The ability of asymmetric dimethylarginine (ADMA) or monomethylarginine (LNMMA) to block endothelium-dependent, nitric oxide-mediated relaxation in rat aorta is inversely related to the efficacy of the relaxant stimulus
Mohammed J. Al-Zobaidy and William Martin* College of Medical, Veterinary and Life Sciences, West Medical Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK.
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Running title: efficacy and NOS inhibition
Abstract Background and purpose: Previous work on rat aorta has shown that L-NMMA and ADMA each enhance vasoconstrictor-induced tone, consistent with blockade of basal nitric oxide activity, whereas they exert little inhibitory effect on acetylcholine-induced relaxation when tone is matched carefully to that of control tissues. The aim of this study was to determine if the ability of L-NMMA or ADMA to inhibit nitric oxide-mediated relaxation was critically determined by the efficacy of the relaxant stimulus.
Experimental approach: The effects of L-NMMA or ADMA were examined on relaxation to a range of agonists producing different maximal responses, namely, acetylcholine, the muscarinic partial agonist, butyrylcholine, and calcitonin gene-related peptide-1 (CGRP-1). The effects of L-NMMA or ADMA were also examined on relaxation to acetylcholine when its apparent efficacy at the M3 muscarinic receptor was reduced using the irreversible receptor blocking agent, phenoxybenzamine.
Key results: Maximal relaxation induced by butyrylcholine or CGRP-1 was lower than to acetylcholine. While acetylcholine-induced relaxation was largely resistant to blockade by LNMMA or ADMA (0.1 or 1 mM), relaxation to butyrylcholine or CGRP-1 was powerfully suppressed. Phenoxybenzamine (3 µM for 30 min) reduced maximal acetylcholine-induced relaxation from ~ 90 to ~ 50 %. When the efficacy of acetylcholine was reduced by phenoxybenzamine, its residual relaxant effect was powerfully inhibited by L-NMMA or ADMA (0.1 or 1 mM).
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Conclusions and implications: Thus, in rat aorta, the ability of L-NMMA or ADMA to block agonist-induced nitric oxide activity is critically determined by the efficacy of the relaxant stimulus.
Key words: acetylcholine; ADMA; asymmetric NG, NG-dimethyl-L-arginine; butyrylcholine; calcitonin gene-related peptide-1; endothelium; nitric oxide; L-NMMA; NG-monomethyl-Larginine; NOS inhibitor; phenoxybenzamine.
Abbreviations: ACh, acetylcholine; ADMA, asymmetric NG, NG-dimethyl-L-arginine; BCh, butyrylcholine; CGRP-1, calcitonin gene-related peptide-1; L-NMMA, NG-monomethyl-Larginine; L-NOARG, NG-nitro-L-arginine; L-NAME, NG-nitro-L-arginine methyl ester; NOS, nitric oxide synthase; PBZ, phenoxybenzamine.
Introduction Three decades have now elapsed since Furchgott and Zawadzki (1980) first discovered that the vascular endothelium produces a powerful vasodilator agent, endothelium derived relaxing factor (EDRF), now identified as nitric oxide (Palmer et al., 1987). It is well accepted that one of the two equivalent guanidino nitrogen (NG) atoms of the amino acid, Larginine, is oxidised to produce nitric oxide in a reaction catalysed by the three isoforms of the enzyme, nitric oxide synthase (NOS), namely, the neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) forms (Palmer et al., 1988; Knowles et al., 1989; Bredt & Snyder, 1990; Olken et al., 1991).
The synthesis of nitric oxide can generally be blocked by a variety of guanidino-substituted analogues of L-arginine, such as NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-
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arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME), that act as competitive, reversible inhibitors of NOS (Palmer et al., 1988; Rees et al., 1989; Rees et al., 1990; Moore et al., 1990; Hobbs et al., 1999). Strikingly, a number of studies have suggested that of these three agents, L-NMMA can exhibit properties that deviate markedly from classical competitive behaviour on all three isoforms of NOS. For example, L-NMMA has been shown to act as an alternative substrate and mechanism-based suicide inhibitor of iNOS in murine macrophages (Olken & Marletta, 1993). Other studies have reported that LNMMA, unlike L-NOARG and L-NAME, does not inhibit nNOS-related, nitrergic nervemediated relaxation in the bovine penile and ciliary arteries and retractor penis muscle (Liu et al., 1991; Martin et al., 1993; Overend & Martin, 2007). In addition, although L-NMMA blocks the eNOS-dependent basal nitric oxide activity that exerts a tonic vasodepressor action in rat aorta, it has little effect on acetylcholine-induced relaxation in this vessel when tone is matched carefully to that of control tissues (Frew et al., 1993). More recent reports show that a pathophysiologically-important endogenous inhibitor of NOS, asymmetric NG, NGdimethyl-L-arginine (ADMA, Vallance et al., 1992), shares the ability of L-NMMA to block preferentially basal over acetylcholine–stimulated activity of nitric oxide in rat aorta (ALZobaidy et al., 2010; 2011).
A possible explanation for the differential effects of L-NMMA and ADMA on basal versus acetylcholine-stimulated activity of nitric oxide in rat aorta may relate to the extent to which eNOS is stimulated under the two conditions. Specifically, the low-level activation of eNOS which underpins basal activity of nitric oxide may, theoretically, be easier to block than the powerful enzyme activation resulting from agonist stimulation. This hypothesis is supported by previous work showing that the relative resistance of acetylcholine-induced relaxation to blockade by L-NAME in rabbit jugular vein when compared to rat aorta results from a higher
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efficacy of the relaxant in the former tissue because of a greater M3 muscarinic receptor reserve (Martin et al., 1992). As a consequence, the aim of this study was to determine if in rat aorta the ability of the methylarginines, L-NMMA and ADMA, to block nitric oxidemediated relaxation is critically determined by the efficacy of the relaxant stimulus. This was investigated by examining the effects of L-NMMA or ADMA on relaxation of rat aorta induced by a range of agonists producing different maximal responses. Their effects were also examined on relaxation to acetylcholine when its apparent efficacy at the M3 muscarinic receptor was reduced using the irreversible receptor blocking agent, phenoxybenzamine (Martin et al., 1992).
Methods Preparation of rat aortic rings and tension recording All animal care and experimental procedures complied with UK Home Office regulations. The preparation of rat aortic rings for tension recording was essentially similar to previous studies (Frew et al., 1993; AL-Zobaidy et al., 2010; 2011). Briefly, female Wistar rats weighing 150-200 g were killed by CO2 overdose. The aorta was removed, cleared of adhering fat and connective tissue and cut into 2.5 mm wide transverse rings using a device with parallel razor blades. The aortic rings were mounted under 10 mN resting tension on stainless steel hooks in 10 ml tissue baths and bathed at 37 °C in Krebs solution containing (mM): NaCl 118, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, glucose 11, and gassed with 95 % O2 and 5 % CO2. Tension was recorded isometrically with Grass FTO3C transducers and displayed on a PowerLab (ADInstruments, Hastings, UK).
Experimental protocols
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Experiments were conducted to assess the effects of L-NMMA or ADMA (0.1 and 1 mM) on endothelium-dependent, nitric oxide-mediated relaxation induced by a range of agonists producing different maximal responses. Specifically, the agents used were the full agonist, acetylcholine, the partial agonist, butyrylcholine (Martin et al., 1992), and the weak relaxant, CGRP-1 (Gray & Marshall, 1992). In these experiments, the NOS inhibitors were added for 1 h prior to contracting the tissues with phenylephrine or, where indicated, endothelin-1. This latter vasoconstrictor was required in experiments in which the irreversible alkylating agent, phenoxybenzamine (Martin et al., 1992), was used to lower the apparent efficacy of acetylcholine or butyrylcholine at the M3 muscarinic receptor (see below), since in these, phenylephrine lost its ability to induce tone because of additional blockade of α1– adrenoceptors. In all experiments, care was taken to induce comparable levels of tone (~50% of max) in control and drug-treated tissues. In order to achieve this, lower concentrations of phenylephrine or endothelin-1 were used in the presence of L-NMMA or ADMA because NOS inhibitors enhance vasoconstrictor tone through removal of basal nitric oxide activity which exerts a tonic vasodilator action (Martin et al., 1986; Rees et al., 1989; Rees et al., 1990). After stabilisation of tone in control and NOS inhibitor-treated tissues, cumulative concentration-response curves were constructed to acetylcholine (1 nM-10 µM), butyrylcholine (100 nM-300 µM), or calcitonin gene-related peptide-1 (0.1 nM-1 µM) and relaxation assessed.
As indicated above, a series of experiments was carried out to reduce the apparent efficacy of the full agonist, acetylcholine, and the partial agonist, butyrylcholine, using the irreversible alkylating agent, phenoxybenzamine (Martin et al., 1992). In these experiments, some tissues were treated with phenoxybenzamine (3 µM for 30 min followed by extensive washout) and others were left as controls. The tissues were then pre-contracted to 50 % of maximal
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endothelin-1-induced tone before constructing cumulative concentration-response curves to acetylcholine or butyrylcholine. Further experiments were conducted to study the effects of L-NMMA or ADMA (both at 0.1 and 1 mM) on relaxation induced by acetylcholine after its efficacy had been reduced by treatment with phenoxybenzamine.
Data analysis Relaxant responses to agonists were expressed as a percentage of the complete relaxation induced by a supramaximal concentration of papaverine (300 µM) added at the end of each experiment. Data are expressed as the mean ± SEM of n separate observations, each from a different tissue. Concentration-response curves were drawn and statistical analysis was performed using one-way analysis of variance followed by Bonferroni's post-test, or by Student's t-test, as appropriate, with the aid of Prism (Graph Pad, San Diego, USA). Values were considered to be statistically different when P was ≤ 0.05.
Drugs and chemicals Acetylcholine chloride, asymmetric NG, NG-dimethyl-L-arginine dihydrochloride (ADMA), butyrylcholine chloride, endothelin-1 (human), NG-monomethyl-L-arginine acetate (LNMMA), papaverine hydrochloride, phenoxybenzamine hydrochloride and phenylephrine hydrochloride were all obtained from Sigma, UK. Calcitonin gene-related peptide-1 (CGRP1, human) was obtained from Calbiochem. All drugs were dissolved and diluted in 0.9 % saline except phenoxybenzamine which was dissolved in 100 % ethanol and calcitonin generelated peptide-1 which was dissolved in 5 % acetic acid (all stocks 10 mM).
Results Relaxation to acetylcholine, butyrylcholine and calcitonin gene-related peptide-1 7
Following induction of ~ 50 % maximal tone using phenylephrine (20-100 nM) in rat endothelium-containing aortic rings, acetylcholine (1 nM-10 µM; Figure 1), butyrylcholine (0.1-300 µM; Figure 2), and CGRP-1 (0.1 nM-1 µM; Figure 3) each induced concentrationdependent relaxation. The maximal relaxation induced by acetylcholine was 93.3 ± 2.8 %. Consistent with its actions as an M3 muscarinic partial agonist, butyrylcholine induced significantly lower maximal relaxation (72.6 ± 5.4 %; P
