Lead acetate deteriorates the improvement effect of L-arginine and tetrahydrobiopterin on endothelin-1 receptors activity in rat aorta

: Endothelin-1 (ET-1) is a potent vasoconstrictor hormone that has been identified as an important factor responsible for the development of cardiovascular dysfunctions. ET-1 exerts its vasoconstrictor activity through two pharmacologically distinct receptors, ETA and ETB that are found in vascular smooth muscle cells (VSMCs) and the vasodilator activity through an ETB receptor located on endothelial cells. This study aimed to show the impact of 1µM L-arginine (LA), 100µM tetrahydrobiopterin (BH 4 ), and their combined effect on ET-1 activity in both lead-treated and lead-untreated rat aortic rings. This means, investigating how endothelial dysfunction reverses the role of nitric oxide precursor and cofactor. In this study, Rat aortic rings have been pre-incubated with BH 4 , LA and their combination. Subsequently, the aortic rings were pre-incubated with 200µM N-Nitro-L-arginine methyl ester (L-NAME) and 0.5µM BQ-123. Then, the vascular response to cumulative doses of rat ET-1 was analyzed in each of the above-mentioned groups (LA, BH 4 , LA & BH 4 , L-NAME, BQ-123), in the presence and absence of lead acetate 1µM Pb (C 2 H 3 O 2 ) 2 . ET-1 efficacy and potency were significantly decreased in the presence of LA, BH 4 , and LA and BH 4 combination in the untreated group, while it significantly increased in the presence of lead. In the second trial of experiments ET-1 efficacy markedly decreased in BQ-123-incubated cells in both lead-treated and untreated aortic rings. In the presence of lead, the efficacy of ET-1 was raised with the use of L-NAME. In conclusion, LA and BH 4 can be considered pharmacological agents to alter the potency of ET-1-induced vasoconstriction and concomitantly lower blood pressure.


Introduction:
Endothelin is a prominent vasoconstrictor that is synthesized and released by vascular endothelial cells, that was first found by Yanagisawa, Inoue 1, 2 ET is known to have a role in leading to many cardiovascular diseases 3 . Jankowich et al 4 described the association of ET-1 with hypertension, cardiac remodeling, aging, chronic kidney disease, pulmonary hypertension and diabetes 5 . It has been demonstrated that endothelin-1 (ET-1) induces pulmonary vasoconstriction through the activation of Ras homolog family member A 6 .
Until now, three isoforms of ET have been identified, namely ET-1, ET-2, and ET-3 7 . ET-1 is the most prominent type of ET and it is produced mainly by endothelial cells, but also by several other cell types in the cardiovascular system. ET-1 exerts its vasoconstrictor effect by activating the G protein ETA and ETB receptors that can be found in (VSMCs). ET-1 exerts the vascular relaxation effect through activation of ETB receptor on endothelial cells. Primary, ET-1 engages endothelial nitric oxide synthase (eNOS) that increases nitric oxide (NO) bioavailability 8 .
Under normal conditions, there is a harmony between the free radical and the antioxidant system in the body, which are both essential to maintain vascular functions 9 . However, during endothelial dysfunctions the production of ET increases, while NO quantity decreases, which is referred to as "an imbalance between vasodilating and vasoconstricting substances produced by (or acting on) endothelial cells" 10,11 . Lead is a common environmental contaminant that can produce several acute and chronic diseases. According to various populational studies, there is a significant association between lead exposure and the risk of development of cardiovascular diseases particularly hypertension 12 . It has been published that the exposure of endothelial cells to lead increases the vascular reactivity to phenylephrine (PE) 13 . However, dropping the vascular reactivity has been identified in different studies 14 . Lead exposure is involved in cardiovascular disease in various mechanisms, according to certain studies, which are stimulating oxidative stress, restrictive NO accessibility, rising the secretion of endothelin, modifying the reninangiotensin system, disturbing vascular smooth muscle Ca2+ signaling, falling endotheliumdependent vasorelaxation, and changing the vascular response to vasoactive agonists. Furthermore, lead exposure has been linked to different cellular disruptions in the heart such as injury in endothelial cells, delaying endothelial repair, restricting endothelial cell growth, suppressing the production of proteoglycan, and accelerating vascular smooth and muscle cell proliferation 15 .
Nitric oxide production needs two main substrates: LA, which acts as a NO precursor, and a cofactor named BH4, which is essential for eNOS dimerization (coupling) 3 . BH4 is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases, ether lipid oxidase, and the three nitric oxide synthase (NOS) isoenzymes. Also, BH4 acts as an antioxidant and neutralizes RNS and ROS 16 . According to our knowledge, no published evidence is yet available to explain how BH4 and LA combination affects ET-1 receptors activity in the intact and lead-treated aortic rings. Therefore, the goal of this study is to see how BH4, LA and their combination affect ET-1 efficacy in lead-treated aortic rings. 25 Male albino rats weighing 200-300 gm were divided into 5 cages and kept in prescribed conditions from February/ 2019 to September/ 2020, according to the laboratory animal care guide.

Materials and Methods: Animals and aortic rings preparation
Intraperitoneal injection of a mixture of ketamine: xylasine 90 mg/kg: 10 mg/kg, has been used to anesthetize rats 17 . The thoracic aorta was immediately extracted, and cleaned from adipose tissues in a cold Krebs solution, then cut into four segments with lengths 2-2.5mm.
The aortic rings were suspended in the organ bath (Automatic organ Bath-Pan lab, Harvard apparatus USA, AD Instrument Power lab 8/35-Australia) that is filled with 10 ml of Krebs solution (mM/L NaCl,119; MgSO4, 1.2; CaCl2, 1.5; NaHCO3; KCl, 4.7; KH2PO4 1.2; glucose 11.1.) that kept up at 37 o C and aerated with a mixture of 95% O2 and 5% CO2. In this, study each aorta was cut into four segments and each sample size represent one segment.
The aortic rings were allowed to equilibrate for 60 to 90 minutes, through that time the Krebs solution was replaced every 15 minutes. The function of the aortic ring was determined through the use of (60mM) KCl, after the response had reached a plateau of the maximum contraction by KCl, aortic rings were washed with a fresh medium and reequilibrated for at least 30 minutes before adding any vasoactive substances. In denuded aortic rings, the endothelium was removed. Then each ring was contracted by 1µM PE and relaxed by 10µM of acetylcholine (ACh) as denudation assessment. ACh is used for endothelial function assessment because an artery with a healthy endothelium responds to acetylcholine by releasing nitric oxide that translates into vasodilation. While, In the presence of artery denudation from the endothelium or if the action of the nitric-oxide synthase enzyme is blocked, the artery responds to acetylcholine with vasoconstriction due to the stimulation of smooth muscle muscarinic receptors not counteracted by the nitric oxide of endothelial origin

Experimental protocol
Prepared aortic rings used to evaluate the effect of lead acetate on vascular rings to ET-1 response in two trials. Cumulative concentration doses of ET-1 (5*10 -11 -10 -7 ) were applied in both Pb (C2H3O2)2(1µM) -treated and untreated rings. 1µM of Pb (C2H3O2)2 was prepared by adding 3.3 mg of Pb (C2H3O2)2 to 10 mL of distilled water, which produce 1mM, and diluted to 1 µM. To assess the influence of endothelium on the response to ET-1, cumulative doses of ET-1 were used in denuded and intact aortic rings. (C2H3O2)2. The intact aortic rings were incubated for 45 minutes with the mentioned substances before the generation of the ET-1 concentration-response curves. Then, to find out the role of Pb (C2H3O2)2 on vascular reactivity in response to ET-1, other aortic rings were incubated with 1µM of Pb (C2H3O2)2 for 45 minutes before using LA, BH4, and their combination.

Experiment 2:
To evaluate the effect of different vasoactive substances in ET-1 elicited vascular contraction, aortic segments were incubated with 200µM L-NAME, and 0.5µM of BQ-123. These drugs were added 20 minutes before the concentrated doses of ET-1 were applied, in the absence and presence of 1µM Pb (C2H3O2)2 . Finally, 10µM ACh was added to lead-treated groups to check the endothelial dysfunction (ED) induced by lead acetate.

Statistical analysis
The contractions stimulated by 60mM KCl were observed and recorded as percentages (%KCl). By means of Graph Pad Prism, the concentrationresponse curves of ET-1 were fitted nonlinearly. To differentiate the effect materials in both control and lead-treated groups of aortic segments, all results were expressed as the differences area under curves (dAUC). Means ± standard error of the mean (SEM) were used for the expression of all data. Independent Students t-test was applied to compare dAUC and potency difference (pD2) between groups. To investigate the distinction between the study groups and control, a two-way analysis of variance (ANOVA) was used, followed by Sidak multiple comparison tests for each mean comparison, Dunnett-test was for comparing the studied groups with the control, and Tukey-test as the pD2 comparison between groups. P-Value ≤ 0.05.

Results
The prepared aortic rings were exposed to cumulative doses of ET-1 (5*10 -11 -10 -7 M). At the end of each experiment on lead acetate-treated aortic rings, impairment of the endothelial has occurred resulting in a lower response to ACh.
The effect of LA, BH4, and their combination on vascular response to ET-1.
1µM of L-arginine was applied to analyze the effect of NO precursor on vascular reaction to ET-1. LA significantly reduced the efficacy and potency of ET-1 in untreated groups Fig.1A. While, in the presence of Pb (C2H3O2)2, LA noticeably boosted the vasoconstriction response to ET-1 in table 1. According to the dAUC graph Fig. 1A, a significant alteration in vascular response to ET-1 was observed with the presence of lead during LA pre-incubation. The studied groups were compared with each other (ANOVA was applied with Tukey test). *, **, *** represents statistical differences at P ≤ 0.05, P ≤ 0.01 P ≤ 0.001versus the corresponding control group, ## Significant differences between groups vs LA group at p ≤ 0.01, and ¤¤¤ Significant differences between groups vs BH4 group at p ≤ 0.001. 100µM of BH4 was applied to determine the influence of lead acetate on vascular actions and the possible role of the NOS cofactor. Fig. 1B showed significant reductions in both efficacy and potency in the absence of lead acetate, while in the lead acetate treated group the concentration-response curve shifted to the left with significant rises in ET-1 potency and maximum response. The dAUC shown in Fig. 1B represented the highly significant change in the presence of lead acetate that can alter vascular behavior.

Open
The results revealed that aortic rings preincubation with LA and BH4 combination causes a significant decrease in the maximum response, as well as a highly significant decrease in potency to ET-1 in the absence of lead acetate in comparison to control, LA and BH4 groups. While no statistical change was observed in ET-1 potency and efficacy in the lead acetate-treated group as shown in Table. 1, the dAUC showed a highly significant change in the vascular response to ET-1, under exposure to both LA and BH4 and in the existence of LA Fig. 1C.

Table 2. The maximum response (Emax) and the potency difference (pD2) to ET-1 in the presence and absence of Pb (C2H3O2)2 in rat aortic rings
The studied groups were compared with each other (ANOVA was applied with Dunnet test). * Significant differences between groups vs control group at p ≤ 0.05, ** Significant differences between groups vs control group at p ≤ 0.01, *** Significant differences between groups vs control group at p ≤ 0.001.

Role of endothelial NOS inhibitor on vascular responsiveness to ET-1.
200µM of L-NAME has been performed to explore the effects of lead acetate on ET-1 vascular actions and the probable impact of eNOS. The result showed that inhibiting eNOS noticeably increases the vasoconstriction response of ET-1 with no significant changes in potency and efficacy in the lead-untreated group. On the contrary, in the presence of Pb (C2H3O2)2, L-NAME exhibited a highly significant rising effect of vascular ET-1 efficacy, although, ET-1 potency remained significantly unchanged. According to the dAUC graph shown in Fig. 2A, L-NAME exerts a higher impact in the presence of lead acetate.

Role of ETA receptor antagonist on vascular responsiveness to ET-1.
0.5µM of BQ-123 was used to investigate the role of lead acetate on ET-1 vascular actions and the role of the ETA receptor. The result, in the presence and absence of AC, showed that BQ-123 significantly reduced the maximum response and potency in Table. 2. The dAUC graph in Fig. 3 showed the great effect of lead on the vascular response to ET-1 as compared with the control group.  The role of endothelium layer on vascular responsiveness to ET-1.
To determine the influence of the endothelial layer on the vascular response of aortic rings to ET-1, cumulative doses of ET-1 were applied to intact and denuded rat aortic rings. ET-1 reactivity following endothelial damage (denudation) did not undergo alterations of the maximum response in both lead-treated and untreated groups, whereas the potency significantly rises in the lead-treated group but remain unchanged in the untreated group as shown in Table. 2. The dAUC values demonstrate a similar attenuating effect in the presence and absence of lead acetate as shown in Fig. 4.

Discussion:
The most important finding of this study revealed that acute lead exposure to isolated rat aortic rings deteriorates the improvement effect of LA and BH4. From the present results, we found that lead acetate reduces ET-1 vascular reactivity which is most probably associated with oxidative stress and hence ED. Although, the exact mechanism by which lead acetate induces ED is not fully understood, the rational reason for our finding may be related to the elevation of hydrogen peroxide and subsequent alteration of K + channel 18 . As demonstrated by this study, pre-incubation of LA, which acts as a substrate for NO production, directly influences NO levels as observed by the decreases of the vascular response to ET-1 in the absence of lead acetate. NO represents a potent vasodilator of the cyclic guanosine monophosphate (cGMP) pathway 19 . In addition, basal NO production can modulate ET-1 activity through endothelial ET-B receptor activity 20 . Interestingly, according to the dAUC shown in enhances the ROS production in the presence of lead. ROS may uncouple the eNOS-catalyzed reduction of molecular oxygen from the oxidation of L-arginine, resulting in the paradoxical production of the ROS superoxide anion instead of the reducing NO. Alternatively, ROS may react with NO directly, reducing its bioavailability 21 . Furthermore, the result suggests that LA in lead acetate-treated aortic rings may deteriorate the vascular reactivity of ET-1 as recorded by the study of Rosano 22 , that concluded that the rise of ROS generation increases ET-1 gene expression 23 . ROS may uncouple the eNOS-catalyzed reduction of molecular oxygen from the oxidation of L-arginine, resulting in the paradoxical production of the ROS superoxide anion instead of the reducing NO. Alternatively, ROS may react with NO directly, reducing its bioavailability In the same manner, BH4 markedly decreases the vascular responses to ET-1 by enhancing NO bioavailability 3,24 , whereas BH4 increases the vascular reactivity to ET-1 in the presence of lead. Reports associated with the impact of BH4 on ET-1 potency are very limited. Also, according to our knowledge, this is the first study that provides evidence that BH4 enhances vascular reactivity in lead-treated and untreated isolated aortic rings. It is well established that lead induces the release of free radicles that potentially oxidize BH4 to BH2; indeed, ONOOproduced by lead acetate can oxidize BH4 spontaneously within minutes at physiologically relevant Concentration 25 .
The vasoconstrictor effect of ET-1 is markedly reduced in lead untreated aortic rings in the presence of LA and BH4 combinations. However, the same result was not obtained in the presence of lead. The present finding is consistent with Milewski study, which recorded that LA and BH4 are the primary substrates in the generation of NO 26 . Moreover, bonding both BH4 to eNOS rises the enzymatic turnover of LA 27 and enhances the stability of the active eNOS dimer 28 . In addition, another study found that pre-incubation of both LA and BH4 stimulated a significant increase in AChinduced relaxation 29 .
However, in lead-treated aortic rings, LA and BH4 in combination significantly increase the vascular responsiveness to ET-1, as previously described, due to two logical reasons. Firstly, it is well known that lead-induced ROS production decreases NO bioavailability 24 , hence ET-1 vasoconstrictor activity increases. Secondly, the oxidation of BH4 to BH2 exerts allosteric action to stabilize the active dimeric form of eNOS 30 .
Several studies demonstrated that NO is an important relaxing factor in conductance arteries 31 . Therefore, in order to investigate the influence of NO on the vascular response to ET-1 in isolated aortic rings, we applied the non-specific NOS inhibitor L-NAME. The obtained results indicated that L-NAME increases the reactivity of ET-1 in both lead-treated and untreated groups although its impact is greater in the presence of lead, as illustrated in dAUC in Fig. 2. L-NAME reduces NO availability and it is well established that NO reduces the vasoconstrictor activity of ET-1 32 , hence NO reduction by L-NAME in isolated aorta potentiates the ET-1 vasoconstriction. On the other hand, the enhancement of ET-1 potency by lead is mainly due to the overproduction of ROS 24 . Additionally, D'Angelo recorded that an increase in ROS may affect downstream ET receptor activation 33 .
It is not surprising that blocking the ETA receptor by BQ-123 in the absence and presence of lead significantly reduced the vascular sensitivity to ET-1. It has been confirmed that vasoconstriction activity of ET-1 generally depends on this receptor subtype 34 and ETA antagonists are able to fully reverse an established ET-1 constrictor response 35 .
In the present study, the removal of the endothelial layer potentiated the vasoreactivity of ET-1, this verifies that the endothelial-independent vasoconstriction of the peptide is through ETA and ETB on VSMCs. This finding is supported by the study of Schiffrin 36 , that demonstrated that ET-1 exerts endothelial-independent vasoconstriction. In the impaired endothelial layer induced by lead, the efficiency of ET-1 was increased possibly through the induction of oxidative stress that further potentiated ET-1 maximum response by removing NO availability and eNOS activity 37 .

Conclusion:
In conclusion, our results demonstrated for the first time that pre-incubation of LA and BH4 alone and in their combination reverse the potency of ET-1-induced vasoconstriction. These effects are markedly changed by lead acetate pre-incubation to ET-1. The results also indicated that the potency of ET-1 partially depends on NO and prostacyclin pathways. Moreover, and according to the data obtained using ETA receptor antagonist (BQ-123) and denudation of the aortic rings suggested that changes in ET-1 reactivity are mostly due to its ET-A receptor which is highly located on VSMCs. In summary, LA and BH4 may be considered pharmacological tools to modulate the potency of ET-1-induced vasoconstriction and concomitantly lower blood pressure.

Authors' contributions statement:
ZK Carried out the experiment, data acquisition and writing the manuscript. Statistical analysis, conception and design of the study was done by IM.

Limitation of the study
There are two major limitations in this study that could be addressed in future research. First, because of time and cost we couldn't estimate ET A and B receptors' gene expression from the isolated aorta by using RT-PCR or western blotting. Second, we highly recommended using different potassium channel blockers which may associate with the LA signal transduction pathway which produces vasodilators in the presence of BH4.