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The idea that asymmetric NG, NG dimethylarginine obtains the ability to block basal-, and not agonist-induced relaxation, has been of major scientific dispute since its discovery recently with ACh-induced relaxation. The aim of the experiment was to determine whether this was true for all agonist-induced relaxations and whether the efficacy of the relaxant agonist is critical in its ability to avoid blockade by ADMA.
The effect of decrease efficacy was assessed by the use different agonists to investigate whether blockade could be induced by ADMA. Phenoxybenzamine (α-adrenergic receptor antagonist) was used, as a partial agonist, to further investigate whether partial displacement of ACh could be blocked by ADMA, since previous studies have noted that only basal activity of nitric oxide (NO) is possible by ADMA.
The efficacy of agonist is critical in the blockade of agonist-induced relaxation by ADMA. Standard conditions resulted in no blockade of NO-mediated, ACh-induced relaxation. However, pre-treatment with phenoxybenzamine not only reduced the efficacy and Emax of ACh-induced relaxation but allowed further blockade of relaxation by ADMA (both at 0.1 and 1mM; Emax 41.3 ± 3.9% and 15.6 ± 2.3%, respectively). Calcitonin gene related peptide (CGRP) and butyrylcholine (BCh) both had a lower Emax (65.6 ± 4.4% and 72.6 ± 5.4%, respectively) initially, allowing sufficient blockade of ADMA without pre-treatment at their receptor sites.
CONCLUSIONS AND IMPLICATIONS
It is important to note from the experiment that ADMA obtains the ability to inhibit agonist-induced relaxation. However, this only occurs when the apparent efficacy of the agonist has been reduced to a significant point, thus allowing sufficient disruption of the L-arginine - NO pathway and the promotion vasoconstriction. Levels of ADMA are associated with many cardiovascular diseases (hypertension etc.) and considering that both basal- and agonist-induced, NO-mediated relaxation can be blocked by ADMA indicates how devastating the vascular dysfunction could be if untreated
ADMA; ACh; NOS inhibitor; vasodilator; NO.
NO, Nitric Oxide; NOS, Nitric Oxide Synthase; ADMA, Asymmetric NG, NG Dimethylarginine; L-NMMA, NG-monomethyl-L-arginine; ACh, acetylcholine; BCh, Butyrylcholine; CGRP, calcitonin gene related pep
Nitric oxide has been regarded as the primary component of vascular relaxation under normal conditions, ever since the discovery that Nitric Oxide (NO) accounted for the same biological activity as expressed by EDRF (Palmer et al, 1987). Other studies have contradicted this theory and believe that more than one agent accounts for the activity of the EDRF (Hoeffner et al, 1989; Boulanger et al, 1989). However, no such experiment has identified another agent which can account for either of the factors expressed in both papers.
Considering that L-NAME, L-NOARG, and L-NMMA (analogues of L-arginine) elicit actions that block this NO-mediated basal relaxation, it can be confidently stated that NO accounts, at least, for the tonically released substance by endothelial cells that induces vasodilation. However, it has been more recently noted that L-NMMA is an endogenously produced NOS inhibitor which works alongside NO to maintain vascular tone. In addition to L-NMMA, asymmetric NG, NG dimethylarginine (ADMA) is also an endogenous NO-synthase (NOS) inhibitor which works to compensate the effects of L-NMMA, when levels are reduced (Leone et al, 1992).
It has recently been suggested that high levels of ADMA are linked to a variety of vascular diseases (hypertension, atherosclerosis and diabetes)by increasing cardiac output and vasoconstriction, in vascular tissue, accounting for the vascular dysfunction that is experienced in these disorders (Achan et al, 2003). However, while it has been understood for the past decade that ADMA is able to inhibit the actions of basal NO-mediated relaxation, meanwhile, more recent experiments have noted that this is not the case for agonist-induced, NO-mediated relaxation (Al-Zobaidy et al, 2010; Martin et al, 1992).
Therefore, the aim of the current experiment was to discover whether a difference in agonist efficacy, to induce relaxation, accounts for the inability of ADMA to block its action.
Preparation of rat aortic rings and tension recording
Rat aortic rings were used primarily for the experiment and the preparation for tension recording was structurally similar to previous experiments (Al-Zobaidy et al, 2010; Frew et al, 1993). Wister Rats (weight also similar to previous studies 100-250g) were killed by stunning and exsanguination. On removal of aorta, 2.5mm rings were prepared and any obstructing fat/connective tissue were removed prior to incubation.
As the experiment was to investigate the effects of ADMA (0.1-1mM) on agonist-induced relaxation in vascular tissue, the endothelial lining of the aortic rings were kept intact. The extracted arotic rings were placed in tissue baths (under 37°C Krebs solution (similar to experiments by Al-Zobaidy et al, 2010 and Frew et al, 1993) and mounted under a resting tension of approximately 9.0-11.0 mN. The tension was recorded and displayed electronically.
Endothelin-1 was used as the spasmogen in all of the experiments.
Previous studies have sucessfully exploited the effects of ADMA's ability to block basal nitric oxide-mediated relaxation and increasing vascular tone, therefore, was not assessed in the present experiment, however, the objective was to determine the ability of ADMA to block ACh-induced relaxation, which has varied throughout the years. Considering some of the more recent studies it is becoming more of a consensus that the efficacy of agonist binding controls the ability of ADMA to block relaxation (Al-Zobaidy et al, 2010; and Martin et al, 1992).
ACh-induced relaxation was exploited in this study by assessing the ability of ADMA (both 0.1 and 1mM) to block relaxation induced by a variety of Ach concentrations (1nM - 10µM). Moreover, by reducing the efficacy of ACh, by pretreatment with phenoxybenzamine, allows one to conclude whether reducing the efficacy, of agonist, would potentiate the blockade by ADMA. In addition, the effects of ADMA to block butyrylcholine (BCh) and calcitonin gene related peptide (CGRP) relaxation was studied to test the hypothesis further, distinguishing whether the efficacy of the agonist is essential in the blockade of nitric oxide-mediated, agonist-induced relaxation by ADMA.
Analysis of Data
Aortic contractions where measured in millinewtons and expressed as the percentage relaxation of the vasocontrictor-induced tone observed. The data is experessed as the mean ± SEM (of % relaxation) of n separate observations, for each individual experiment. Graphs were executed as % relaxation against the log concentration of agonist in microsoft excel, with a statistical analysis using one-way ANOVA and Bonferroni's post-hoc test using computer aided SPSS software (IBM softwares, Hampshire, Portsmouth, UK).
Effects of ADMA (0.1 - 1mM) on ACh-induced relaxation
A basal tone of 8.9 ± 0.6 mN was observed in rat aorta and was increased by ET-1 administration to 16.9 ± 0.2 mN. However, in the presence of ACh a concentration-dependent relaxation was observed with maximal relaxation occurring at 10µM (Emax 81.7 ± 3.8%; pEC50 -7.53 ± 1.9).
In the presence of ADMA (0.1mM, 1 hour) there was a slight increase in contraction (from 16.9 ± 0.2 to 19.9 ± 0.7 mN). However, the overall induced-relaxation, by ACh, was similar to the control with a maximal relaxation being observed at 3µM (Emax 84.3 ± 1.9%; pEC50 = -7.14 ± 2.8). Similar effects were observed at 1mM ADMA with a maximal relaxation of 83.8 ± 2.8% (pEC50 -7.17 ± 3.14) being recorded and an initial contraction of 18.4 ± 0.2 mN. It can therefore be concluded that, under normal conditions, as expected ADMA does not obtain the ability to block nitric oxide-mediated relaxation, induced by ACh and our findings correlate with previous studies (Martin et al., 1992). Figure 1 displays the concentration-dependent relaxation curves observed in each experiment and each graph is almost superimposable to the one before, again highlighting the inability of ADMA to block relaxation induced by ACh. However, a quick note would be that in the initial ACh concentrations the graphs begins to shift to the right and it could be plausible that there is some competitive antagonism occurring between both agents.
Figure 1: Effects of ADMA (0.1 - 1mM) on vascular relaxation induced by Ach. The findings from the experiment support previous reports that ADMA has no effect on nitric oxide-mediated, ACh-induced relaxation. The initial agonist concentrations (in the presence of ADMA) show a slight shift in the graph to the right. However, the overall responsiveness of ADMA is virtually ineffective in each experiment with maximal relaxation being observed at approximately 3µM of ACh (Emax 84.3 ± 1.9%). The statistical analysis of this experiment was insignificant with p>0.05.
Effects of ADMA (0.1mM) on relaxation induced by CGRP
CGRP alone produced maximal relaxation at a concentration of 300nM, however, this was initially less than that induced by ACh alone (Emax 65.6 ± 4.4%; pEC50 -8.61 ± 0.01). In the presence of 0.1mM ADMA, agonist-induced relaxation was almost completely abolished (Emax 15.2 ± 4.2%). A pEC50 value could not be obtained, electronically, from the graph for CGRP-induced relaxation in the presence of 0.1mM ADMA. Already, we can see that as the efficacy of agonist-induce vascular relaxation is reduced, so too is the drugs ability to withstand blockade by ADMA. CGRP can only induce about half the response that is induced by ACh, and so is less efficacious, thus, allowing ADMA to displace the action of CGRP and promote vasoconstriction in the aortic tissue. The result from the CGRP-induced experiment is shown in figure 2, and highlights the significance of blockade by ADMA.
Figure 2: The maximal response elicited by CGRP is achieved at a concentration of 300nM. The Emax 65.6 ± 4.4% is significantly reduce by the treatment of 0.1mM ADMA (1 hour) to 15.2 ± 4.2% (where p<0.01), thus, at the level of 0.05 the population means are significantly different and a Bonferroni significance of 1 indicates that the difference between the means are significant.
Effects of ADMA (0.1 - 1mM) on BCh-induced relaxation
ET-1 induced a low level contraction (16.0 ± 0.5mN) which was effectively blocked by administration of BCh (11.8 ± 0.3mN at 300µM). This was the point when agonist-induced relaxation had reached the maximal level (Emax 72.6 ± 5.4%; pEC50 -5.1 ± 1.9) indicating that, under normal conditions, the efficacy of the agonist to induce relaxation was also effectively lower than that of ACh.
In the presence of 0.1mM ADMA the maximal relaxation was reduced (Emax 49.5 ± 4.9%; pEC50 -4.9 ± 6.7) and was almost completely blocked by 1mM ADMA (Emax 10.5 ± 3.8%; pEC50 -4.86 ± 4.9; figure 3). Therefore, this reinforces the idea that the ability of ADMA to overcome agonist-induced relaxation is defined by the efficacy of the relaxant.
The significance of means in the experiment is such that p<0.05 for 1 mM ADMA. The Bonferroni post-hoc highlights that the significance lies between the control and the 1mM ADMA levels with there being no significant correlation between the control and 0.1mM ADMA.
Figure 3: BCh acts at the muscarinic M3 receptor and effectively blocks the vasoconstrictor tone induced by ET-1. The maximal relaxation (Emax 72.6 ± 5.4%) is reached at a agonist concentration of 300µM. The presence of 0.1mM ADMA inhibits BCh-induced relaxation (Emax 49.5 ± 4.9%) and is significantly reduced by 1mM (Emax 10.5 ± 3.8%; Bonferroni significance level of 1). ** indicates p<0.05 significant blockade of agonist-induced relaxation by ADMA.
Effects of ADMA (0.1 - 1mM) on ACh-induced relaxation when pre-treated with phenoxybenzamine
The effects of phenoxybenzamine were used to investigate whether the lowering of agonist efficacy would allow sufficient blocking of the induced relaxation by ADMA. The addition of phenoxybenzamine alone, reduces both the potency and efficacy of the agonist and therefore, the level of relaxation is immediately reduced (Emax 63.3± 3.5%; pEC50 -7.2 ± 1.8; figure 4). The reduction in efficacy allowed sufficient blockade of NOS at both 0.1 and 1mM concentrations of ADMA (Emax 41.3 ± 3.9% and 15.6 ± 2.3%, respectively; figure 4). This reinforces the hypothesis: does the apparent efficacy of agonist-induced relaxation affect the ability of ADMA to sufficiently block the NO-mediated relaxation? P=0.00071 indicating there is a very strong significant difference between the means of the graphs. Bonferroni's post-hoc analysis expressed significant value of 1 for both the phenoxybenzamine present with 0.1 and 1mM ADMA.
Figure 4: The presence of phenoxybenzamine is enough on its own to reduce the efficacy of ACh-induced relaxation to an Emax 63.3 ± 1.8%, through a partial agonist action. This reduction in efficacy allows ADMA to interfere with the NOS production of NO and block the relaxation induced by ACh, which under normal conditions would not occur. ** p=0.011 indicates that compared to the control there is a significant blockade of action by 0.1mM ADMA. *** p=0.00053 indicates that the greatest significant blockade of relaxation is observed at the 1mM ADMA.
Discussion and conclusions
From the experiment one can conclude that with a reduction in the efficacy of the relaxant, the ability of ADMA to inhibit NOS is increased. The study acts as a follow-up to some of those previous studies (Al-Zobaidy et al, 2010 and Martin et al, 1992) to determine whether the apparent efficacy of the agonist is critical in enabling ADMA to inhibit agonist-induced relaxation. Previous studies explored the effects of ACh-induced realxation and noted that pre-treatment with phenoxybenzamine reduced the affinity and efficacy of ACh (Martin et al, 1992). This runs in parellel with the findings from the current experiment where the ACh-induced relaxation (Emax 87.9 ± 2.6%) was effectively block by pre-treatment with phenoxybenzamine. However, with the addition of ADMA (0.1 and 1mM) vaso-relaxation was inhibited in the experiment (Emax 41.3 ± 3.9%, and Emax 15.6 ± 2.3%, respectively). The efficacy of the agonist-induced binding was reduced from -7.02 ± 7.4 to -5.45 ± 2.34 with pre-treatment with phenoxybenzamine alone, resulting in immediate reduction in maximal relaxation (Emax 63.3 ± 3.5%). Once the efficacy had been effectively reduced, the ability of ADMA to block the action of agonist was increased significantly (p<0.05).
Although this seems like a legitimate conclusion to make it does not fully indicate whether the initial agonist binding affinity is critical to the reducing the desired efficacy. This is because the phenoxybenzamine concentration had been effectively reduced (where 2mM was dissolved in absolute ethanol and diluted in distilled water (Martin et al, 1992), thus allowing the agent to act as a partial agonist. This stimulates the tonic sympathetic constrictor tone, actively produced under normal conditions, to overcome some of the induced-relaxation produced by ACh. However, when treated with both pheonxybenzamine and ADMA there was inhibition of eNOS, along with increased stimulation the tonic sympathetic vasoconstrictor-induced tone (from phenoxybenzamine acting as the partial agonist), that allowed sufficient blockade of ACh-induced relaxation. Although this does show that ADMA can block the relaxation observed when the efficacy of ACh has been reduced (through stimulating the sympathetic constrictor tone also), it does not provide evidence of whether blockade of the M3 receptor (to reduce efficacy and affinity of ACh-induced relaxation) could potentiate the action of ADMA in the experiment. Some of the results are conflicting with other studies (Feng et al, 1998) who believed to have inhibited ACh-induced relaxation by ADMA administration. However, as noted by Al-Zobaidy et al, 1992, it is thought that this is due to human errors that an overcontraction (as a results of competative antagonism of ADMA) is the likely cause of relaxation blockade rather than through a disruption in the NOS pathway.
Furthermore, the understanding that sympathetic tone, in addition to increased ADMA, defines the reason why vasoconstriction is a major factor in those suffering from cardiovascular diseases (such as hypertension, diabetes and atherosclerosis). If levels of ADMA are increased then the presence of the tonically-induced constriction of the blood vessels rise to deterimental levels, as seen in these disease states. It proposes the idea that future therapeutic innervention could arise from blockade of either the tonic release of sympathetic vasoconstrictors and/or from inhibiting the blockade of NO-mediated vasodilation, induced by ADMA. Whether this would make a good drug target or even begin to combat the problems associated with such diseases is one of many ideas proposed by current researchers and futher detailed studies would be required to determine the effects of ADMA blockade, or even if the discovery of a new compound that would have an efficacy as high, or higher, than that of ACh, would be beneficial to inducing relaxation in hypertensive patients.
From the other experiments it can been seen that BCh (acting also at the M3 muscarinic receptors) is a partial agonist and has a lower efficacy than ACh at its target, in the absence of ADMA (Emax 72.6 ± 5.4%). Although the apparent efficacy is not effectively different the action of ADMA is still significantly larger. The ability of ADMA to block agonist-induced relaxation is almost completely abolished at 1mM. However, kin the presence of agonist CGRP, the efficacy is reduced to <70% relaxation (at maximum) and the ability of ADMA to almost completely block the relaxation is increased by 10-fold.
So, to conclude it is a valuable lesson to understand that the efficacy of agonist-induced relaxation is essential in preventing blockade from endogenous NOS inhibitor ADMA. Although, previous studies have noted that ADMA is only effective against basal-induced, and not agonist-induced, NO-mediated vascular relaxation it seems to be the case that this is because of the efficacy of the agonist used (ACh; Al-Zobaidy et al, 2010; and Martin et al, 1992). However, Martin et al, 1992, did not that the use of phenoxybenzamine (as a partial agonist) reduced the relaxation induced by ACh. The current study, acting as a follow-up, highlights that the reduced efficacy (through increase sympathetic vasconstriction) allows the eNOS that is blocked by ADMA to over power the induced relaxation. The use of both BCh and CGRP also shows that as the percentage of relaxation is reduced (possibly to <80%) then ADMA blockade surfaces, and a rise in vasoconstriction occurs. Therefore, the set of experiments carried out work in a very concise and particulate way to allow us to accept our hypothesis that ADMA blockade of NOS, in vascular vessels of rat aorta, is critically dependent on the efficacy of the relaxant agonist