Chronic lead exposure through pollution or occupational hazard may cause various health problems in humans, including cardiovascular disorders such as hypertension (1-4), but some human studies show no correlation between lead exposure and hypertension (5,6). The influence of lead exposure on the cardiovascular system remains unclear. In hypertensive rats exposed to lead, endothelial function might be impaired, since some endothelial factors, such as increased endothelin-3 or decreased plasma cyclic GMP (as reflection of nitric oxide, NO) have been reported (7). The endothelial monolayer cell is a source of vasodilator mediators, including endothelium-derived relaxing (EDRF; most likely NO), prostacyclin, and an as yet unidentified endothelium-derived hyperpolarizing factor (EDHF) (8-10). We investigated the effect of chronic lead exposure on endothelium-dependent responses by detecting mechanical, electrophysiological, and tissue levels of cyclic GMP changes in rat arteries.
MATERIALS AND METHODS
Male Wistar rats aged (7 weeks, and weighing 260.1 ± 5.4 g (Kyudo, Kumamoto, Japan) were assigned randomly to two different protocols. Animals in lead-exposed groups received drinking water containing lead acetate (50 ppm, Wako Pure chemicals, Osaka, Japan); controls received distilled water. Both groups were fed standard diet (CE-2, Nihon CLEA, Tokyo, Japan) and were housed for 1 or 3 months in a chronic care facility before being killed. Indirect systolic blood pressure (SBP) and heart rate (HR) were measured by the tail cuff method in unanesthetized animals (MK-1000, Muromachi Kikai, Tokyo, Japan).
The rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally (i.p.) (Abbott Laboratories, North Chicago, IL, U.S.A.). The main branches of the superior mesenteric artery and thoracic aorta were excised and placed in modified Krebs-Ringer bicarbonate solution (4°C) [(in mM): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, Ca-EDTA 0.026, and glucose 11.1, aerated with 95% O2/5% CO2 (control solution)]. Blood vessels were cleaned of surrounding tissues and cut into rings 4-5 mm long. In some rings, the endothelium was removed mechanically by inserting the tip of a forceps into the lumen of the blood vessel and rolling the rings back and forth on a paper towel wetted with control solution. Whole blood in each animal was stored at -80°C for determination of blood lead. Plasma was separated from the red blood cells by centrifugation at 2,500 rpm for 20 min at 4°C and stored at -80°C for determination of δ-amino-levulinic acid (δ-ALA). The concentration of blood lead was measured by flameless atomic absorption spectrometer (Z-9000, Hitachi, Tokyo, Japan) after whole blood was diluted 10-fold with 0.1 N HNO3 containing 1% Triton X-100. The plasma δ-ALA was determined by fluorimetric high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) (11). All experiments were performed in the presence of indomethacin (10-5M) to inhibit the formation of vasoactive prostanoids.
Organ chamber studies
The arterial and mesenteric arterial rings were suspended between two stirrups in organ chambers (60 ml) filled with control solution (gassed with 95% O2/5% CO2 and maintained at 37°C). One of the stirrups was anchored to the inside of the organ chamber; and the other was connected to a force transducer (TB-611T, Nihon Kohden, Tokyo, Japan) to record changes in isometric tension. In all experiments, the rings were stretched in a stepwise manner to the optimal point of their length-tension curve (resting tensions of rings from the two regions; 1.0 g, which induced maximal contraction with 20 mM KCl) and were allowed to equilibrate for 45 min. The preparations were contracted with phenylephrine (PE 3 × 10-7M for the aorta, and 10-6M for the mesenteric artery, which produced ≈80% of the maximal contraction in preliminary experiments), and a concentration-response curve to acetylcholine (Ach) (10-8-10-6M) was obtained in rings incubated either with or without NG-nitro-L-arginine methyl ester (L-NAME 10-4M).
Rings of the superior mesenteric artery were cut open along their longitudinal axis and pinned on the bottom of an organ chamber (2-ml capacity) with the endothelial side facing up. The arteries were superfused with control solution (37°C, 3 ml/min) for 45 min before the recording was started. The membrane potentials of arterial smooth muscle cells (SMC) were measured with glass microelectrodes (FHC, ME, U.S.A.) filled with 3 M KCl solution (tip resistance, 40-80 MΩ). The electrical signal was recorded from the intimal side of the vessels (12), and was amplified by a recording amplifier (MEZ-7101, Nihon Kohden). The membrane potential was monitored continuously on an oscilloscope (VC-10, Nihon Kohden). Experiments were performed with addition of L-NAME (10-4M).
Tissue content of cyclic GMP
A radioimmunoassay (RIA) technique was used to determine the tissue content cyclic GMP. The rings were frozen quickly by liquid nitrogen and stored -80°C. At the time of assay, rings were homogenized in 1 ml 6% trichloroacetic acid at 4°C and centrifuged three times at 3,000 rpm for 10 min. The supernatant was removed from each sample, placed in a glass tube, and extracted three times with 6 ml water-saturated ethyl ether. The samples were then assayed with a cyclic GMP RIA kit (Yamasa Shoyu, Chiba, Japan). Levels of cyclic GMP in tissues were expressed in femtomoles per milligram of wet weight of tissue.
The main drugs used were PE, Ach, indomethacin, and L-NAME (all from Sigma, St. Louis, MO, U.S.A.), and lead acetate (Wako Pure Chemicals). Indomethacin was dissolved in distilled water together with equimolar concentration of Na2CO3. The other drugs were prepared with distilled water.
The data are mean ± SEM and were analyzed by Student's t test (paired or unpaired) or analysis of variance followed by Fisher's protected least significant difference test; n represents the number of animals examined, and <0.05 was regarded as statistically significant.
Table 1 summarizes the data on body weight, SBP, HR, blood lead and plasma δ-ALA in lead-exposed (1 or 3 months) and age-matched control rats. There was no statistical significance in body weight, HR, or SBP between lead-exposed and age-matched control groups. In both 1- and 3-month lead-exposed rats, the blood lead levels were more greatly increased than those in age-matched control (p < 0.05). Plasma levels of δ-ALA were significantly more increased in 1-month lead-exposed animals than in control animals (p < 0.05).
Organ chamber studies
Thoracic aorta. In rings with endothelium, there was no significant difference in contraction to PE (3 × 10-7M) between 1-month lead-exposed and age-matched control groups either with or without addition of L-NAME (Fig. 1A and B). In rings from 3-month lead-exposed and age-matched controls rats, the contraction to PE was markedly increased in those from the exposed groups both with and without addition of L-NAME (p < 0.05) (Fig. 1A and B). In tissue without endothelium, there was no significant difference in contraction to PE between the two groups, regardless of the duration of the exposure (Fig. 1C).
Ach (10-8-10-6M) caused concentration-dependent relaxation in rings with endothelium from both lead-exposed and control rats. There was no significant difference in relaxations between lead-exposed and age-matched control groups, regardless of the duration of the exposure (Fig. 1A).
In rings with endothelium plus L-NAME or in rings without endothelium, the relaxations to Ach were almost abolished (<3% of maximum relaxation as a percentage of contraction to PE), and no significant difference was observed between those from lead-exposed and age-matched control animals (Fig. 1B and C).
Mesenteric artery. In rings with endothelium, there was no significant difference in contraction to PE (10-6M) between lead-exposed and control groups, regardless of the duration of exposure (Fig. 2A). In the presence of L-NAME, the contraction to PE was markedly increased in rings from the lead-exposed groups than in those from controls, regardless of duration of exposure (p < 0.05) (Fig. 2B).
In the absence of L-NAME, Ach (10-8-10-6M) produced concentration-dependent relaxation in rings with endothelium contracted with PE. There was no significant difference in relaxation between lead-exposed and control groups regardless of duration of exposure (Fig. 2A).
With addition of L-NAME, concentration-dependent relaxation to Ach was significantly decreased as compared to relaxation in the absence of L-NAME (p < 0.05) (Fig. 2A and B). The relaxation to Ach (10-7 and 10-6M) was markedly decreased in rings from the 3-month lead-exposed group in the presence of L-NAME as compared with those from age-matched control rats (Fig. 2B). Relaxation to Ach (10-6M) was also significantly decreased in rings from the 3-month lead-exposed groups as compared with those from the 1-month lead-exposed group (p < 0.05) (Fig. 2B).
There was no significant difference in resting membrane potentials of the arteries among the four protocol groups (Table 2). In the presence of L-NAME, Ach (10-8-10-6M in cumulative application) produced concentration-dependent hyperpolarization of the SMC (Fig. 3A; typical traces). There was no significant difference in hyperpolarizations between tissues from 1-month lead-exposed and those from control rats, whereas in tissues from those housed for 3 months hyperpolarization to Ach (10-6M) was significantly decreased in lead-exposed animals (p < 0.05) (Fig. 3B).
Production of the cyclic GMP
There was no significant difference in the basal content of cyclic GMP in the aortas with endothelium between lead-exposed and age-matched controls regardless of duration of lead exposure (Fig. 4).
Our experiments confirm that L-NAME, an inhibitor of NO synthase, completely blocks endothelium-dependent relaxation to Ach in the rat aorta and, in part, in the mesenteric artery (13). Our results further demonstrate that both endothelium-dependent hyperpolarization and L-NAME-resistant relaxation decrease with chronic lead exposure in the mesenteric artery, whereas L-NAME-sensitive relaxation does not decrease in either aorta or mesenteric artery. L-NAME-resistant relaxation could be best explained by EDHF (8-10). Although EDHF has not yet been identified, EDHF increases the K+, conductance of the vascular SMC membrane by opening K+ channels (8,10,14). These results imply that inhibition of EDHF may account, at least in part, for the impaired L-NAME-resistant relaxation in lead-treated animals.
One of the possible mechanisms of the impairment of endothelium-dependent hyperpolarization by chronic lead exposure includes cytotoxicity of lead to the endothelium. If chronic lead exposure inhibits the endothelial cell function, as by vasorelaxation, through selective membrane receptors of EDRF exocytosis or calcium-calmodulin complex (EDRF product pathway) (15), the vasodilation response or cyclic GMP in smooth muscle should be decreased. However, no significant decrease was induced either in relaxation to Ach or in cyclic GMP levels in the aorta by chronic lead treatment.
Second, decreased release of NO might explain decrease in the electrical response, since a previous study showed that cyclic GMP in plasma and urine concentration (as a reflection of NO) decreased in rats with chronic lead exposure (7). NO can cause membrane hyperpolarization in rat mesenteric arteries (16). However, in the present experiments, reduced release and/or production of EDRF/NO was not likely to be responsible for the decreased hyperpolarization associated with lead exposure, since L-NAME-sensitive relaxation and plasma levels of cyclic GMP were not decreased in the lead-exposed groups.
Third, lead might inhibit production or release of EDHF. One of the endothelium-derived cytochrome P450 metabolites of arachidonic acid, 5,6-epoxy-eicosatrienoic acid, displays the characteristics of EDHF (17), since cytochrome P450 inhibitors (e.g., clotorimazole, metyrapone, and SKF525a) abolish the EDHF-mediated relaxation elicited by Ach (18). Exposure to lead causes a disturbance of cytochrome P450 in rat liver (19). In rat liver microsomes, treatment with ionic lead decreases total cytochrome P450 to 60-80% of control and inhibits the cytochrome P4501A2 expression (19). Together these findings suggest that production of EDHF, possibly a cytochrome P450-metabolite, is inhibited by lead exposure. Our results also indicate that > 1-month lead exposure is required to cause a decrease in the Ach-induced hyperpolarization.
In addition to affecting endothelial function, chronic lead exposure may also affect SMC function; i.e., sensitivity to EDHF of the smooth muscle might be changed in the lead-exposed animals. Because EDHF is believed to cause hyperpolarization by activating either the Na-K pump (9) or potassium channels (8) in the vascular smooth muscle, lead might affect these targets. Such a hypothesis is supported by observations that lead has the ability to inhibit sodium/potassium-activated adenosine triphosphatase (Na-K-ATPase) in hog cerebral cortex (20) and that lead inhibits voltage-dependent potassium channels in rat brain (21) and voltage-activated calcium channels in rat dorsal root ganglion neuron (22). However, whether these direct effects of lead ion can explain decreased electrical response in chronic lead-exposed vascular smooth muscle is not known.
In earlier observations in lead-treated rats, not only a positive correlation between blood lead level and systolic BP (7,23) but a decrease in plasma cyclic GMP was observed (7). In our study, despite increasing blood lead levels of lead-exposed rats (mean blood lead level was 13.3 and 10.8 μg/dl for 1- and 3-month groups, respectively), SBP did not increase significantly and basal level of cyclic GMP in the aorta was not changed by lead exposure. This discrepancy might be due to the concentration of lead used, since in the former study 100 ppm lead resulted in a blood lead concentration of 29.4 ± 4.1 μg/dl in 3-month lead-exposed rats (7). These findings also suggest that the endothelium is involved in lead-induced hypertension through a decrease in NO, whereas EDHF does not play a major role in such hypertension.
The present study also confirms that aortic rings from the low-lead-treated rats exhibit increased force-generating ability to PE, as does rat tail artery (24). The mechanisms of increased reactivity associated with chronic lead treatment are not known. Endothelium-derived contracting factor (25) probably is not responsible for the phenomenon, since all experiments were performed in the presence of indomethacin. The increased vascular contractility might be due to a direct action of lead on SMC, since the tissue content of radio-active calcium is increased after lead exposure (23) and since lead induces endothelium-independent and calcium-dependent contraction with protein kinase C activation in rabbit mesenteric artery (26). In contrast, lead does not contract the endothelium-free ventral aorta of the shark (27).
Our results indicate that chronic lead exposure might inhibit endothelium-dependent hyperpolarization and L-NAME-sensitive relaxations in rat mesenteric artery without affecting EDRF/NO-induced relaxation, suggesting that chronic lead exposure might be an inhibitor of EDHF in rat mesenteric artery.
Acknowledgment: We thank Dr. Masayoshi Ichiba and Kaori Takahashi for technical help.
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