Withania somnifera ameliorates lead-induced augmentation of adrenergic response in rat portal vein.

OBJECTIVES: Present study was undertaken to elucidate the ameliorating potential of Withania somnifera root extract (WRE) against lead-induced augmentation of adrenergic response in rat portal vein.
MATERIALS AND METHODS: In-vitro studies were conducted on effect of lead alone and lead+WRE on rat-isolated portal vein while in-vivo studies were done in three groups of 12 rats each; Group-II and III received 0.5% lead acetate and 1.0% WRE + 0.5% lead acetate, respectively, in drinking water for 12 weeks whereas group-I served as control. Adrenaline and noradrenaline levels in brain and blood were determined by HPLC assay while vascular reactivity of portal vein to lead and WRE was determined by measuring the isometric tension.
RESULTS: Following in-vitro exposure, lead did not alter the contractile effect of phenylephrine. In-vivo studies revealed that contractile effect of lead on portal vein was significantly potentiated and it was antagonized by prazosin (10(-7) M) and WRE (1%). WRE treatment significantly reduced elevated blood noradrenaline (37.80%) and restored noradrenaline level in brain (39.39%) in lead-exposed animals. These values were almost comparable to the control group. But it failed to significantly affect the blood and brain adrenaline levels.
CONCLUSIONS: Results suggest that following pre-exposure of rats to WRE, lead-induced augmentation of alpha 1-adrenoceptors mediated response was reversed possibly by regulating catecholamine release from nerve endings. Thus, WRE may be useful in therapeutic management of lead-induced hypertension.

Introduction

Lead is one of the most widely studied occupational and environmental toxicant and exposure to it at lower concentrations is associated with long-term adverse effects on different body systems including blood pressure,[12] renal functions,[3] and cognition.[4] Lead influences the contractility of vascular smooth muscles both directly and indirectly. Vascular smooth muscle cells, in general, are sensitive to lead cytotoxicity due to its higher accumulation in cells. The mechanism of lead-induced contractile effect on different blood vessels varies; in rat aorta, lead enters into the smooth muscle cells through non-L-type Ca2+ channels and acts like calcium on the contractile machinery[5] and interferes with synthesis or action of endothelium-derived contractile or relaxant factors.[67] Interaction of lead with PKC in endothelium-independent but calcium-dependent manner is reported to increase lead-induced vascular contractility in rabbit mesenteric artery and results in hypertension.[8] Decrease in cellular Na+-K+-ATPase activity leading to rise in intracellular Na+ increased influx of Ca2+ through stimulation of Na+/Ca2+ exchanger mechanism and augmentation in vascular responsiveness to catecholamines is also associated with lead-induced hypertension.[9]

Small doses of lead have been reported to intensify α-adrenergic response and diminish β-adrenergic response in blood vessels, and increase positive-chronotropic action of isoproterenol.[10] Reactive oxygen species (ROS)-mediated inactivation of endogenous nitric oxide (NO), increased circulatory level of noradrenaline along with reduction in β-adrenergic receptor density, elevated plasma renin, angiotensin II, and aldosterone levels have also been proposed to be associated with lead-induced hypertension.[11]

Withania somnifera has been documented to possess several pharmacological activities in ancient literature. Root extract has been found to reduce the toxicity of heavy metals[1213] and possess hypotensive activity. However, no information is available on its modulatory effect on vascular activity in lead-exposed animals and human beings. Therefore, in view of the paucity of detailed information on impact of lead in modulating vascular contractility through adrenergic receptors, this study was undertaken.

Materials and Methods

Healthy adult male Sprague Dawley rats (90-150 g) were obtained from the Laboratory Animal House of the Institution and maintained under standard management and feeding conditions.

For in-vitro studies, animals were sacrificed following pentobarbitone sodium administration (40 mg/kg b.wt.; i.p.) and the main branch of hepatic portal vein was isolated in a petri dish containing aerated Tyrode solution of the following composition (mM/L): NaCl- 137; KCl- 2.7; CaCl2- 1.8; MgCl2- 0.1; NaHCO3- 11.9; NaH2PO4- 0.4 and d-Glucose- 5.55 and having a pH of 7.4. For in-vivo studies, rats were divided into three groups of 12 animals each. Group-I rats were kept as control and received acetic acid solution (2.2 ml/L of drinking water) only. Rats of group-II and III received 0.5% lead acetate containing 2750 ppm lead[14] and 1.0% W. somnifera root extract (WRE) + 0.5% lead acetate[13] (w/v), respectively, in drinking water continuously for 12 weeks. One milliliter of 5N HCl per liter of lead acetate solution was added to prevent the precipitation of insoluble lead salt.[15] The animals had a free access to drinking water and no additional drinking water was offered to the animals.

After 12 weeks of lead exposure, animals were sacrificed and portal veins were collected in the similar manner as mentioned earlier. Brain and blood samples from rats of all the three groups were collected for determining the adrenaline and noradrenaline levels. Institutional Animals Ethics Committee had approved the experimental protocol.

Chemicals: Lead acetate trihydrate (Loba Chemie), pentobarbitone sodium (Rhone Poulenc), EGTA (HiMedia), glutathione reduced (Merck) and Na2EDTA (HiMedia) were used in the present study. Phenylephrine, prazosin, nifedipine, adrenaline, noradrenaline were procured from Sigma, USA. All other chemicals used were of high purity grade. Drug solutions were prepared either fresh or from the stock solutions prepared in triple glass distilled water and stored at 4°C. Further dilutions of the required concentration were made in freshly prepared PSS on the day of use.

Preparation of plant extract: Roots of W. somnifera were powdered and soaked in distilled water for 24 h with intermittent stirring at 40°C with the help of magnetic stirrer. The infusion thus obtained was filtered through muslin cloth and centrifuged at 400 g for 15 min to obtain the clear supernatant. The supernatant, thus, collected was dried (40°C) and lyophilized. The extract was stored in refrigerator till used.

Recording of isometric tension: After careful removal of the adjacent connective tissue and fats, hepatic portal vein was mounted in a thermostatically controlled (37°C ± 0.5°C) organ bath (20 ml capacity) containing Tyrode solution (pH 7.4) continuously aerated with carbogen (95% O2 + 5% CO2) and allowed to equilibrate for 40 min under a constant resting tension of 0.5 g. During the equilibration period, the bath fluid was changed every 10 min. Isometric tensions were recorded using the force displacement transducer (T-305) connected to a Physiograph (Biodevice, India).

Estimation of catecholamines: Adrenaline and noradrenaline levels in plasma and brain of rats of all the experimental groups were determined employing HPLC method of Semerdjian-Rouquier et al.[16] using high performance liquid chromatogram (Shimadzu Corporation, Japan) having C18 reversed phase column (125 mm × 5 μm I.D.) and L-ECD6A electrochemical detector.

Catecholamines were extracted from the plasma as per the method of Chang et al., 1997.[17] Briefly, 2 ml blood was added to 1 ml aqueous solution containing 90 mg ethylene glycol-bis-alpha-amino ethyl ether-N,N,N’,N’-tetra-acetic acid (EGTA) and 60 mg reduced glutathione (GSH). Tubes were then centrifuged at 3000 rpm for 10 min and the supernatant (plasma) was collected and stored at –80°C till further use. Plasma samples (0.5 ml) were deproteinized by adding 10 μl of 70% perchloric acid and centrifuged at 5000 rpm for 10 min. Supernatant was filtered through 0.22 μm filter. The filtrate was diluted six fold in running buffer and the diluted filtrate (20 μl) was injected into the loop of HPLC. In case of brain tissue, 2 g of tissue sample was homogenized in 5 ml of pre-cooled 70% nitric acid (0.1 M). The mixture was then centrifuged at 28,000 rpm for 10 min and the supernatant was stored at –80°C till further use. After filtering through 0.22 m filter, 20 l of filtrate was injected into the loop of HPLC as mentioned earlier for the estimation of catecholamines. The mobile phase comprised of 0.1 M K2HPO4 and 0.1 M citric acid buffer (1:6 v/v; pH 4.7) containing 5% methanol and 0.1 mM Na2EDTA was used. The flow rate was maintained at 1 ml/min at 30°C. The chromatogram data were analyzed using Chromatopak software. The r2 value of standard curves and retention time of adrenaline and noradrenaline were found to be 0.99, 1.46 min and 0.96, 1.30 min., respectively.

Data analysis: Results are expressed as mean ± SE. Two-way ANOVA followed by Bonferroni post-hoc test was employed for statistical analysis of concentration-response curves while one way ANOVA followed by Newman — Keul post-hoc test was used for analysis of plasma and brain adrenaline and noradrenaline levels. P < 0.05 was considered to be statistically significant. EC50 or IC50 (concentration that produce or inhibit 50% of maximal response of agonist, respectively) and Emax (maximal response) were determined through nonlinear regression analysis using Graph Pad Prism 4.0, USA.

Results

In-vitro Studies

Effect of lead and WRE on isolated rat portal vein

Effect on basal tone and autorhythmicity: Following equilibration, rat portal vein exhibited autorhythmicity. Lead acetate did not show any appreciable contractile response up to a concentration of 3×10-4 M. However, at higher concentration (10-3 M to 3×10-2 M), lead acetate elicited concentration-dependent contraction [Figure 1a]. Prazosin and WRE failed to produce any appreciable change in lead-induced vascular contractility [Figure 1b and 1c]. Compared to the pD2 value of lead (2.27 ± 0.01), the pD2 values of lead in the presence of prazosin and WRE were found to be 2.24 ± 0.02 and 2.24 ± 0.02, respectively, and these did not significantly differ from that of lead treatment alone.

Figure 1

Physiographic recordings showing the effect of lead acetate alone (PbA; 1A) and in the presence of 0.1 μM prazosin (Prz; 1B) or 1 mg/ml WRE (1C) on autorhythmicity of rat isolated portal vein

Effect of nifedipine and Ca: To elucidate the role of extracellular and intracellular calcium release in lead-induced contraction on portal vein, contractile response of 10 mM lead was recorded either in the presence of nifedipine (1 μM) or Ca2+ free EGTA solution (mM/L; NaCl- 137; KCl- 2.7; MgCl2 - 0.1; NaHCO3- 11.9; NaH2PO4- 0.4, d-Glucose- 5.55 and EGTA-3.0; pH of 7.4.), respectively. There was almost complete loss of spontaneity following preincubation of the tissue either with nifedipine or Ca2+ free EGTA solution. As revealed in the Figure 2, in the presence of nifedipine (1 μM), lead acetate-induced contraction was significantly (P<0.05) reduced from 26.36 ± 1.26 g/g of tissue (n = 5) to 2.04 ± 0.54 g/g of tissue (n = 5), while it produced nearly comparable contractile response (25.07 ± 1.03 g/g of tissue) following pre-exposure to Ca2+ free EGTA solution.

Figure 2

Bar diagram showing the effect of nifedipine (1 μM) or Ca[2]+ free EGTA solution on in vitro lead acetate (10 mM) – induced contraction on isolated rat portal vein. Data presented are mean ± SEM of five animals. *P<0.05 vs lead alone

Effect of lead on phenylephrine-induced response: To determine whether lead alters the contractile effect of phenylephrine, concentration-dependent responses to phenylephrine in the absence (control) and presence of lead acetate (0.1 mM, 15 min) or lead in combination with WRE (1 mg/ml, 15 min) were recorded. As shown in the Figure 3, the maximal contraction (Emax) of phenylephrine was nonsignificantly altered following preincubation of the tissue with lead acetate alone (10.45 ± 1.25 g/g of tissue) or in the combination of lead acetate plus WRE (10.15 ± 1.02 g/g of tissue) compared to control (11.14 ± 1.75 g/g of tissue). No significant changes were also observed in pD2 values of phenylephrine in the presence of lead acetate alone (5.76 ± 0.07) or lead acetate plus WRE (5.93 ± 0.10) in comparison to control (5.82 ± 0.10).

Figure 3

Cumulative concentration response curves showing the effect phenylephrine (PE) in the absence and presence of in-vitro exposure of lead acetate (0.1 mM) alone or lead acetate (0.1 mM) + WRE (1 mg/ml) on isolated rat portal veins. Data presented are mean ± SEM of six animals

In-vivo Studies

Contractility of portal vein in lead alone and lead with WRE treated rats

Effect on normal autorhythmicity: Exposure of rats of group II to low dose of lead (0.5% in drinking water) resulted in significant (P<0.001) increase in the basal amplitude (4.22 ± 0.31 g/g of tissue) and frequency (6.17 ± 0.38 per min) of spontaneity in isolated portal vein in comparison to that of control group (2.13 ± 0.14 g/g of tissue and 3.50 ± 0.18 per min, respectively). However, compared to lead-treated rats, basal amplitude of autorhythmicity of portal vein (2.59 ± 0.61 g/g of tissue) was significantly (P<0.05) lower in animals treated with lead acetate +WRE (group III) and these values were almost similar to those in the control group. The basal frequency of the group-III rats was calculated to be 4.92 ± 0.64 per minute.

Effect on KCl-induced contractions: KCl (80 mM) elicited a submaximal contraction of 8.6 ± 0.8 g/g of tissue on isolated portal vein of the control group rats and it was not significantly different from that in the presence of lead alone (11.3 ± 1.9 g/g of tissue) or lead in combination with WRE (8.5 ± 1.0 g/g of tissue).

Effect on phenylephrine-induced contraction: Phenylephrine (10-8 M to 10-4 M) produced contractile effect on the portal vein of rats of all the three groups. At higher concentration (10-6 M to 3×10-4 M), the amplitude of spontaneity was reduced and even the spontaneity completely vanished in all the experimental groups. Lead significantly (P<0.05–0.01) potentiated the contractile responses to phenylephrine and the DRC of phenylephrine was shifted leftward in group II rats [Figure 4]. Though, WRE treatment in lead-exposed animals failed to produce any significant change in phenylephrine-induced contraction compared to the control group but the rightward shift of the DRC of phenylephrine in group III was found to be significant (P<0.05–0.01) compared to lead treated group [Figure 4]. The Emax value of phenylephrine in group II rats (18.63 ± 3.58 g/g tissue) was found to be significantly higher (P<0.05) compared to that of 9.71 ± 0.80 g/g of tissue (n = 6) in the control group and 8.76 ± 1.49 g/g of tissue in the WRE and lead treated group (group III). Similarly the pD2 value of phenylephrine in group III (5.95 ± 0.16, n = 6) was found to be significantly lower than in the lead-treated group (6.42 ± 0.11) and the latter value was significantly higher (P<0.001) compared to the value of the control group (5.79 ± 0.08).

Figure 4

Cumulative concentration response curves of phenylephrine on isolated rat portal veins of control, lead acetate (PbA; 0.5%) and PbA (0.5%) + WRE (1%) treated animals. Data presented are mean ± SEM of six animals. *P<0.05 vs PE alone

Prazosin (10-7 M), an α1-adrenergic receptor blocker, significantly (P<0.05–0.01) blocked the effect of phenylephrine as evidenced by rightward shift of the DRC of phenylephrine in all the three groups (group I, II, and III) [Figure 5].

Figure 5

Cumulative concentration response curves showing the effect of phenylephrine (PE) in the absence and presence of prazosin (PrZ; 10-7 M) on isolated rat portal vein of different experimental groups of rats. Data presented are mean ± SEM of six animals. *P<0.05 vs PE alone

KCl (80 mM) produced submaximal contraction in isolated rat portal vein preparations. KCl-induced contraction was compared with the phenylephrine-induced responses on portal vein of different groups. Concentration versus per cent response curves of phenylephrine alone and in the presence of prazosin as per cent of the KCl responses considering KCl response as 100%, also represented almost similar trend or pattern of responses to phenylephrine.

Plasma and brain levels of catecholamine: Compared to the control group, lead-treated rats of group II exhibited 53% higher while those of lead + WRE (Gr-III) treated rats showed 29% higher plasma adrenaline level. Although slight increase in brain adrenaline levels (8%) was also observed in group-II and III rats but these values did not differ significantly from those in the control group [Figure 6].

Figure 6

Effect of 12 weeks exposure of lead acetate (PbA, 0.5%) alone or in combination with WRE (1%) in drinking water on plasma (5A) and brain (5B) catecholamine levels in rats. Data presented are mean ± SEM of 9-10 animals. **P<0.001 vs control and ##P< 0.01 vs PbA

Contrary to adrenaline, noradrenaline (NA) level was significantly (P<0.05–0.001) elevated in plasma (0.87 ± 0.07 μg/L) but reduced in brain (0.33 ± 0.05 μg/g) of lead-treated rats (group II) as compared to that control group (0.38 ± 0.04 μg/L and 0.57 ± 0.05 μg/g, respectively) [Figure 6]. Treatment with WRE significantly (P<0.01) reduced the level of NA in plasma (0.51 ± 0.08 μg/L) of group III animals to the level almost comparable to that in control group. Similarly, WRE also markedly restored the level of brain NA (0.46 ± 0.07 μg/g) [Figure 6].

Discussion

Concentration-dependent contractile effect of lead acetate on isolated rat portal vein in the present study is in conformity with the findings in rat aorta[5] except that comparatively higher concentrations of lead (1-30 mM) were required to contract rat portal vein. Our observations of in-vitro studies in portal vein revealed that lead-induced contractile effect was not altered by prazosin. Lead did not affect the sensitivity of portal vein to phenylephrine, thus, suggesting that lead does not act directly through α-adrenoceptors. But significant reduction in the contractile effect of lead in the presence of nifedipine and almost comparable response in Ca2+ free EGTA solution suggests the role of extracellular calcium entry through L-type Ca2+ channels and intracellular calcium release mechanisms in lead-induced contraction.

Lead is known to alter sympathetic activity of the cardiovascular system by direct action on vascular smooth muscle but the effect does not seem to be acute in rat tail arteries.[17] In this study too, lead alone or in combination with WRE failed to alter the DRC of phenylephrine in in-vitro studies on isolated portal vein. Therefore, it may not be unreasonable to infer that instant exposure of portal vein to lead does not seem to be related to alterations in sympathetic transmission and WRE does not directly antagonize the effect of lead.

In the present investigation, subchronic exposure of rats to lead for 12 weeks, at a dose level which did not cause any appreciable change in body weight and behavior of rats, significantly (P<0.05–0.001) increased the frequency and amplitude of spontaneity of portal vein. Our findings suggest that portal vein may be one of the target tissues of lead and this observation is in agreement with the earlier report on rat tail artery.[17]

Significant potentiation of phenylephrine-induced contraction and its antagonism by prazosin following exposure to low lead levels suggests augmented α1-adrenergic response of portal vein. This observation is in conformity with the earlier findings in different vascular smooth muscles of rats.[1718] Elevation in the plasma NA level also supports the claim that lead exposure increases intracellular Ca2+ concentration through escalation in NA level or by augmenting sensitivity of voltage sensitive Ca2+ channels.

Possibility of modulation of α-drenoceptors activity by lead in the present study cannot be ruled out as the DRC of phenylephrine exhibited significant shift towards left in the lead-treated group. Decreased neuronal uptake of catecholamines is known to contribute toward enhanced vascular reactivity in lead treated rats but it does not seem to be true in the this study as phenylephrine is not taken up by nerve endings[19] and changes in the neuronal uptake activity are usually not accompanied by change in Emax of vascular preparation.[20] Interestingly in this study, the Emax and pD2 values of phenylephrine were decreased in the presence of lead, thus, suggesting that the target site of lead and phenylephrine may differ.

Potassium chloride elicits membrane depolarization by causing influx of extracellular Ca2+,[21] while phenylephrine-induced contraction is due to initial release of intracellular pool of Ca2+ followed by extracellular Ca2+ entry.[22] Following exposure of rats to low levels of lead for 12 weeks, there was appreciable (31.40%) but insignificant increase in KCl-induced tension in the lead-treated group compared to that in the control group. In the WRE and lead treated group, such an increase in tension was not observed as the tension in this group was 24.78% lower compared to that in group II. Therefore, enhanced Ca2+ influx in the rat portal vein through voltage gated calcium channels does not seem to be involved rather the possibility of an increase in intracellular pool of activator Ca2+ in lead-treated animals cannot be ruled out. This is in conformity with earlier findings in rat tail artery,[17] but it differs from rat aorta.[5]

Following treatment of rats with WRE, significant reversal of the effect of lead on portal vein (group III) was evident as there was rightward shift of the DRC of lead and phenylephrine. Thus, an apparent antagonistic effect of WRE in rats of group III compared to that of group II suggests the possible inhibition of release of NA from adrenergic nerve endings as there was significant increase in blood level of noradrenaline in lead-treated rats of group II. Vyskocil et al.,[23] also reported almost similar increase in noradrenaline level in blood in developing male and female rats following five months exposure to lead acetate in drinking water @ 0.5%. Contrary to the increase in blood noradrenaline, a significant decrease in brain noradrenaline in lead treated rats was observed as has been reported following long-term exposure of rats to lead.[24] Possibility of decrease in brain noradrenaline level in the present study may be due to decreased synthesis or enhanced release from adrenergic nerve endings in brain and its binding with α2-adrenoceptors on peripheral sympathetic nerves and thus the possibility of further release of noradrenaline by auto-inhibitory mechanism cannot be ruled out. Accordingly, decrease in the concentration of noradrenaline in brain may lead to stimulation of peripheral adrenergic nerves to release more noradrenaline into plasma.[19] A promising potential of WRE in reversing the catecholamine levels in blood and brain to almost normal levels in lead-exposed rats was a fascinating observation. Such an effect of WRE due to decreased catecholamines release from adrenal medulla and/or adrenergic nerve endings cannot be ruled out as W. somnifera is known to possess antistress and adaptogenic properties. Such catecholamine lowering potential of WRE might be the basis of its use in the management of hypertension.[25]

Based on the results of present study, it is inferred that prolonged exposure to lead at low levels augments α1-adrenergic response in portal vein by increasing the responsiveness of these receptors along with possibly augmented release of neurotransmitters from adrenergic nerve endings. Treatment with W. somnifera root extract significantly reversed the effect of lead on the vascular system possibly by modulating α1-adrenergic system and, thus, it seems to have therapeutic potential for management of lead-induced hypertension especially in the persons having the risk of occupational hazards.