MMA, ADMA and SDMA as inhibitors of, and hArg as substrate for NO synthesis
In 1992, Vallance et al. [
3] reported that ADMA and MMA, but not SDMA, inhibited iNOS activity in J774 macrophage cytosol (by 18% at 5 µM ADMA), and that ADMA (EC
50, 26 µM) contracted endothelium-intact rat aortic rings. In the same study, ADMA infusion (25 µmol/kg/h) raised systolic blood pressure by nearly 15% at a plasma concentration of about 10 µM in anaesthetized Guinea pigs, whereas ADMA infusion (8 µmol for 5 min into the brachial-artery) decreased forearm blood-flow by 28% in healthy humans [
3]. The authors stated in their article that free ADMA and MMA, but not free SDMA, inhibited NO synthesis in vitro and in vivo. Since then, the report by Vallance et al. [
3] received much attention, is a much-quoted and landmark paper concerning the particular importance of ADMA, but not of SDMA, as an endogenous inhibitor of NO synthesis [
3]. The focus on ADMA given subsequently by the scientific community is a plausible explanation for numerous studies that eventually revealed free ADMA as a cardiovascular risk factor in humans, while free SDMA was left entirely in the shadow of ADMA. Thus, it took many years until SDMA has attracted particular attention, beyond its importance as a uremic toxin, and has also emerged as a cardiovascular risk factor, in some studies hand-in-hand with ADMA [
1,
4,
5]. This is a big surprise when considering that free SDMA has been and still is generally considered not to be an inhibitor of NO synthesis. One may therefore ask the question, whether the scientific community has uncritically adopted previous observations [
3] and ignored or overlooked important information.
It is indeed very surprising, that, despite the substantial interest in ADMA and its undoubtful importance in the cardiovascular system, only very scare information is available about the inhibitory potency of free ADMA towards all known NOS isoforms, i.e., eNOS, nNOS, iNOS, and the underlying mechanisms of the inhibition of NOS activity [
6]. A likely reason could be the generalization of the results reported by Vallance et al. [
3] with respect to the rather very weak inhibitory action of free ADMA and their uncritical extension to eNOS, and the almost complete neglect of free SDMA in the subsequent years.
By using a recombinant nNOS, we found that not only free ADMA, MMA,
NG-nitro-
l-arginine (NNA) and
NG-nitro-
l-arginine methyl ester (
l-NAME), but also free SDMA inhibit nNOS activity (discussed in Ref. [
6]). At a molar basis (1 µM), the order of the inhibitory potency towards nNOS activity was approximately 6:5:4:1:1 for NNA:ADMA:NMA:L-NAME:SDMA. Thus, compared to ADMA, SDMA is a considerably less potent inhibitor of nNOS activity. Free ADMA is a potent (IC
50, 1.5 µM) noncompetitive inhibitor of recombinant nNOS activity, but a very weak (IC
50, 12 µM) competitive inhibitor of eNOS activity [
6]. To the best of our knowledge, there are no reported data on the inhibitory potency of SDMA towards eNOS activity. Presumably, free SDMA is a weak inhibitor of eNOS, most likely weaker than ADMA. The quite weak inhibitory potency of ADMA and presumably of SDMA towards eNOS activity is not compatible with the key importance attributed to these methylated L-Arg derivatives in the human circulation.
Free
l-hArg has been demonstrated to be converted to NO by recombinant eNOS, nNOS and iNOS; yet,
l-hArg was found to have a much lower affinity to these enzymes when compared with
l-Arg [
7]. Because of this and the very low circulating and tissue
l-hArg concentrations, notably in relation to free
l-Arg, free
l-hArg cannot be considered as an important substrate and much less as an inhibitor of eNOS.
Are ADMA, SDMA and hArg inducers of oxidative stress?
ADMA is generally associated with elevated oxidative stress, although no solid evidence has been provided thus far. In patients suffering from type 2 diabetes mellitus (T2DM), no association between free ADMA and oxidative stress was observed [
8]. Treatment of the T2DM patients with an angiotensin II receptor antagonist did reduce the oxidative stress level, but did not alter the plasma ADMA concentration [
8]. With regard to an association of SDMA with oxidative stress, the body of evidence is even much thinner. To the best of our knowledge, there is only in vitro indication that free SDMA and ADMA may very weakly increase “oxidative burst” in
N-formyl-methionine-leucine-phenylalanine (fMLP)-stimulated monocytes [
9].
In women with normal (Group 1) or impaired (Group 2, normal outcome; Group 3, intrauterine growth restriction; Group 4, preeclampsia) placental function [
10], a moderate correlation (
r = 0.58,
P = 0.0009) was found between ADMA and SDMA plasma concentrations only in the healthy pregnant women (Table
1). This correlation is of the same order of magnitude (
r = 0.45,
P < 0.001) reported by Zobel et al. [
5] for T2DM patients. In our study, neither SDMA nor ADMA plasma concentration correlated with the plasma concentration of the oxidative stress biomarker
cis-epoxyoleic acid (
cis-EpOA) (Table
1) [
10]. This finding suggests that neither SDMA nor ADMA are associated with oxidative stress in normal or abnormal pregnancy.
Table 1
Correlation (by Spearman) between the plasma concentrations of ADMA, SDMA and cis-EpOA in women with normal (Group 1) or impaired placental function (Groups 2, 3, 4)
Group 1 | r = 0.58, P = 0.0009, n = 30 | r = 0.05, P = 0.79, n = 30 | r = − 0.07, P = 0.72, n = 29 |
Group 2 | r = 0.05, P = 0.85, n = 16 | r = 0.42, P = 0.11, n = 16 | r = − 0.42, P = 0.10, n = 16 |
Group 3 | r = 0.29, P = 0.33, n = 14 | r = − 0.20, P = 0.51, n = 13 | r = 0.10, P = 0.75, n = 13 |
Group 4 | r = 0.26, P = 0.47, n = 10 | r = 0.31, P = 0.39, n = 10 | r = − 0.18, P = 0.63, n = 10 |
Groups 2,3,4 | r = 0.15, P = 0.35, n = 40 | r = 0.08, P = 0.64, n = 39 | r = − 0.21, P = 0.21, n = 39 |
In vitro in HUVEC and in vivo in the rat (i.p. injection up to 400 mg/kg bodyweight), exogenous free
l-hArg was found not to induce oxidative stress [
11].
Paraoxonases (PON) possess several biological functions, yet their primary role is still speculative. PON-1 is thought to be associated with lipid peroxidation. In patients suffering from T2DM and dyslipidemia, 12-weeks treatment with rosuvastatin was reported to increase serum PON-1 activity and to lower plasma ADMA concentration [
12]. Yet, a correlation between PON-1 and ADMA seems not to have occurred in that study at baseline or after rosuvastatin treatment.
NG-Methylated and guanidinated proteins versus free ADMA, SDMA and hArg
Free
l-hArg is synthesized from free
l-Arg and
l-lysine by
l-arginine:glycine amidinotransferase (AGAT), with
l-ornithine being the second reaction product. AGAT also catalyzes the reaction of
l-Arg and glycine to form guanidinoacetate (GAA), the precursor of the energy-related creatine.
l-hArg is considered to be a non-proteinogenic amino acid. Thus far, there are no reports that proteins comprise
l-hArg residues due to physiological post-translational modifications. Yet, it has been demonstrated that synthetic peptides that contain alkylated hArg or Arg residues in position 6 possess strong biological activity, presumably via interaction of the positively charged hArg moiety with the negatively charge phosphate group of the phospholipid cell membrane [
13]. The most feasible possibility for the formation of
l-hArg-containing proteins could be guanidination of the terminal amine group of
l-Lys residues, analogous to the widely occurring and abundant
Nε-methylation.
In accordance with the prevailing doctrine, free MMA, ADMA and SDMA are released by regular proteolysis of
NG-methylated proteins. Identity, concentration and potential roles of the majority of those proteins in the renal, cardio- and cerebrovascular systems are largely unknown. It is worth mentioning that
NG-methylated proteins may play a role in insulin signaling in L6 skeletal muscle cells [
14]. Should
NG-methylated proteins and guanidinated proteins possess intrinsic biological activity, there arise several questions. Do
NG-methylated and guanidinated proteins act antagonistically? Are free ADMA, SDMA and
l-hArg risk markers or risk factors in human disease such as diabetes mellitus? Does the concentration of free ADMA, SDMA and
l-hArg in the circulation reflect the tissue concentration of
NG-methylated and guanidinated proteins? These issues warrant deeper investigations.