Rainer H. Böger

Supplementation of hypercholesterolaemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production

L-arginine, the precursor of endogenous nitric oxide (NO), has been shown to enhance endothelial function and to reduce intimal plaque area in cholesterol (Chol)-fed rabbits. We have studied endogenous NO production in such animals in vitro (endothelium-dependent relaxations) and in vivo (assessed by urinary NO3- excretion) before and during chronic oral administration of L-arginine and inhibitor of NO synthesis, L-NAME. Vascular superoxide anion (O2-) production of aortic rings was measured under basal conditions and following exposure to phorbol-myristate-acetate (PMA). Cholesterol feeding reduced endothelium-dependent relaxations and decreased urinary NO3- excretion. These effects were potentiated by administration of L-NAME. L-arginine partly restored endothelium-dependent relaxations and increased NO3- excretion. PMA-stimulated O2- production was increased in aortic rings from rabbits given cholesterol ( +159 +/- 28%; mean +/- S.E.M.) or cholesterol + L-NAME ( +149 +/- 37%) as compared with controls ( -22 +/- 7%). In rabbits given cholesterol + L-arginine, O2- production was decreased to control levels ( +14 +/- 17%; P < 0.05). We conclude that the systemic synthesis of NO is impaired in cholesterol-fed rabbits, as indicated by the decreased urinary excretion of NO3-. Enhanced O2- production may further contribute to the decreased biological activity of NO in hypercholesterolaemia. L-arginine restores endothelial function in hypercholesterolaemia by enhancing NO production and by protecting NO from early breakdown by O2-.

Urinary 8-iso-Prostaglandin F2α as a Risk Marker in Patients With Coronary Heart Disease A Matched Case-Control Study

Background—Oxidative stress is involved in the pathophysiology of atherosclerosis, diabetes mellitus, hypertension, obesity, and cigarette smoking, all of these being risk factors for coronary heart disease (CHD). We tested the hypothesis that risk factors of CHD are associated with abundant systemic oxidative stress. Methods and Results—We conducted a case-control study with 93 CHD patients and 93 control subjects frequency-matched by age and sex. Urinary excretion of the F2-isoprostane 8-iso-prostaglandin (PG) F2&agr; and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr;, were measured by gas chromatography–tandem mass spectrometry. Body mass index, systolic blood pressure, and C-reactive protein were elevated in CHD patients (P <0.01). Urinary 8-iso-PGF2&agr; and 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr; also differed, from 77 (interquartile range, 61–101) to 139 (93–231) pmol/mmol creatinine and from 120 (91–151) to 193 (140–275) pmol/mmol in control subjects and case subjects, respectively (P <0.001). 8-iso-PGF2&agr; and its metabolite were highly correlated (Spearman’s &rgr;=0.664, P <0.001). HDL cholesterol was diminished in CHD patients (P <0.001). All of these characteristics predicted CHD in univariate analysis. In a multivariate model, the odds ratios were increased only for 8-iso-PGF2&agr; (≥131 pmol/mmol, P <0.001) and C-reactive protein (>3 mg/L, P <0.01), ie, by 30.8 (95% CI, 7.7–124) and 7.2 (1.9–27.6), respectively. 8-iso-PGF2&agr; was found to be a novel marker in addition to known risk factors of CHD, ie, diabetes mellitus, hypercholesterolemia, hypertension, and smoking. Urinary excretion of 8-iso-PGF2&agr; correlated with the number of risk factors for all subjects (P <0.001 for trend). Conclusions—8-iso-PGF2&agr; is a sensitive and independent risk marker of CHD.

Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox.

L‐Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K M values of these enzymes. Therefore, additional L‐arginine should not enhance NO formation. In vivo, however, increasing L‐arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L‐arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC50 1.5 μM), are responsible for the L‐arginine paradox is presented.

Elevation of asymmetrical dimethylarginine may mediate endothelial dysfunction during experimental hyperhomocyst(e)inaemia in humans.

Hyperhomocyst(e)inaemia is associated with endothelial dysfunction in animals and humans. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inaemia are poorly understood, but may involve impaired bioavailability of endothelium-derived nitric oxide (NO). We hypothesized that acute elevation of homocyst(e)ine by oral methionine loading may stimulate the formation of asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, due to a transmethylation reaction during the formation of homocyst(e)ine from methionine. We studied nine healthy human subjects (five males, four females) aged 29+/-2 years. Flow-mediated vasodilation (FMD) in the brachial artery (endothelium-dependent) and vasodilation induced by nitroglycerine (endothelium-independent) were measured with high-resolution ultrasound before and 8 h after oral methionine (100 mg/kg in cranberry juice) or placebo (cranberry juice), on separate days and in random order. Plasma homocyst(e)ine and ADMA concentrations were measured by specific HPLC methods. After a methionine bolus, elevation of homocyst(e)ine (28.4+/-3.5 micromol/l) was associated with an increased plasma concentration of ADMA (2.03+/-0.18 micromol/l) and reduced FMD (1.54+/-0.92%). Placebo had no effect on these parameters. There was a significant inverse linear relationship between ADMA concentration and FMD (r=-0.49; P<0.05), which was stronger than the relationship between the homocyst(e)ine concentration and FMD (r=-0.36; not significant). We conclude that acute elevation of the homocyst(e)ine concentration impairs vascular endothelial function by a mechanism in which an elevated concentration of ADMA may be involved. This finding may have importance for understanding the mechanism(s) leading to homocyst(e)ine-associated vascular disease, and its potential treatment.

Dietary l-arginine and α-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms

Vascular oxidative stress brought about by superoxide radicals and oxidized low-density lipoproteins (oxLDL) is a major factor contributing to decreased NO-dependent vasodilator function in hypercholesterolemia and atherosclerosis. We investigated whether chronic administration of L-arginine (2% in drinking water) or of alpha-tocopherol (300 mg/day) improves endothelium-dependent vasodilator function and systemic NO production, reduces vascular oxidative stress, and reduces the progression of atherosclerosis in cholesterol-fed rabbits with pre-existing hypercholesterolemia. Systemic NO production was assessed as urinary nitrate excretion; oxidative stress was measured by urinary 8-iso-PGF2alpha excretion in vivo, by lucigenin-enhanced chemiluminescence of isolated aortic rings ex vivo, and by copper-mediated LDL oxidation in vitro. Endothelium-dependent relaxation was almost completely abrogated in cholesterol-fed rabbits. Urinary nitrate excretion was reduced by 46+/-10%, and 8-iso-PGF2alpha excretion was increased by 61+/-18% as compared to controls (each P <0.05). Vascular superoxide radical release stimulated by PMA ex vivo was increased by 273+/-93% in this group, and the lag time of LDL oxidation was reduced by 35+/-6% (each P <0.05). Treatment with L-arginine and alpha-tocopherol reduced intimal lesion formation (by 68+/-6 and 4+/-11%, respectively; P <0.05) and improved endothelium-dependent relaxation. Both treatments also normalized urinary 8-iso-PGF2alpha excretion. L-Arginine increased urinary nitrate excretion by 43+/-13% (P <0.05) and reduced superoxide radical release by isolated aortic rings to control levels, which was unaffected by vitamin E treatment. By contrast, vitamin E dramatically increased the resistance of isolated LDL to copper-mediated oxidation in vitro by 178+/-7% (P <0.05), which was only marginally prolonged by L-arginine. Intimal thickening was reduced by both treatments. We conclude that both L-arginine and alpha-tocopherol reduce the progression of atherosclerotic plaques in cholesterol-fed rabbits. However, while L-arginine increases NO formation and reduces superoxide release, alpha-tocopherol antagonizes mainly oxLDL-related events in atherogenesis. Thus, both treatments reduce urinary isoprostane excretion and improve endothelium-dependent vasodilation via different mechanisms.

Dietary l-arginine decreases myointimal cell proliferation and vascular monocyte accumulation in cholesterol-fed rabbits

L-arginine, the precursor of endogenous nitric oxide (NO), has been shown to enhance endothelial function and to reduce the progression of atherosclerosis in cholesterol-fed rabbits. In the present study, we investigated whether myointimal cell proliferation is enhanced in hypercholesterolaemic rabbit aorta and whether chronic treatment of the rabbits with L-arginine or with the NO synthase inhibitor L-NAME influences this proliferative response and vascular monocyte accumulation. Rabbits were fed 1% cholesterol or normal rabbit chow for 12 weeks. Subgroups of cholesterol-fed rabbits were treated with oral L-arginine (2.25%) or L-NAME (3 mg/dl) in drinking water. Myointimal cell proliferation was quantified in aortic segments by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation into nuclear DNA; vascular monocyte accumulation was assessed by immunohistochemistry using a monoclonal anti-macrophage/monocyte antibody (RAM-11). Plasma levels of L-arginine and the endogenous NO synthase inhibitor, ADMA, were quantified by high-performance liquid chromatography (HPLC). Cholesterol feeding increased the aortic intima/media (I/M) ratio, which was not measurable in the control group, to 1.9 +/- 0.3. This was paralleled by enhanced cell proliferation (cholesterol, 2.4 +/- 0.2%; P < 0.05; control, 0.02 +/- 0.001% BrdU-positive cells per 72 h) and vascular monocyte accumulation. Double immunostaining for BrdU and alpha-actin showed that about two thirds of the proliferating cells were smooth muscle cells. ADMA levels increased from 0.8 +/- 0.1 micromol/l to 2.2 +/- 0.2 micromol/l in cholesterol-fed rabbits, but were unchanged by L-arginine or L-NAME treatment. Myointimal proliferation and intima/media ratios were correlated with ADMA plasma levels. Dietary L-arginine reduced monocyte accumulation by 85 +/- 2% (P < 0.05 vs cholesterol), myointimal cell proliferation (1.8 +/- 0.3% per 72 h; P < 0.05) and intimal thickening (I/M ratio: 0.7 +/- 0.2), whereas the inhibitor of NO synthase, L-NAME, further increased cell proliferation to 3.1 +/- 0.4% per 72 h (P < 0.05). No significant difference was observed in vascular monocyte infiltration between the cholesterol and L-NAME groups. We conclude that cell proliferation and vascular monocyte accumulation are enhanced in hypercholesterolaemic rabbit aorta. These atherogenic effects can be attenuated by dietary L-arginine. Decreased NO formation might underlie the enhanced monocyte accumulation and cell proliferation in hypercholesterolaemic rabbit aorta. The observed inhibition of cell proliferation adds to our understanding of the antiatherosclerotic effects of L-arginine in vivo.

Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the determination of nitric oxide synthase activity is described. The method is based on the gas chromatographic-mass spectrometric measurement of L-[15N2]arginine-derived [15N]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [15N]nitrite formation by purified neuronal nitric oxide synthase revealed K(M) values of 3.1 microM by Hanes and 4.6 microM by Lineweaver-Burk for L-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 micromol [15N]nitrite min(-1) mg(-1) NOS, respectively. N(G)-Nitro-L-arginine and N(G),N(G)-dimethylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was also assessed in vivo by i.v. injection of L-[15N2]arginine in a rat and determination of plasma [15N]nitrite and [15N]nitrate. The assay described in this work allows for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.

NG-nitro-L-arginine- and indomethacin-resistant endothelium-dependent relaxation in the rabbit renal artery : effect of hypercholesterolemia

Studies were designed to compare the N(G)-nitro-L-arginine- and indomethacin-resistant, endothelium-dependent relaxation to acetylcholine in isolated renal artery rings from normal and cholesterol-fed rabbits. It was assumed that the resistant part in response to acetylcholine is mediated by the endothelial-derived hyperpolarizing factor (EDHF). Rabbits were fed normal (n = 15) or cholesterol enriched chow (n = 13, 1% cholesterol for 4 weeks, 0.5% for 12 weeks). In organ chamber experiments, renal artery rings were precontracted with 0.1-1 microM phenylephrine or 35 mM KCl, and relaxed with acetylcholine (0.001-10 microM) in the presence of 10 microM indomethacin. Studies were performed in the presence or absence of: 100 microM N(G)-nitro-L-arginine (L-NOARG) to inhibit the nitric oxide pathway, 100 nM charybdotoxin (CTX) or 1 mM tetrabutylammonium (TBA) to inhibit Ca2+-activated K+ channels, and 100 microM SKF 525a to inhibit cytochrome P450 monoxygenase pathway. In normal arteries, L-NOARG partially inhibited acetylcholine-induced relaxation. The resistant part was almost abolished when the arteries were depolarized with KCl, or when L-NOARG was combined with either CTX, TBA or SKF 525a. In arteries from hypercholesterolemic animals, the relaxation to acetylcholine was only slightly impaired as compared to normal animals. However, in comparison to arteries from normal animals, the L-NOARG-resistant part of acetylcholine-induced endothelium-dependent relaxation was enhanced. It is speculated that differences in the balance between nitric oxide (NO)- and EDHF-mediated control of vascular tone may maintain acetylcholine-induced vasodilatation of the renal artery in hypercholesterolemia.

URINARY NO3− EXCRETION AS AN INDICATOR OF NITRIC OXIDE FORMATION IN VIVO DURING ORAL ADMINISTRATION OF l‐ARGININE OR l‐NAME IN RATS

1 Endothelium‐derived nitric oxide (NO), a major modulator of vascular tone, is synthesized from the terminal guanidino nitrogen of l‐arginine. This reaction is inhibited by analogues of l‐arginine, such as N‐nitro‐l‐arginine methyl ester (l‐NAME). Many of the biological effects of NO are mediated by the second messenger cGMP. NO is rapidly oxidized to NO3− which, like cGMP, is eliminated via excretion into the urine. In a placebo controlled study, we investigated whether oral bolus administration of l‐arginine and l‐NAME affects the urinary excretion rates of NO3− and cGMP in Munich Wistar Frömmter (MWF) rats. 2 Twenty MWF rats were kept in metabolic cages and received l‐arginine (3 g/kg body weight), l‐NAME (50mg/kg), or placebo (0.9% saline) in randomized order. Urine samples were sequentially collected for 10 h and analysed for creatinine, NO3− and cGMP. 3 l‐Arginine induced a slight, but prolonged increase in urine flow, whereas l‐NAME induced an early, transient increase in urine flow which was followed by a decrease. Creatinine clearance decreased by 65% after l‐NAME, but was not affected by l‐arginine or placebo. 4 Urinary NO3− and cGMP excretion rates transiently increased after l‐arginine (NO3−: + 29%; cGMP: + 16%) for 4–5h, whereas l‐NAME induced an immediate, pronounced and lasting inhibition of urinary NO3− and cGMP excretion (NO3−:‐76%; cGMP:‐46%). Urinary NO3− and cGMP excretions were significantly correlated (r = 0.755; P< 0.001). 5 Urinary excretion rates of NO3− and cGMP, expressed as μmol/h, were correlated to urine flow (mL/h; r = 0.617 and 0.649, respectively; both P<0.05), whereas after correction by urinary creatinine (μmol/mmol creatinine) no correlation with urine flow was observed, indicating that these excretion rates were independent of renal excretory function. Thus we conclude that changes in the urinary excretion rates of NO3− and cGMP represent changes in NO production rates in vivo when expressed in relation to urinary creatinine. Urinary NO3− and cGMP excretion is modulated by acute NO synthase inhibition or substrate provision.

Chronic dietary supplementation with l-arginine inhibits platelet aggregation and thromboxane A2 synthesis in hypercholesterolaemic rabbits in vivo

OBJECTIVES L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. METHODS The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha (major urinary metabolite of PGI2) and 2,3-dinor-TXB2 (major urinary metabolite of thromboxane A2) were used as indicators of platelet-endothelial cell interactions in vivo. Rabbits were fed 1% cholesterol (Cholesterol group, N = 8), 1% cholesterol plus 2.25% L-arginine (Cholesterol + L-arginine, N = 8), or normal rabbit chow (Control, N = 4) for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. RESULTS Urinary 2,3-dinor-TXB2 excretion significantly increased in the cholesterol group (p < 0.05), and endogenous NO formation (measured as urinary nitrate excretion) decreased (p < 0.05). Both parameters were significantly correlated with each other (R = 0.48, p < 0.01). L-arginine partly restored urinary nitrate excretion and significantly reduced TXA2 production to values even below those in the control group (p < 0.001). Urinary 2,3-dinor-6-keto-PGF1 alpha excretion increased in early hypercholesterolaemia and returned to control values in the second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF1 alpha excretion was attenuated by L-arginine. Platelet aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. CONCLUSIONS Cholesterol-feeding enhances platelet aggregation and TXA2 formation, and stimulates platelet-endothelial cell interaction in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI2/TXA2 ratio. It thus beneficially influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo.