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EFFECTS OF SUBACUTE LEAD ACETATE ADMINISTRATION ON NITRIC OXIDE AND CYCLOOXYGENASE PATHWAYS IN RAT ISOLATED AORTIC RING
Pharmacological Research, Vol. 46, No. 1, 2002 doi:10.1016/S1043-6618(02)00035-X, available online at http://www.idealibrary.com on
EFFECTS OF SUBACUTE LEAD ACETATE ADMINISTRATION ON NITRIC OXIDE AND CYCLOOXYGENASE PATHWAYS IN RAT ISOLATED AORTIC RING GHOLAMREZA KARIMI a , ALI KHOSHBATEN b , MOHAMMAD ABDOLLAHI c , MOHAMMAD SHARIFZADEH c , KHODADAD NAMIRANIAN d and AHMAD REZA DEHPOUR d,∗ a Department of Pharmacology and Toxicology, School of Pharmacy, Mashad University of Medical Sciences, Mashad, Iran, b Faculty of Medicine, Baghiatallah University of Medical Sciences, Tehran, Iran, c Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical School, Tehran, Iran, d Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Accepted 6 March 2002
Low level exposure to lead increases blood pressure in human and rats. In this study, we investigated the contribution of the nitric oxide (NO) and cyclooxygenase pathways of aortic rings of 28-day lead-treated and control rats, to the responsiveness to phenylephrine and acetylcholine. There were no differences in phenylephrine contractions between the two groups. N(ω)-nitro-l-Arginine-methyl ester (l-NAME), a NO synthase inhibitor, caused attenuation in contraction response to phenylephrine in the aortic rings of the lead-treated rats, while endothelium-denudation caused attenuation in those of controls. This may be due to either endothelium-derived vasoconstrictor(s) (such as reactive oxygen species or endothelins) or a source of NO in smooth muscle cells. There is a left-shift in acetylcholine relaxation response. Indomethacin incubation caused a left-shift in relaxation response to acetylcholine in controls but without any effect on lead-treated ones. Indomethacin incubation caused attenuation in contraction to phenylephrine in both groups. The relaxation response to sodium nitroprusside is not different between the two groups, suggesting that smooth muscle relaxation component is intact. However, the relaxation response to glyceryl trinitrate is impaired in aortic rings of lead-treated rats. It can be concluded that NO and cyclooxygenase pathways are altered in aortic rings of lead-treated rats, with possible involvement of endothelium-derived vasoconstrictors. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: lead, subacute exposure, cyclooxygenase pathway, nitric oxide, rat aortic ring.
INTRODUCTION Chronic exposure to low levels of lead is universal in industrialized countries [1]. Continuous exposure to low levels of lead has been shown to result in increased blood pressure in both human and animals [2–5]. Many mechanisms are proposed as the cause of leadinduced hypertension, such as increased activity of renin– angiotensin–aldosterone axis [6]; inhibition of calcium extrusion in the cell membrane, and lowering the calcium binding capacity in intracellular calcium stores, leading to an increase in intracellular calcium [7]; altered kallikrein–kinnin system causing decreased plasma levels ∗ Corresponding
author. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. E-mail: [email protected]
1043-6618/02/$ – see front matter
of bradykinin [8, 9]; increased plasma catecholamines [10]; and direct smooth muscle protein kinase C activation [11, 12]. Endothelium regulates vascular tone through the release of several vasodilators (nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF)) and vasoconstrictors (endothelins, vasoconstrictor prostanoids (TXA2 and PGH2 ), and oxygen reactive species). Hypertension is reported to be associated with endothelium dysfunction in both human and animal studies [13]. Endothelium is also reported to play some role in the lead-induced hypertension. Increased level of endothelin-3 and an increase in the reactive oxygen species, both endothelial-derived vasoconstrictors, are reported in lead hypertensive rats [14, 15]. Decreased urinary nitrite/nitrate and decreased plasma cGMP, a reflection of NO activity, were also demonstrated in lead-exposed hypertensive rats [14–16]. © 2002 Elsevier Science Ltd. All rights reserved.
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We have recently reported interaction between lead and arginine/NO pathway in the rat kidney [17] and submandibular gland [18]. On a similar basis, we had previously reported that the cadmium inhibitory effects on salivary gland function are modulated by the NO system [19], suggesting the general effects of metallic ions on the NO system. However, none of the mentioned reports studied the NO pathway in the responsiveness of isolated vascular elements of lead hypertensive animals. Cyclooxygenase metabolites modulate many effects on the vascular system, such as vasodilatation (prostacyclin) and vasoconstriction (PGH2 and TXA2 ). Some studies suggested the effect of lead on arachidonic acid metabolism. Dietary lead increased the tissue concentration of arachidonic acid in the chicken [20], and organic lead increased the arachidonic acid release in the HL-60 cell lines [21]. Carsia et al. reported the increase in membrane arachidonic acid of isolated aortic smooth muscle cells, propagated in media containing lead [22]. So, it seems that cyclooxygenase pathway of vascular system might be altered in lead-induced hypertension. In the present study on lead-induced hypertension, we aimed to investigate the effect of NO pathway, by blocking NO synthase (NOS) with N(ω)-nitro-l-arginine-methyl ester (l-NAME), and of cyclooxygenase pathway, by blocking with indomethacin, on the responsiveness of the isolated aortic rings of lead-treated rats to a vasoconstrictor agent (phenylephrine) and an endothelium-dependent vasodilator (acetylcholine). We also assess the vasorelaxation response to two other important vasodilators, sodium nitroprusside and glyceryl trinitrate (GTN).
MATERIALS AND METHODS
Animals Adult male Sprague–Dawley rats, weighing 200–250 g were used. The animals were handled according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH US publication 86-23, revised 1985). One group of animals, known as lead-treated group, were fed standard laboratory chow and were given 100 ppm lead acetate (0.01%) in their drinking water for 28 days (lead-treated group). The other group of rats, which served as controls, were treated the same as the lead-treated group, except they were given tap water. They were housed in an environment at 20–25 ◦ C with a 12-h light:12-h dark cycle starting at 08:00 h. Rats were weighed before entering into study and also at the time of sacrifice. Six to seven animals were included in lead-treated and control groups each.
Measurement of systolic blood pressure Conscious rats were placed in a restrainer and allowed to rest inside the cage for 15 min prior to blood pressure measurement. The rat tail was placed inside the tail cuff and the cuff was inflated and released a few times to allow
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the animal to be conditioned to the procedure. Systolic blood pressure values (four consecutive readings) were recorded by a rat tail sphygmomanometer (PE 300, Narco Biosystems, Inc., Houston, TX, USA) attached to a student physiograph (MK III-S, Narco Biosystems, Inc., Houston, TX, USA) and averaged for presentation.
Determination of lead in blood Lead concentration of whole blood was measured using an atomic absorption spectrophotometer (Shimadzu 680A, with graphite furnace, Shimadzu, Japan). Whole blood lead values were expressed as microgram per liter [17–19].
Preparation of rat thoracic aortic rings As previously described [23, 24], animals were killed by a blow to head and thoracic aorta was immediately removed and placed in physiological salt solution (containing (in mM): NaCl, 130; KCl, 4.7; CaCl2 , 1.6; MgSO4 , 1.17; KH2 PO4 , 1.18; NaHCO3 , 14.9; dextrose, 5.5; and EDTA-Ca,Na2 , 0.03) bubbled with mixture of 95% O2 and 5% CO2 . After cleaning the tissue free of fat and other adhering tissues, the vessels were cut into 3 mm long rings, with special care to avoid damaging the endothelium. The preparations were mounted on a pair of stainless-steel hooks; one of each was fixed to an L-shaped rod inside the chamber and the other to an isometric force transducer (F-60, Narco Biosystems, Inc., TX, USA) connected to a polygraph (MK III-S, Narco Biosystems, Inc., TX, USA). Tissues were allowed to equilibrate under an optimum final force of 1.5 g for a period of 60 min in a water-jacketed tissue bath (10 ml) containing oxygenated physiologic salt solution at 37 ◦ C (final pH of 7.4), renewing the buffer every 15 min. After stabilization, the preparations were contracted twice with KCl (40 mM) and the last contraction was taken as the reference value for analysis. The presence of functional endothelium was tested by the relaxation response to 1 µM ACh in phenylephrine-precontracted rings. In all experiments, each ring was used only once.
Experimental protocol All of the following experiments were conducted on the aortic rings of both lead-treated and control rats. First. Contraction response was assessed by adding cumulative concentration of phenylephrine (0.1 nM to 10 µM). The concentration–contraction curves were studied for: (1) intact aortic rings; (2) endothelium-denuded aortic rings; (3) aortic rings incubated with l-NAME (100 µM) for 20 min; (4) aortic rings incubated with indomethacin (20 µM) for 20 min; and (5) aortic rings incubated for 20 min with l-NAME (100 µM) and indomethacin (20 µM). Endothelium-denuded rings were prepared by removing endothelium by rubbing the lumen of aortic ring with a cotton thread, and the removal was confirmed by the loss of relaxation to 1 µM acetylcholine.
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Fig. 1. Dose–response curves of the contraction of aortic rings of the control and 28-day lead-treated rats to phenylephrine. The panels show: (a) the effect of endothelium-denudation; (b) the effect of incubation with l-NAME (100 µM); (c) the effect of incubation with indomethacin (20 µM); and (d) the effect of incubation with l-NAME/indomethacin, on the phenylephrine contraction. ((hollow *) P < 0.05 compared to the value of the intact ring of control rats and (*) P < 0.05 compared to the value of the intact ring of lead-treated rats). (䊊) Intact aortic rings of control rats; (䊉) intact aortic rings of lead-treated rats; (䊐) endothelium-denuded aortic rings of control rats; (䊏) endothelium-denuded aortic rings of lead-treated rats; (䉫) aortic rings of control rats incubated with l-NAME (100 µM); (䉬) aortic rings of lead-treated rats incubated with l-NAME; () aortic rings of control rats incubated with indomethacin (20 µM); (䉱) aortic rings of lead-treated rats incubated with indomethacin; (×) aortic rings of control rats incubated with l-NAME/indomethacin; (+) aortic rings of lead-treated rats incubated with l-NAME/indomethacin).
Second. Relaxation response to acetylcholine, an endothelium-dependent vasorelaxant, was assessed by adding cumulative concentration of acetylcholine (10 nM to 10 µM) on the aortic rings precontracted with phenylephrine 1 µM. The similar concentration–relaxation curves were also done on: (1) the aortic rings incubated with indomethacin (20 µM for 20 min); (2) aortic rings incubated with l-NAME (100 µM for 20 min). Third. Relaxation response curves to sodium nitroprusside and glyceryl trinitrate (GTN) were also constructed by adding cumulative doses of sodium nitroprusside (1 nM to 10 µM) or GTN (1 nM to 10 µM) to phenylephrine-precontracted aortic rings in separate experiments.
Chemicals The following drugs were used: acetylcholine chloride, phenylephrine hydrochloride, l-NAME, indomethacin (purchased from Sigma Chemical Co., St. Louis, MO, USA); sodium nitroprusside, glyceryl trinitrate (GTN) and lead acetate (purchased form Merck Chemical Co., Germany). Chemicals were dissolved in distilled water, except for indomethacin, which is dissolved in absolute ethanol (0.2 µmol in 10 µl). Ethanol (10 µl) has no effect on the resting tension, phenylephrine-induced contraction, or any of the vasorelaxation responses.
Statistics and calculations Contraction to the second exposure of 40 mM KCl was taken as 100%, and the contractile responses to
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Table I Characteristics of the phenylephrine-induced contraction response and relaxation responses induced by acetylcholine, sodium nitroprusside and glyceryl trinitrate, in aortic rings of control and lead-treated rats Control group
Lead-treated group
Rmax
pEC50
Rmax
pEC50
Phenylephrine-induced vasoconstriction Intact Endothelium-denuded In presence of l-NAME In presence of indomethacin In presence of l-NAME and indomethacin
133.47 ± 5.55 172.89 ± 4.79a 126.41 ± 2.76 96.19 ± 5.32a 120.30 ± 2.60
7.31 ± 0.04 7.57 ± 0.05 7.50 ± 0.03 7.14 ± 0.06 7.05 ± 0.04
133.32 ± 8.69 145.19 ± 6.45 145.12 ± 3.17b 83.18 ± 6.90a 136.19 ± 3.74
7.20 ± 0.04 7.37 ± 0.06 8.06 ± 0.03c,d 6.95 ± 0.03a 7.51 ± 0.05a,b
Acetylcholine-induced vasorelaxation Intact In presence of indomethacin Sodium nitroprusside-induced vasorelaxation Glyceryl trinitrate-induced vasorelaxation
54.77 ± 3.33 55.45 ± 2.40 122.56 ± 11.30 135.69 ± 5.37
7.07 ± 0.04 7.31 ± 0.04a 7.66 ± 0.06 7.87 ± 0.05
55.28 ± 5.46 61.79 ± 0.93 103.76 ± 4.83 108.2 ± 2.93b
7.32 ± 0.03b 7.34 ± 0.03 7.73 ± 0.09 7.58 ± 0.09
Values are means ± SEM (n = 6–7 for both group). a P < 0.05 compared to the values of the intact aortic rings of the respective experiment. P < 0.05 compared to respective values of the control group. c P < 0.01 compared to respective values of the control group. d P < 0.01 compared to the values of the intact aortic rings of the respective experiment.
b
phenylephrine were expressed as a percentage of this contraction. Relaxation was expressed as the percentage of the initial precontraction with 1 µM phenylephrine. The concentration (EC50 ) of the agonist exhibiting 50% of the maximal response (Rmax ) was calculated with a curve-fitting software (GraphPad Prism version 3.00, Graph Pad Inc., San Diego, USA) and expressed as negative log molar (pEC50 ) value. All data are expressed as means ± SEM. The interaction of lead treatment and incubation with mentioned drugs (l-NAME and indomethacin) was studied by two-way analysis of variance (ANOVA) and were mentioned where no significant interaction exists. Difference between the values was tested for significance by unpaired Student’s t-test. Differences between more than two mean values were tested by one-way ANOVA, followed by Student– Newman–Keuls’ multiple comparisons as post hoc test. Differences were considered significant at P < 0.05.
RESULTS
Blood pressure changes There was a significant increase in systolic blood pressure of rats treated with 100 ppm lead acetate in respect to controls after 28 days (121.5±2.17 vs 95.8±3.13 mmHg, P < 0.05). At the time of sacrifice, the blood lead concentration of lead-treated group was significantly higher than that of control animals, reaching a mean value of 258.4 ± 23.2 µg/l, compared to controls of 114.5±31.4 µg/l (P < 0.05). There was no significant difference between the mean body weight of two groups, at the beginning and at the time of sacrifice.
Phenylephrine-induced contraction Phenylephrine, an α 1 -adrenoceptor agonist, caused a concentration-dependent contraction in the isolated aortic rings. There was no significant difference between the
pEC50 and Rmax of the control and lead-treated animals (Fig. 1 and Table I). Endothelium-denuded aortic rings of control rats showed greater Rmax compared to intact aortic rings of controls (P < 0.05), but did not show any difference in the pEC50 . There was no significant difference between the pEC50 and Rmax of the endothelium-denuded and endothelium-intact aortic rings of the lead-treated group [Fig. 1(a)]. Inhibition of NO synthesis by l-NAME (100 µM) had no significant effect on the pEC50 and Rmax of the aortic rings of the control rats. But on the aortic rings of lead-treated group, l-NAME incubation caused significant increase in pEC50 (P < 0.05) with no significant effect on Rmax compared to respective intact aortic rings. Also there was a significant difference between the pEC50 (P < 0.01) and Rmax (P < 0.05) of the aortic rings of the lead-treated animals incubated with l-NAME, compared to respective control ones [Fig. 1(b)]. There was no significant interaction between indomethacin incubation (20 µM) and lead treatment on Rmax . Indomethacin incubation caused a significant decrease in the Rmax and pEC50 of the aortic ring of lead-treated animals, compared to intact respective ones (P < 0.05). However there was significant decrease in Rmax with no significant change in pEC50 of indomethacin-incubated aortic ring of controls compared to intact ones. In indomethacin-incubated rings, the pEC50 and Rmax of the lead-treated group were less than those of control respectives, but these differences did not reach the statistical significance [Fig. 1(c)]. Except for a significant increase in pEC50 of lead-treated group (P < 0.05), there was no significant difference between Rmax and pEC50 of l-NAME/indomethacin incubated aortic rings of lead-treated and control rats compared to intact respective ones. The Rmax of the l-NAME/ indomethacin incubated lead-treated group were not significantly different from the incubated rings of the
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controls, but the pEC50 showed significant increase (P < 0.05) [Fig. 1(d)].
Acetylcholine-induced relaxation responses Phenylephrine (1 µM) caused submaximal contraction (about 90% of Rmax of the respective group) in aortic rings. Acetylcholine caused a concentration-dependent relaxation in the precontracted aortic rings. There was a mild but significant leftward shift in the relaxation curve of the aortic rings of lead-treated animals, compared to the controls (no difference in Rmax but significant increase in pEC50 (P < 0.05)) (Fig. 2 and Table I).
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Indomethacin incubation had no significant effect on the Rmax and pEC50 of the acetylcholine-induced relaxation in aortic rings of the lead-treated animals, but there was some mild left-shift in relaxation-curve, characterized by significant differences in lower doses of acetylcholine response. There was a significant increase in pEC50 (P < 0.05), but no significant difference was observed in the Rmax of the indomethacin-incubated aortic rings of control rats, compared to indomethacin-incubated rings of controls [Fig. 2(a)]. Incubation with l-NAME (100 µM for 20 min) caused significant attenuation of acetylcholine-induced relaxation of aortic rings. These suggest that relaxation response to acetylcholine in aortic ring is mediated mainly via NO, in agreement with previous study [25]. There was no significant interaction of lead treatment with l-NAME incubation on Rmax . There was no difference between the curves of acetylcholine-induced relaxation of l-NAME-incubated aortic rings of lead-treated with the respective rings of control rats. Due to significant attenuation of relaxation by l-NAME incubation, the pEC50 was not calculated for these groups [Fig. 2(b)].
Sodium nitroprusside-induced relaxation response Sodium nitroprusside induced a concentration-dependent relaxation in the aortic rings (Fig. 3). There was no significant difference in sodium nitroprusside-induced relaxation pEC50 and Rmax of the aortic rings between the lead-treated and control rats.
Glyceryl trinitrate-induced relaxation response GTN induced a concentration-dependent relaxation in the aortic rings, which showed significant decrease in Rmax in aortic rings of lead-treated compared to control rats (P < 0.05), with no significant change in pEC50 (Fig. 4).
Fig. 2. Acetylcholine-induced relaxation response curves of aortic rings of control and 28-lead-treated rats. Panel (a) shows the effect of incubation with indomethacin (20 µM); and (b) shows the effect of incubation with l-NAME (100 µM). () P < 0.05 compared to the value of the intact ring of control rats; (hollow *) P < 0.05 compared to the value of the intact ring of control rats; and (*) P < 0.05 compared to the value of the intact ring of lead-treated rats). (䊊) Intact aortic rings of control rats, (䊉) intact aortic rings of lead-treated rats; () aortic rings of control rats incubated with indomethacin (20 µM); (䉱) aortic rings of lead-treated rats incubated with indomethacin; (䉫) aortic rings of control rats incubated with l-NAME (100 µM); (䉬) aortic rings of lead-treated rats incubated with l-NAME).
Fig. 3. Relaxation response curve to sodium nitroprusside in control (䊊) and 28-day lead-treated (䊉) rats.
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Fig. 4. Relaxation response curve to glyceryl trinitrate in control (䊊) and 28-day lead-treated (䊉) rats; (*) P < 0.05 compared to the value of the aortic rings of control rats).
DISCUSSION In this study, we used rats, which were treated with 100 ppm lead acetate in their drinking water for 28 days. This amount of lead is considered as low level of exposure, similar to the level seen in the environment, although the result values are not directly comparable to those of humans. Previous studies have shown that exposure for longer duration (three and more months) to low level (100 ppm) of lead, not high level (5000 ppm), results in hypertension in rats [14, 26]. Our results show that subacute (28-day) exposure will also result in increased systolic blood pressure. In this study, we demonstrated that the contraction response to phenylephrine is not different between control and lead-treated groups. This is in agreement with previous study [26]. But, blocking NO synthesis caused a significant accentuation of phenylephrine-induced contraction only in aortic rings of lead-treated rats; while the endothelium-denudation made a significant accentuation only in controls. Two possible mechanism may explain the mentioned results. The first possible mechanism is that the endothelium may secrete vasoconstrictor substances in lead-treated rats. The candidates for this vasoconstrictor substance are endothelin, reactive oxygen species, and prostanoid vasoconstrictor [14, 16, 27]. When NO synthesis is inhibited, the vasoconstrictor will cause a left-shift in phenylephrine-induced contraction, and when endothelium is removed, the left-shift disappears. The second possible explanation is that another source of NO production may exist outside the endothelium, possibly in the smooth muscle cells, of the aortic rings of the lead-treated rats. Gonick et al. demonstrated the increased inducible NOS (iNOS) in the kidney cortex of lead-treated rats [15]. It may be possible that increased iNOS exists in smooth muscle of aorta of lead-treated rats, providing a source of NO production, which is blocked
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by l-NAME, but persists in endothelium-denuded rings. Further studies are needed to explore these explanations. In this study we also demonstrated that relaxation response to sodium nitroprusside of isolated aortic ring was the same in control and 28-day lead-treated rats. It may be thus concluded that smooth muscle relaxation pathway is not altered by 28-day exposure to lead. But acetylcholine-induced vasorelaxation was mildly shifted to left in lead-treated group. Purdy et al. showed that the responses to the aortic vasodilators (acetylcholine and sodium nitroprusside) are, however, unchanged in 3-month lead-treated rats [26]. The increased acetylcholine relaxation response may be due to either increased release of endothelial vasodilators or decreased release of vasoconstrictors. However, endothelial NOS is reported to be inhibited by lead in brain microvascular endothelium cells [28]; and EDHF is reported to decrease in lead hypertension in rats [29]. Also as will be discussed later, there is also evidence for the shift of prostanoids synthesis toward vasoconstrictors. Finally, this mild difference may be due to technical procedures or difference in pathophysiological mechanisms between the 28-day and 3-month lead-treated rats, which deserves further studies with more specific interventions. In the present study, we showed that indomethacinincubation caused attenuation of phenylephrine contraction in both control and lead-treated groups. While indomethacin also caused a left-shift in acetylcholine relaxation curves of controls, it exerted no effect on lead-treated group. These results provided evidence for that the vasoconstrictor prostanoids might contribute to vascular responsiveness, in general. The lack of effect of indomethacin on relaxation in lead-treated group, is probably due to either simultaneous increase in the vasoconstrictor prostanoids, or a decrease in vasodilator prostanoids, leading to a balance. Blocking both NO and cyclooxygenase pathways caused a right shift in the phenylephrine-induced contraction of the aortic rings of lead-treated rats; but to a lesser degree than the right-shift caused by l-NAME alone. This provides further evidence for the altered production of the prostanoids during lead hypertension. Previous studies suggested that lead is able to increase the free arachidonic acid in the cell [20, 21]. Lead is also reported to stimulate arachidonic acid release from cultured bovine retinal endothelial cells [30]. The activation of arachidonic acid metabolism will result in the release of cyclooxygenase metabolites, known to act as vasoconstrictor [31]. More studies, using specific inhibitors of the cyclooxygenase pathways and measuring metabolite are needed to explore the changes of cyclooxygenase metabolites in lead hypertension. As we showed in the present study, relaxation response to GTN is impaired in aortic rings of lead-treated rats, but the relaxation response to sodium nitroprusside is not different between the two groups. GTN releases either nitrite ion via bioactivation by gluthathione S-transferase, or NO by unknown enzymatic activation [32]. Thus the impaired relaxation to GTN is possibly due to decreased
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activation. Lead is reported to bind to sulfhydryl group of enzyme through body [33], and may interfere with GTN bioactivation. This effect of lead may explain the impaired responsiveness to GTN in lead-treated group. GTN bioactivation is suggested to be inhibited by NO [34]. If we assume that the increased NO production exists in the aortic smooth muscle of lead-treated rats, we can explain the decreased GTN bioactivation. Until further studies explore these possibilities, these are as a matter of speculations. In conclusion, the possibility of altered cyclooxygenase pathway in lead hypertension exists. There is also evidence for the role of endothelium in lead hypertension, possibly by producing vasoconstrictors. The exact nature of these vasoconstrictors needs further detailed studies. ACKNOWLEDGEMENT This work is supported in part by the research deputy of Tehran University of Medical Sciences. REFERENCES 1. Elias RW. Lead exposures in the human environment. In: Maffey KR, ed. Dietary and environmental lead. New York: Elsevier, 1985: 79–107. 2. Beevers DG, Erskine E, Robertson M, Hawthorne VM. Blood lead and hypertension. Lancet 1976; 2: 1–3. 3. Harlan WR. The relationship of blood levels to blood pressure in U.S. population. Environ Health Perspect 1988; 78: 9–13. 4. Victery W. Symposium on lead blood pressure relationships. Environ Health Perspect 1988; 78: 139–55. 5. Cheng Y, Schwartz J, Sparrow D, Aro A, Weiss ST, Hu H. Bone lead and blood lead levels in relation to baseline blood pressure and the prospective development of hypertension: the Normative Aging Study. Am J Epidemiol 2001; 153: 164–71. 6. Goodfriend TL, Ball DL, Elliot ME. Lead increase aldosterone production by rat adrenal cells. Hypertension 1995; 25: 785–9. 7. Piccinini F, Favalli L, Chiari MC. Experimental investigations on the contraction induced by lead in arterial smooth muscle. Toxicology 1977; 8: 43–51. 8. Carmignani M, Boscolo P, Poma A, Volpe AR. Kininergic system and arterial hypertension following chronic exposure to inorganic lead. Immunopharmacology 1999; 44: 105–10. 9. Carmignani M, Volpe AR, Boscolo P, Qiao N, Di Gioacchino M, Grilli A, Felaco M. Catecholamine and nitric oxide systems as targets of chronic lead exposure in inducing selective functional impairment. Life Sci 2000; 68: 401–15. 10. Chang HR, Chen SS, Tsao DA, Cheng JT, Ho CK, Yu HS. Reduced vascular beta-adrenergic receptors and catecholamine response in rats with lead induced hypertension. Arch Toxicol 1997; 71: 778–81. 11. Markovac J, Goldstein GW. Lead activates protein kinase C in immature rat brain microvessels. Toxicol Appl Pharmacol 1988; 96: 14–23. 12. Watts SW, Chai S, Webb CR. Lead acetate-induced contraction in rabbit mesenteric artery: interaction with calcium and protein kinase C. Toxicology 1995; 99: 55–65. 13. Taddei S, Virdis A, Ghiadoni L, Salvetti G, Salvetti A. Endothelial dysfunction in hypertension. J Nephrol 2000; 13: 205–10.
37 14. Khalil-Manesh F, Gonick HC, Weiler EWJ. Lead induce hypertension: possible role of endothelial factors. Am J Hypertens 1993; 6: 723–9. 15. Gonick HC, Ding Y, Bondy SC, Ni Z. Lead induce hypertension: interplay of nitric oxide and reactive oxygen species. Hypertension 1997; 30: 1487–92. 16. Khalil-Manesh F, Gonick HC, Weiler EWJ, Purdy RE. Effect of chelation treatment with DMSA on lead related blood pressure changes. Environ Res 1994; 65: 86–99. 17. Dehpour AR, Essalat M, Ala S, Ghazi-Khansari M, Ghafourifar P. Increase by NO synthase inhibitor of lead-induced release of N-acetyl-beta-d-glucosaminidase from perfused rat kidney. Toxicology 1999; 132: 119–25. 18. Abdollahi M, Dehpour AR, Shafayee F. l-Arginine/nitric oxide pathway and interaction with lead acetate on rat submandibular gland function. Pharmacol Toxicol 2000; 87: 198–203. 19. Abdollahi M, Dehpour A, Kazemian P. Alteration by cadmium of rat submandibular gland secretory function and the role of the l-arginine/nitric oxide pathway. Pharmacol Res 2000; 42: 591–7. 20. Knowles SO, Donaldson WE. Dietary modification of lead toxicity: effects of fatty acid and eicosanoid metabolism. Comp Biochem Physiol 1990; 95: 99–104. 21. Kafer A, Zoltzer H, Krug HF. The stimulation of arachidonic acid metabolism by organic lead and tin compounds in human HL-60 leukemia cells. Toxicol Appl Pharmacol 1992; 116: 125–32. 22. Carsia RV, Forman D, Hock CE, Nagele RG, McIlroy PJ. Lead alters growth and reduces angiotensin II receptor density of rat aortic smooth muscle cells. Proc Soc Exp Biol Med 1995; 210: 180–90. 23. Dehpour AR, Aghadadashi H, Ghafourifar P, Roushanzamir F, Ghahremani MH, Meysamee F, Rassaee N, Koucharian A. Effect of chronic lithium administration on endothelium-dependent relaxation in rat aorta. Clin Exp Pharmacol Physiol 2000; 27: 55–9. 24. Rastegar H, Jorjani M, Roushanzamir F, Ahmadiani A, Namiranian K, Dehpour AR. Time-dependent relaxation of acetylcholineinduced relaxation in the rings of cholestatic rats. Pharmacol Res 2001; 44: 519–25. 25. Castillo C, Reyes G, Escalante B, Lopez P, Castillo EF. Endothelium-dependent vasodilatation in rat aorta is mainly mediated by nitric oxide. Proc West Pharmacol Soc 1997; 40: 39–40. 26. Purdy RE, Smith JR, Ding Y, Oveisi F, Vaziri ND, Gonick HC. Lead-induced hypertension is not associated with altered vascular reactivity in vitro. Am J Hypertens 1997; 10: 997–1003. 27. Ding Y, Gonick HC, Vaziri ND. Lead promotes hydroxyl radical generation and lipid peroxidation in cultured aortic endothelial cells. Am J Hypertens 2000; 13: 552–5. 28. Balzka ME, Harry GJ, Luster MI. Effects of lead acetate on nitrite production by murine brain endothelial cell cultures. Toxicol Appl Pharmacol 1994; 126: 191–4. 29. Oishi H, Nakashima M, Totoki T, Tomokuni K. Chronic lead exposure may inhibit endothelium-dependent hyperpolarizing factor in rats. J Cardiovasc Pharmacol 1996; 28: 558–63. 30. Bressler J, Forman S, Goldstein GW. Phospholipid metabolism in neural microvascular endothelial cells after exposure to lead in vitro. Toxicol Appl Pharmacol 1994; 126: 352–60. 31. Cosentino F, Sill JC, Katusic ZS. Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension 1994; 23: 229–35. 32. Katzung BG, Chatterjee K. Vasodilator and the treatment of angina pectoris. In: Katzung BG, ed. Basic and clinical pharmacology. 6th edn. Norwalk, CT, USA: Appleton and Lange, 1998: 179–96. 33. Ellenhorn MJ. Medical toxicology. 2nd edn. New York: Elsevier, 1988: 1032–4. 34. Kojda G, Patzner M, Hacker A, Noack H. Nitric oxide inhibits vascular bioactivation of glyceryl trinitrate: a novel mechanism to explain preferential venodilation of organic nitrates. J Pharmacol Exp Ther 1998; 53: 547–54.
EFFECTS OF SUBACUTE LEAD ACETATE ADMINISTRATION ON NITRIC OXIDE AND CYCLOOXYGENASE PATHWAYS IN RAT ISOLATED AORTIC RING GHOLAMREZA KARIMI a , ALI KHOSHBATEN b , MOHAMMAD ABDOLLAHI c , MOHAMMAD SHARIFZADEH c , KHODADAD NAMIRANIAN d and AHMAD REZA DEHPOUR d,∗ a Department of Pharmacology and Toxicology, School of Pharmacy, Mashad University of Medical Sciences, Mashad, Iran, b Faculty of Medicine, Baghiatallah University of Medical Sciences, Tehran, Iran, c Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical School, Tehran, Iran, d Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Accepted 6 March 2002
Low level exposure to lead increases blood pressure in human and rats. In this study, we investigated the contribution of the nitric oxide (NO) and cyclooxygenase pathways of aortic rings of 28-day lead-treated and control rats, to the responsiveness to phenylephrine and acetylcholine. There were no differences in phenylephrine contractions between the two groups. N(ω)-nitro-l-Arginine-methyl ester (l-NAME), a NO synthase inhibitor, caused attenuation in contraction response to phenylephrine in the aortic rings of the lead-treated rats, while endothelium-denudation caused attenuation in those of controls. This may be due to either endothelium-derived vasoconstrictor(s) (such as reactive oxygen species or endothelins) or a source of NO in smooth muscle cells. There is a left-shift in acetylcholine relaxation response. Indomethacin incubation caused a left-shift in relaxation response to acetylcholine in controls but without any effect on lead-treated ones. Indomethacin incubation caused attenuation in contraction to phenylephrine in both groups. The relaxation response to sodium nitroprusside is not different between the two groups, suggesting that smooth muscle relaxation component is intact. However, the relaxation response to glyceryl trinitrate is impaired in aortic rings of lead-treated rats. It can be concluded that NO and cyclooxygenase pathways are altered in aortic rings of lead-treated rats, with possible involvement of endothelium-derived vasoconstrictors. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: lead, subacute exposure, cyclooxygenase pathway, nitric oxide, rat aortic ring.
INTRODUCTION Chronic exposure to low levels of lead is universal in industrialized countries [1]. Continuous exposure to low levels of lead has been shown to result in increased blood pressure in both human and animals [2–5]. Many mechanisms are proposed as the cause of leadinduced hypertension, such as increased activity of renin– angiotensin–aldosterone axis [6]; inhibition of calcium extrusion in the cell membrane, and lowering the calcium binding capacity in intracellular calcium stores, leading to an increase in intracellular calcium [7]; altered kallikrein–kinnin system causing decreased plasma levels ∗ Corresponding
author. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. E-mail: [email protected]
1043-6618/02/$ – see front matter
of bradykinin [8, 9]; increased plasma catecholamines [10]; and direct smooth muscle protein kinase C activation [11, 12]. Endothelium regulates vascular tone through the release of several vasodilators (nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF)) and vasoconstrictors (endothelins, vasoconstrictor prostanoids (TXA2 and PGH2 ), and oxygen reactive species). Hypertension is reported to be associated with endothelium dysfunction in both human and animal studies [13]. Endothelium is also reported to play some role in the lead-induced hypertension. Increased level of endothelin-3 and an increase in the reactive oxygen species, both endothelial-derived vasoconstrictors, are reported in lead hypertensive rats [14, 15]. Decreased urinary nitrite/nitrate and decreased plasma cGMP, a reflection of NO activity, were also demonstrated in lead-exposed hypertensive rats [14–16]. © 2002 Elsevier Science Ltd. All rights reserved.
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We have recently reported interaction between lead and arginine/NO pathway in the rat kidney [17] and submandibular gland [18]. On a similar basis, we had previously reported that the cadmium inhibitory effects on salivary gland function are modulated by the NO system [19], suggesting the general effects of metallic ions on the NO system. However, none of the mentioned reports studied the NO pathway in the responsiveness of isolated vascular elements of lead hypertensive animals. Cyclooxygenase metabolites modulate many effects on the vascular system, such as vasodilatation (prostacyclin) and vasoconstriction (PGH2 and TXA2 ). Some studies suggested the effect of lead on arachidonic acid metabolism. Dietary lead increased the tissue concentration of arachidonic acid in the chicken [20], and organic lead increased the arachidonic acid release in the HL-60 cell lines [21]. Carsia et al. reported the increase in membrane arachidonic acid of isolated aortic smooth muscle cells, propagated in media containing lead [22]. So, it seems that cyclooxygenase pathway of vascular system might be altered in lead-induced hypertension. In the present study on lead-induced hypertension, we aimed to investigate the effect of NO pathway, by blocking NO synthase (NOS) with N(ω)-nitro-l-arginine-methyl ester (l-NAME), and of cyclooxygenase pathway, by blocking with indomethacin, on the responsiveness of the isolated aortic rings of lead-treated rats to a vasoconstrictor agent (phenylephrine) and an endothelium-dependent vasodilator (acetylcholine). We also assess the vasorelaxation response to two other important vasodilators, sodium nitroprusside and glyceryl trinitrate (GTN).
MATERIALS AND METHODS
Animals Adult male Sprague–Dawley rats, weighing 200–250 g were used. The animals were handled according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH US publication 86-23, revised 1985). One group of animals, known as lead-treated group, were fed standard laboratory chow and were given 100 ppm lead acetate (0.01%) in their drinking water for 28 days (lead-treated group). The other group of rats, which served as controls, were treated the same as the lead-treated group, except they were given tap water. They were housed in an environment at 20–25 ◦ C with a 12-h light:12-h dark cycle starting at 08:00 h. Rats were weighed before entering into study and also at the time of sacrifice. Six to seven animals were included in lead-treated and control groups each.
Measurement of systolic blood pressure Conscious rats were placed in a restrainer and allowed to rest inside the cage for 15 min prior to blood pressure measurement. The rat tail was placed inside the tail cuff and the cuff was inflated and released a few times to allow
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the animal to be conditioned to the procedure. Systolic blood pressure values (four consecutive readings) were recorded by a rat tail sphygmomanometer (PE 300, Narco Biosystems, Inc., Houston, TX, USA) attached to a student physiograph (MK III-S, Narco Biosystems, Inc., Houston, TX, USA) and averaged for presentation.
Determination of lead in blood Lead concentration of whole blood was measured using an atomic absorption spectrophotometer (Shimadzu 680A, with graphite furnace, Shimadzu, Japan). Whole blood lead values were expressed as microgram per liter [17–19].
Preparation of rat thoracic aortic rings As previously described [23, 24], animals were killed by a blow to head and thoracic aorta was immediately removed and placed in physiological salt solution (containing (in mM): NaCl, 130; KCl, 4.7; CaCl2 , 1.6; MgSO4 , 1.17; KH2 PO4 , 1.18; NaHCO3 , 14.9; dextrose, 5.5; and EDTA-Ca,Na2 , 0.03) bubbled with mixture of 95% O2 and 5% CO2 . After cleaning the tissue free of fat and other adhering tissues, the vessels were cut into 3 mm long rings, with special care to avoid damaging the endothelium. The preparations were mounted on a pair of stainless-steel hooks; one of each was fixed to an L-shaped rod inside the chamber and the other to an isometric force transducer (F-60, Narco Biosystems, Inc., TX, USA) connected to a polygraph (MK III-S, Narco Biosystems, Inc., TX, USA). Tissues were allowed to equilibrate under an optimum final force of 1.5 g for a period of 60 min in a water-jacketed tissue bath (10 ml) containing oxygenated physiologic salt solution at 37 ◦ C (final pH of 7.4), renewing the buffer every 15 min. After stabilization, the preparations were contracted twice with KCl (40 mM) and the last contraction was taken as the reference value for analysis. The presence of functional endothelium was tested by the relaxation response to 1 µM ACh in phenylephrine-precontracted rings. In all experiments, each ring was used only once.
Experimental protocol All of the following experiments were conducted on the aortic rings of both lead-treated and control rats. First. Contraction response was assessed by adding cumulative concentration of phenylephrine (0.1 nM to 10 µM). The concentration–contraction curves were studied for: (1) intact aortic rings; (2) endothelium-denuded aortic rings; (3) aortic rings incubated with l-NAME (100 µM) for 20 min; (4) aortic rings incubated with indomethacin (20 µM) for 20 min; and (5) aortic rings incubated for 20 min with l-NAME (100 µM) and indomethacin (20 µM). Endothelium-denuded rings were prepared by removing endothelium by rubbing the lumen of aortic ring with a cotton thread, and the removal was confirmed by the loss of relaxation to 1 µM acetylcholine.
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Fig. 1. Dose–response curves of the contraction of aortic rings of the control and 28-day lead-treated rats to phenylephrine. The panels show: (a) the effect of endothelium-denudation; (b) the effect of incubation with l-NAME (100 µM); (c) the effect of incubation with indomethacin (20 µM); and (d) the effect of incubation with l-NAME/indomethacin, on the phenylephrine contraction. ((hollow *) P < 0.05 compared to the value of the intact ring of control rats and (*) P < 0.05 compared to the value of the intact ring of lead-treated rats). (䊊) Intact aortic rings of control rats; (䊉) intact aortic rings of lead-treated rats; (䊐) endothelium-denuded aortic rings of control rats; (䊏) endothelium-denuded aortic rings of lead-treated rats; (䉫) aortic rings of control rats incubated with l-NAME (100 µM); (䉬) aortic rings of lead-treated rats incubated with l-NAME; () aortic rings of control rats incubated with indomethacin (20 µM); (䉱) aortic rings of lead-treated rats incubated with indomethacin; (×) aortic rings of control rats incubated with l-NAME/indomethacin; (+) aortic rings of lead-treated rats incubated with l-NAME/indomethacin).
Second. Relaxation response to acetylcholine, an endothelium-dependent vasorelaxant, was assessed by adding cumulative concentration of acetylcholine (10 nM to 10 µM) on the aortic rings precontracted with phenylephrine 1 µM. The similar concentration–relaxation curves were also done on: (1) the aortic rings incubated with indomethacin (20 µM for 20 min); (2) aortic rings incubated with l-NAME (100 µM for 20 min). Third. Relaxation response curves to sodium nitroprusside and glyceryl trinitrate (GTN) were also constructed by adding cumulative doses of sodium nitroprusside (1 nM to 10 µM) or GTN (1 nM to 10 µM) to phenylephrine-precontracted aortic rings in separate experiments.
Chemicals The following drugs were used: acetylcholine chloride, phenylephrine hydrochloride, l-NAME, indomethacin (purchased from Sigma Chemical Co., St. Louis, MO, USA); sodium nitroprusside, glyceryl trinitrate (GTN) and lead acetate (purchased form Merck Chemical Co., Germany). Chemicals were dissolved in distilled water, except for indomethacin, which is dissolved in absolute ethanol (0.2 µmol in 10 µl). Ethanol (10 µl) has no effect on the resting tension, phenylephrine-induced contraction, or any of the vasorelaxation responses.
Statistics and calculations Contraction to the second exposure of 40 mM KCl was taken as 100%, and the contractile responses to
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Table I Characteristics of the phenylephrine-induced contraction response and relaxation responses induced by acetylcholine, sodium nitroprusside and glyceryl trinitrate, in aortic rings of control and lead-treated rats Control group
Lead-treated group
Rmax
pEC50
Rmax
pEC50
Phenylephrine-induced vasoconstriction Intact Endothelium-denuded In presence of l-NAME In presence of indomethacin In presence of l-NAME and indomethacin
133.47 ± 5.55 172.89 ± 4.79a 126.41 ± 2.76 96.19 ± 5.32a 120.30 ± 2.60
7.31 ± 0.04 7.57 ± 0.05 7.50 ± 0.03 7.14 ± 0.06 7.05 ± 0.04
133.32 ± 8.69 145.19 ± 6.45 145.12 ± 3.17b 83.18 ± 6.90a 136.19 ± 3.74
7.20 ± 0.04 7.37 ± 0.06 8.06 ± 0.03c,d 6.95 ± 0.03a 7.51 ± 0.05a,b
Acetylcholine-induced vasorelaxation Intact In presence of indomethacin Sodium nitroprusside-induced vasorelaxation Glyceryl trinitrate-induced vasorelaxation
54.77 ± 3.33 55.45 ± 2.40 122.56 ± 11.30 135.69 ± 5.37
7.07 ± 0.04 7.31 ± 0.04a 7.66 ± 0.06 7.87 ± 0.05
55.28 ± 5.46 61.79 ± 0.93 103.76 ± 4.83 108.2 ± 2.93b
7.32 ± 0.03b 7.34 ± 0.03 7.73 ± 0.09 7.58 ± 0.09
Values are means ± SEM (n = 6–7 for both group). a P < 0.05 compared to the values of the intact aortic rings of the respective experiment. P < 0.05 compared to respective values of the control group. c P < 0.01 compared to respective values of the control group. d P < 0.01 compared to the values of the intact aortic rings of the respective experiment.
b
phenylephrine were expressed as a percentage of this contraction. Relaxation was expressed as the percentage of the initial precontraction with 1 µM phenylephrine. The concentration (EC50 ) of the agonist exhibiting 50% of the maximal response (Rmax ) was calculated with a curve-fitting software (GraphPad Prism version 3.00, Graph Pad Inc., San Diego, USA) and expressed as negative log molar (pEC50 ) value. All data are expressed as means ± SEM. The interaction of lead treatment and incubation with mentioned drugs (l-NAME and indomethacin) was studied by two-way analysis of variance (ANOVA) and were mentioned where no significant interaction exists. Difference between the values was tested for significance by unpaired Student’s t-test. Differences between more than two mean values were tested by one-way ANOVA, followed by Student– Newman–Keuls’ multiple comparisons as post hoc test. Differences were considered significant at P < 0.05.
RESULTS
Blood pressure changes There was a significant increase in systolic blood pressure of rats treated with 100 ppm lead acetate in respect to controls after 28 days (121.5±2.17 vs 95.8±3.13 mmHg, P < 0.05). At the time of sacrifice, the blood lead concentration of lead-treated group was significantly higher than that of control animals, reaching a mean value of 258.4 ± 23.2 µg/l, compared to controls of 114.5±31.4 µg/l (P < 0.05). There was no significant difference between the mean body weight of two groups, at the beginning and at the time of sacrifice.
Phenylephrine-induced contraction Phenylephrine, an α 1 -adrenoceptor agonist, caused a concentration-dependent contraction in the isolated aortic rings. There was no significant difference between the
pEC50 and Rmax of the control and lead-treated animals (Fig. 1 and Table I). Endothelium-denuded aortic rings of control rats showed greater Rmax compared to intact aortic rings of controls (P < 0.05), but did not show any difference in the pEC50 . There was no significant difference between the pEC50 and Rmax of the endothelium-denuded and endothelium-intact aortic rings of the lead-treated group [Fig. 1(a)]. Inhibition of NO synthesis by l-NAME (100 µM) had no significant effect on the pEC50 and Rmax of the aortic rings of the control rats. But on the aortic rings of lead-treated group, l-NAME incubation caused significant increase in pEC50 (P < 0.05) with no significant effect on Rmax compared to respective intact aortic rings. Also there was a significant difference between the pEC50 (P < 0.01) and Rmax (P < 0.05) of the aortic rings of the lead-treated animals incubated with l-NAME, compared to respective control ones [Fig. 1(b)]. There was no significant interaction between indomethacin incubation (20 µM) and lead treatment on Rmax . Indomethacin incubation caused a significant decrease in the Rmax and pEC50 of the aortic ring of lead-treated animals, compared to intact respective ones (P < 0.05). However there was significant decrease in Rmax with no significant change in pEC50 of indomethacin-incubated aortic ring of controls compared to intact ones. In indomethacin-incubated rings, the pEC50 and Rmax of the lead-treated group were less than those of control respectives, but these differences did not reach the statistical significance [Fig. 1(c)]. Except for a significant increase in pEC50 of lead-treated group (P < 0.05), there was no significant difference between Rmax and pEC50 of l-NAME/indomethacin incubated aortic rings of lead-treated and control rats compared to intact respective ones. The Rmax of the l-NAME/ indomethacin incubated lead-treated group were not significantly different from the incubated rings of the
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controls, but the pEC50 showed significant increase (P < 0.05) [Fig. 1(d)].
Acetylcholine-induced relaxation responses Phenylephrine (1 µM) caused submaximal contraction (about 90% of Rmax of the respective group) in aortic rings. Acetylcholine caused a concentration-dependent relaxation in the precontracted aortic rings. There was a mild but significant leftward shift in the relaxation curve of the aortic rings of lead-treated animals, compared to the controls (no difference in Rmax but significant increase in pEC50 (P < 0.05)) (Fig. 2 and Table I).
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Indomethacin incubation had no significant effect on the Rmax and pEC50 of the acetylcholine-induced relaxation in aortic rings of the lead-treated animals, but there was some mild left-shift in relaxation-curve, characterized by significant differences in lower doses of acetylcholine response. There was a significant increase in pEC50 (P < 0.05), but no significant difference was observed in the Rmax of the indomethacin-incubated aortic rings of control rats, compared to indomethacin-incubated rings of controls [Fig. 2(a)]. Incubation with l-NAME (100 µM for 20 min) caused significant attenuation of acetylcholine-induced relaxation of aortic rings. These suggest that relaxation response to acetylcholine in aortic ring is mediated mainly via NO, in agreement with previous study [25]. There was no significant interaction of lead treatment with l-NAME incubation on Rmax . There was no difference between the curves of acetylcholine-induced relaxation of l-NAME-incubated aortic rings of lead-treated with the respective rings of control rats. Due to significant attenuation of relaxation by l-NAME incubation, the pEC50 was not calculated for these groups [Fig. 2(b)].
Sodium nitroprusside-induced relaxation response Sodium nitroprusside induced a concentration-dependent relaxation in the aortic rings (Fig. 3). There was no significant difference in sodium nitroprusside-induced relaxation pEC50 and Rmax of the aortic rings between the lead-treated and control rats.
Glyceryl trinitrate-induced relaxation response GTN induced a concentration-dependent relaxation in the aortic rings, which showed significant decrease in Rmax in aortic rings of lead-treated compared to control rats (P < 0.05), with no significant change in pEC50 (Fig. 4).
Fig. 2. Acetylcholine-induced relaxation response curves of aortic rings of control and 28-lead-treated rats. Panel (a) shows the effect of incubation with indomethacin (20 µM); and (b) shows the effect of incubation with l-NAME (100 µM). () P < 0.05 compared to the value of the intact ring of control rats; (hollow *) P < 0.05 compared to the value of the intact ring of control rats; and (*) P < 0.05 compared to the value of the intact ring of lead-treated rats). (䊊) Intact aortic rings of control rats, (䊉) intact aortic rings of lead-treated rats; () aortic rings of control rats incubated with indomethacin (20 µM); (䉱) aortic rings of lead-treated rats incubated with indomethacin; (䉫) aortic rings of control rats incubated with l-NAME (100 µM); (䉬) aortic rings of lead-treated rats incubated with l-NAME).
Fig. 3. Relaxation response curve to sodium nitroprusside in control (䊊) and 28-day lead-treated (䊉) rats.
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Fig. 4. Relaxation response curve to glyceryl trinitrate in control (䊊) and 28-day lead-treated (䊉) rats; (*) P < 0.05 compared to the value of the aortic rings of control rats).
DISCUSSION In this study, we used rats, which were treated with 100 ppm lead acetate in their drinking water for 28 days. This amount of lead is considered as low level of exposure, similar to the level seen in the environment, although the result values are not directly comparable to those of humans. Previous studies have shown that exposure for longer duration (three and more months) to low level (100 ppm) of lead, not high level (5000 ppm), results in hypertension in rats [14, 26]. Our results show that subacute (28-day) exposure will also result in increased systolic blood pressure. In this study, we demonstrated that the contraction response to phenylephrine is not different between control and lead-treated groups. This is in agreement with previous study [26]. But, blocking NO synthesis caused a significant accentuation of phenylephrine-induced contraction only in aortic rings of lead-treated rats; while the endothelium-denudation made a significant accentuation only in controls. Two possible mechanism may explain the mentioned results. The first possible mechanism is that the endothelium may secrete vasoconstrictor substances in lead-treated rats. The candidates for this vasoconstrictor substance are endothelin, reactive oxygen species, and prostanoid vasoconstrictor [14, 16, 27]. When NO synthesis is inhibited, the vasoconstrictor will cause a left-shift in phenylephrine-induced contraction, and when endothelium is removed, the left-shift disappears. The second possible explanation is that another source of NO production may exist outside the endothelium, possibly in the smooth muscle cells, of the aortic rings of the lead-treated rats. Gonick et al. demonstrated the increased inducible NOS (iNOS) in the kidney cortex of lead-treated rats [15]. It may be possible that increased iNOS exists in smooth muscle of aorta of lead-treated rats, providing a source of NO production, which is blocked
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by l-NAME, but persists in endothelium-denuded rings. Further studies are needed to explore these explanations. In this study we also demonstrated that relaxation response to sodium nitroprusside of isolated aortic ring was the same in control and 28-day lead-treated rats. It may be thus concluded that smooth muscle relaxation pathway is not altered by 28-day exposure to lead. But acetylcholine-induced vasorelaxation was mildly shifted to left in lead-treated group. Purdy et al. showed that the responses to the aortic vasodilators (acetylcholine and sodium nitroprusside) are, however, unchanged in 3-month lead-treated rats [26]. The increased acetylcholine relaxation response may be due to either increased release of endothelial vasodilators or decreased release of vasoconstrictors. However, endothelial NOS is reported to be inhibited by lead in brain microvascular endothelium cells [28]; and EDHF is reported to decrease in lead hypertension in rats [29]. Also as will be discussed later, there is also evidence for the shift of prostanoids synthesis toward vasoconstrictors. Finally, this mild difference may be due to technical procedures or difference in pathophysiological mechanisms between the 28-day and 3-month lead-treated rats, which deserves further studies with more specific interventions. In the present study, we showed that indomethacinincubation caused attenuation of phenylephrine contraction in both control and lead-treated groups. While indomethacin also caused a left-shift in acetylcholine relaxation curves of controls, it exerted no effect on lead-treated group. These results provided evidence for that the vasoconstrictor prostanoids might contribute to vascular responsiveness, in general. The lack of effect of indomethacin on relaxation in lead-treated group, is probably due to either simultaneous increase in the vasoconstrictor prostanoids, or a decrease in vasodilator prostanoids, leading to a balance. Blocking both NO and cyclooxygenase pathways caused a right shift in the phenylephrine-induced contraction of the aortic rings of lead-treated rats; but to a lesser degree than the right-shift caused by l-NAME alone. This provides further evidence for the altered production of the prostanoids during lead hypertension. Previous studies suggested that lead is able to increase the free arachidonic acid in the cell [20, 21]. Lead is also reported to stimulate arachidonic acid release from cultured bovine retinal endothelial cells [30]. The activation of arachidonic acid metabolism will result in the release of cyclooxygenase metabolites, known to act as vasoconstrictor [31]. More studies, using specific inhibitors of the cyclooxygenase pathways and measuring metabolite are needed to explore the changes of cyclooxygenase metabolites in lead hypertension. As we showed in the present study, relaxation response to GTN is impaired in aortic rings of lead-treated rats, but the relaxation response to sodium nitroprusside is not different between the two groups. GTN releases either nitrite ion via bioactivation by gluthathione S-transferase, or NO by unknown enzymatic activation [32]. Thus the impaired relaxation to GTN is possibly due to decreased
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activation. Lead is reported to bind to sulfhydryl group of enzyme through body [33], and may interfere with GTN bioactivation. This effect of lead may explain the impaired responsiveness to GTN in lead-treated group. GTN bioactivation is suggested to be inhibited by NO [34]. If we assume that the increased NO production exists in the aortic smooth muscle of lead-treated rats, we can explain the decreased GTN bioactivation. Until further studies explore these possibilities, these are as a matter of speculations. In conclusion, the possibility of altered cyclooxygenase pathway in lead hypertension exists. There is also evidence for the role of endothelium in lead hypertension, possibly by producing vasoconstrictors. The exact nature of these vasoconstrictors needs further detailed studies. ACKNOWLEDGEMENT This work is supported in part by the research deputy of Tehran University of Medical Sciences. REFERENCES 1. Elias RW. Lead exposures in the human environment. In: Maffey KR, ed. Dietary and environmental lead. New York: Elsevier, 1985: 79–107. 2. Beevers DG, Erskine E, Robertson M, Hawthorne VM. Blood lead and hypertension. Lancet 1976; 2: 1–3. 3. Harlan WR. The relationship of blood levels to blood pressure in U.S. population. Environ Health Perspect 1988; 78: 9–13. 4. Victery W. Symposium on lead blood pressure relationships. Environ Health Perspect 1988; 78: 139–55. 5. Cheng Y, Schwartz J, Sparrow D, Aro A, Weiss ST, Hu H. Bone lead and blood lead levels in relation to baseline blood pressure and the prospective development of hypertension: the Normative Aging Study. Am J Epidemiol 2001; 153: 164–71. 6. Goodfriend TL, Ball DL, Elliot ME. Lead increase aldosterone production by rat adrenal cells. Hypertension 1995; 25: 785–9. 7. Piccinini F, Favalli L, Chiari MC. Experimental investigations on the contraction induced by lead in arterial smooth muscle. Toxicology 1977; 8: 43–51. 8. Carmignani M, Boscolo P, Poma A, Volpe AR. Kininergic system and arterial hypertension following chronic exposure to inorganic lead. Immunopharmacology 1999; 44: 105–10. 9. Carmignani M, Volpe AR, Boscolo P, Qiao N, Di Gioacchino M, Grilli A, Felaco M. Catecholamine and nitric oxide systems as targets of chronic lead exposure in inducing selective functional impairment. Life Sci 2000; 68: 401–15. 10. Chang HR, Chen SS, Tsao DA, Cheng JT, Ho CK, Yu HS. Reduced vascular beta-adrenergic receptors and catecholamine response in rats with lead induced hypertension. Arch Toxicol 1997; 71: 778–81. 11. Markovac J, Goldstein GW. Lead activates protein kinase C in immature rat brain microvessels. Toxicol Appl Pharmacol 1988; 96: 14–23. 12. Watts SW, Chai S, Webb CR. Lead acetate-induced contraction in rabbit mesenteric artery: interaction with calcium and protein kinase C. Toxicology 1995; 99: 55–65. 13. Taddei S, Virdis A, Ghiadoni L, Salvetti G, Salvetti A. Endothelial dysfunction in hypertension. J Nephrol 2000; 13: 205–10.
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