung vascular resistance we perfused rat lungs using a technique similar to that used for the rat tail vascular bed (20).Rats were anesthetized with sodium pentobarbital (65 mg/kg, ip) and received 500 units of heparin, ip.After 10 min the thorax was opened and a cannula was placed inside the pulmonary artery to perfuse the lungs.The heart and lungs were then excised and placed in a temperature-controlled tissue bath.The heart was not separated from the lungs to avoid lesion of structures.Thus, the nutrient solution was infused through both lungs by the pulmonary artery, returning to the left atria and ventricle.The pulmonary artery was perfused with Krebs-Henseleit (KH) bicarbonate buffer solution, pH 7.4, bubbled with 5% CO 2 -95% O 2 (27.2 mM NaHCO 3 , 119 mM NaCl, 1 mM NaH 2 PO 4 , 1.2 mM MgSO 4 , 2.5 mM CaCl 2 .2H 2 O, 11 mM glucose, 5.4 mM KCl and 0.03 mM EDTA), at 36 ± 0.5 o C, using a peristaltic pump at a constant flow of 10 ml/min.The pulmonary perfusion pressure was measured with a TP-200T-Nihon-Kohden pressure transducer (connected to an MP-100 FUNBEC preamplifier) placed between the pump and the arterial cannula and recorded continuously on a polygraphic (ANAMED, AM-820) recorder.
Since lungs appeared to be in good condition indicated by a clear aspect and floating in the tissue bath, after a 10-to 15-min equilibration period the experimental protocol was initiated.The mean perfusion pressure of the pulmonary artery (PMPP) was measured under control conditions and followed for 50 min during continuous infusion of normal Krebs (N = 5) or Krebs containing 5 µM HgCl 2 (1360 ng/ml) (N = 5); PMPP was then measured at 5, 10, 15, 20, 30, 40 and 50 min.A similar protocol was repeated in lungs perfused without HgCl 2 .Since PMPP increased during Hg 2+ infusion pulmonary edema occurred which was confirmed by the observation of a shining lung surface and the complete sinking of the lungs into the solution.
Effects of mercury on cholinergic activity.Effects of mercury on cholinergic activity were evaluated using 2 protocols.Protocol 1: effects of HgCl 2 on the hypotensive response elicited by Ach.Nine rats were anesthetized with sodium pentobarbital (65 mg/kg, ip) and implanted with arterial and venous cannulae.Increasing doses of Ach (0.035, 0.105, 0.35, 1.05, 3.5 or 10.5 µg) were administered iv before and 30 min after iv injection of HgCl 2 (5 mg/kg).The magnitude of the peak decrease in mean blood pressure (MBP) elicited by all doses of Ach was recorded.Protocol 2: effects of HgCl 2 on heart and plasma cholinesterase activity.Two groups of rats were anesthetized with pentobarbital and prepared with cannulae as described above.Six rats received an injection of HgCl 2 (5 mg/kg, iv), while 5 control rats received iv injections of saline.Fifteen minutes after the injections the rats were killed by decapitation and heart and blood samples were collected.The cardiac tissue was immediately homogenized in 0.32 M saccharose plus 10 mM Tris-HCl solution, pH 7.5 (1 g of tissue per 10 ml of solution) and centrifuged at 1,000 rpm for 10 min.Plasma and tissue pseudocholinesterase activity (µM h -1 mg protein -1 ) was measured using the method of Ellman et al. (21).Protein was determined by the Coomassie blue method (22) with bovine serum albumin as a standard.

Drugs used
Ach, HgCl 2 , urethane and pentobarbital were purchased from Sigma Chemical Co., St. Louis, MO; heparin was purchased from Roche Pharmaceuticals, São Paulo, SP, Brazil.

Data analysis
Data are reported as mean ± SEM.Comparisons between means were made using a repeated-measures ANOVA or the Student t-test.A significant ANOVA was followed by a Tukey test to compare the means.The level of significance was set at P<0.05.

Hemodynamic studies
Figure 1 shows that 1 min after HgCl 2 iv injection there was a small increase in RVSP and a small decrease in LVSP.RVSP showed minor changes up to the 20th minute while LVSP did not change until the 40th minute.Then RVSP began to increase continuously, attaining an increment of 60% after 80 min.LVSP tended to decrease attaining a small but significant decrement of 20% only after 80 min.RVSP and LVSP of control rats did not change throughout the experiment (Table 1).The diastolic pressure of both RV and LV suddenly increased after HgCl 2 injection (Figure 2) and showed a continuous tendency to increase thereafter.

Perfused lungs
Since there was an increase in RV systolic and diastolic pressures after Hg 2+ treatment the possibility of a pulmonary vasoconstrictor effect produced by the metal was considered.Isolated lungs were then perfused with a constant flow (10 ml/min) to investigate this possibility.After the beginning of continuous HgCl 2 infusion PMPP increased continuously (Figure 3) while no changes were observed in untreated lungs (Table 1).If we plot PMPP and RVSP it can  be seen that both variables increase in parallel up to 30 min after the beginning of continuous HgCl 2 infusion (Figure 3).It is interesting to point out that about this time the PMPP begins to fall and this is coincident with signs of pulmonary edema (32 ± 2.9 mmHg).Indeed, signs of pulmonary edema (dyspnea, tachypnea, pulmonary rales) also occurred in anesthetized rats at a not too different RVSP (41.6 ± 1.61 mmHg).This usually happened 50 min after the beginning of HgCl 2 administration and was followed by intense dyspnea and death of the rats at 80 min.

Effects of mercury on cholinergic activity
Intravenous administration of Ach elicited dose-related decreases in MBP and HR (Figure 4).Administration of HgCl 2 reduced the magnitude of the hypotensive response to all doses of Ach (Figure 4).Regarding heart rate, only at the dose of 3.5 µg was the bradycardic response significantly attenuated.All the other bradycardic responses were similar to the pretreatment response.
To determine whether Hg 2+ decreased the hypotensive response to Ach by increasing cholinesterase activity we measured the specific activity of cholinesterase in the plasma and heart of Hg 2+ -treated and salinetreated rats.Hg 2+ treatment significantly increased cholinesterase activity in heart and plasma by 32 and 65%, respectively (Table 2).

Discussion
The present results support the view that the reduction of ABP caused by acute administration of Hg 2+ in vivo depends mainly on the depression of cardiac mechanical function.This depression resulted from the development of diastolic dysfunction of both ventricles plus pulmonary hypertension.
Previous reports showed that one of the main cardiovascular changes occurring in response to the acute administration of Hg 2+ in vivo was a reduction in ABP (1,2).The reduction of ABP could result from the deterioration of cardiac mechanical function (1,2,4,5,7) and perhaps interactions of Hg 2+ and cholinergic mechanisms could account for the ABP reduction since atropine blocks the Hg 2+ -induced hypotension (2).The purpose of our study was to determine whether any or all of these possible mechanisms were responsible for the reduction of ABP caused by Hg 2+ .

Hemodynamic effects of Hg 2+
One of the main mechanisms which may account for the hypotensive response to Hg 2+ is a direct effect reducing myocardial performance (1,2,4,5,7).Acute HgCl 2 poisoning in Langendorff-perfused rat hearts reduced the left ventricular systolic pressure and heart rate and delayed atrioventricular conduction (2,3).In addition, other studies from our laboratory using tetanic contractions of isolated papillary muscles also suggested that the force reduction could result from the effect of HgCl 2 on contractile proteins (4).
Only an important reduction in ABP was described for acute cardiovascular toxic effects in vivo (1,2).At least two aspects may contribute to this fall in ABP.Firstly, since Hg 2+ affects the cardiac contractile activity in isolated preparations the reduction in ABP observed in vivo could result from an altered mechanical cardiac function.The second could be a depressed vascular smooth muscle contraction produced by Hg 2+ which may reduce ABP.This is unlikely since previous findings showed that Hg 2+ induces vasoconstriction (18).These observations raised two possibilities to explain the reduction in ABP: cardiac mechanical failure and/or pulmonary hypertension caused by pulmonary vasoconstriction.These questions were investigated by studying the acute effects of the metal in anesthetized rats, measuring LV and RV systolic and diastolic pressures, and for Ach vs Ach-free, both before and after Hg 2+ treatment and + P<0.01 for differences between each Ach effect before and after Hg 2+ treatment.on the isolated perfused lung.
For the anesthetized rat the results did not show appreciable changes of LVSP during the first 40 min after HgCl 2 administration.Only after this period of time was a small reduction of LVSP observed.The results suggest that this dose of HgCl 2 , in vivo, does not produce an important depression of LVSP and cannot explain the reduction of ABP, as we observed before (2).In contrast, RVSP increased slowly during the first 30 min and then more steeply.Results also indicated that RV pressure development was preserved and the progressive increment of RVSP could be the result of increased pulmonary vascular resistance.Hg 2+ has been reported to constrict the rat tail artery (18) and other types of smooth muscle such as guinea pig ileum and vas deferens (23).In guinea pig ileum and vas deferens, Hg 2+ increases basal tone at low concentrations (10 -9 -10 -8 M) by stimulating autonomic neuromuscular transmission.At higher concentrations (10 -4 M), Hg 2+ produces smooth muscle contraction by a direct action.
To answer this question, we perfused isolated lungs at constant flow to measure pulmonary resistance.Again, the increase in vascular resistance produced by HgCl 2 was demonstrable and this increased pulmonary resistance could explain the fall in ABP by reducing cardiac output.Although this was not the main goal of the present work, this finding deserves additional comments.Magos et al. (24) reported that the lung is an important site in the removal of elemental Hg 2+ from the blood stream.According to these authors, after an intravenous dose of elemental Hg 2+ dissolved in aqueous buffer 10 to 20% of the metal may be exhaled.Thereafter, if Hg 2+ increases pulmonary vascular resistance, special attention should be given to diagnostic procedures requiring Hg 2+ salts, like lung scintigraphy.
Another interesting aspect resulting from HgCl 2 administration was the important increase of the RV and LV diastolic pressures.
Two main mechanisms could explain these findings.First, this could result from an increase in the afterload of both ventricles.However, although being true for the RV, since pulmonary resistance increased, this is unlikely for the LV because the afterload for this chamber decreased as its diastolic pressure increased.The second mechanism could explain the increment of diastolic pressure for both ventricular chambers.This could be the result of a calcium overload since Hg 2+ reduces the activity of the calcium (14,15) and sodium pumps (8-13) and increases calcium release from SR (4,16,17).The occurrence of a calcium overload may impair relaxation and cardiac filling during diastole, thereafter reducing cardiac output and then ABP.

Effects of Hg 2+ on cholinergic activity
Previous studies using atropine showed that the decrease in MBP elicited by HgCl 2 could be attenuated by prior administration of atropine (2).Several reports presented results which suggested interactions between Hg 2+ and cholinergic activities.Burg and coworkers (25) suggested that Hg 2+ binds to muscarinic receptors in rat brain preparations.Interactions of HgCl 2 and CH 3 HgOH with muscarinic receptor subtypes in the rat brain were also described by Castoldi et al. (26).Kostial and Landeka (27) suggested that Hg 2+ might stimulate the release of Ach from nerve terminals.Candura et al. (28) presented similar findings which suggested that small concentrations of Hg 2+ stimulate cholinergic transmission while high concentrations inhibit it.Inhibition of cholinergic transmission was also reported by Moberg et al. (23).Mercury may also interfere with cholinergic transmission by blocking Ca 2+dependent Ach release and enhancing Ca 2+independent release in nerve terminals (29).These facts could explain why atropine antagonized the effects of Hg 2+ .
The administration of HgCl 2 attenuated, instead of enhancing, the hypotensive and bradycardic responses elicited by exogenous Ach.The explanation for these findings was the observation that Hg 2+ increased cholinesterase activity in both plasma and heart.The increased activity of the enzyme may have degraded the exogenously administered Ach before it reached the receptor.However, many reports regarding cholinesterase activity suggested that Hg 2+ strongly inhibits this enzyme (30)(31)(32)(33) instead of activating it.Our results do not permit a clear explanation for this controversy but some results in the literature may help to explain it.Previous reports showed that the effects of Hg 2+ on enzyme activities may differ according to the enzyme affected (34,35) and even within the different organs in the same animal (36).
Other reports even suggested that acetylcholinesterase or cholinesterase are not affected by Hg 2+ (37,38).Another aspect to be considered is the difference between our protocol and others reported in the literature.Ours was an acute in vivo protocol while all others were performed over a longer period of time, usually days, or in vitro.We may speculate that the activity of plasma and heart cholinesterase may increase just after Hg 2+ injection and progressively decrease with the continuation of the toxic effects of the metal.Another explanation for the attenuation of the hypotensive and bradycardic responses to exogenous Ach by Hg 2+ may be the impairment of cholinergic transmission.The same explanation is valid for the effects of atropine since atropine may interfere with the interactions between Hg 2+ and the muscarinic receptors.However, taken together, these findings indicate that the interactions with cholinergic mechanisms may not be a pivotal mechanism to explain the reduction in ABP produced by Hg 2+ .
In summary, our results confirm previous findings showing that acute administration of Hg 2+ decreases ABP in rats.The present results suggest for the first time that HgCl 2 may produce cardiac diastolic failure and pulmonary hypertension in vivo, which may be the main mechanism producing the significant ABP reduction caused by HgCl 2.

Figure 1 -
Figure 1 -Effects of HgCl 2 on the time course changes of systolic pressure (SP) in the right ventricle (RV) (circles) and left ventricle (LV) (triangles) of anesthetized rats.Zero time is the control condition of the RV or LV systolic pressure obtained before HgCl 2 administration (5 mg/kg).Each symbol indicates the mean ± SEM. *P<0.05,one-way ANOVA used to compare the time course changes of RV or LV systolic pressure to their respective controls.Observe that there were only minor changes in LV systolic pressure, whereas changes in RV systolic pressure were more significant.

Figure 2 -Figure 3 -
Figure 2 -Effects of HgCl 2 , 5 mg/kg, on the time course changes in diastolic pressures (DP) of the right ventricle (RV) (circles) and left ventricle (LV) (triangles) of anesthetized rats.Zero time is the control condition of the RV or LV diastolic pressure obtained before HgCl 2 administration.Each symbol represents the mean ± SEM. *P<0.05,one-way ANOVA used to compare the time course changes of RV or LV diastolic pressures to its respective control.Observe that DPs, in contrast to the systolic pressure changes, increased progressively for both ventricular chambers.

Figure 4 -
Figure4-Effects of increasing concentrations of acetylcholine (Ach) on mean blood pressure (MBP) and heart rate (HR) before (control) and after HgCl 2 (Hg 2+ ) treatment (5 mg/ kg).a, b, c, d, e and f correspond to increasing concentrations of Ach (0.035, 0.105, 0.35, 1.05, 3.5 and 10.5 µg, respectively) injected iv.Each column represents the mean and the bars one SEM.Two-way ANOVA indicated a significant decrease in MBP and HR.*P<0.005 for Ach vs Ach-free, both before and after Hg 2+ treatment and + P<0.01 for differences between each Ach effect before and after Hg 2+ treatment.

Table 1 -
Time course changes of left ventricular (LV), right ventricular (RV), systolic (SP) and diastolic (DP) pressures of untreated rats and pulmonary mean perfusion pressure (PMPP) of untreated isolated perfused lungs.