Guy Whitley

Asymmetric Dimethylarginine Causes Hypertension and Cardiac Dysfunction in Humans and Is Actively Metabolized by Dimethylarginine Dimethylaminohydrolase

Objective—Plasma levels of an endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), are elevated in chronic renal failure, hypertension, and chronic heart failure. In patients with renal failure, plasma ADMA levels are an independent correlate of left ventricular ejection fraction. However, the cardiovascular effects of a systemic increase in ADMA in humans are not known. Methods and Results—In a randomized, double-blind, placebo-controlled study in 12 healthy male volunteers, we compared the effects of intravenous low-dose ADMA and placebo on heart rate, blood pressure, cardiac output, and systemic vascular resistance at rest and during exercise. We also tested the hypothesis that ADMA is metabolized in humans in vivo by dimethylarginine dimethylaminohydrolase (DDAH) enzymes. Low-dose ADMA reduced heart rate by 9.2±1.4% from 58.9±2.0 bpm (P <0.001) and cardiac output by 14.8±1.2% from 4.4±0.3 L/min (P <0.001). ADMA also increased mean blood pressure by 6.0±1.2% from 88.6±3.4 mm Hg (P <0.005) and SVR by 23.7±2.1% from 1639.0±91.6 dyne · s · cm−5 (P <0.001). Handgrip exercise increased cardiac output in control subjects by 96.8±23.3%, but in subjects given ADMA, cardiac output increased by only 35.3±10.6% (P <0.05). DDAHs metabolize ADMA to citrulline and dimethylamine. Urinary dimethylamine to creatinine ratios significantly increased from 1.26±0.32 to 2.73±0.59 after ADMA injection (P <0.01). We estimate that humans generate approximately 300 &mgr;mol of ADMA per day, of which approximately 250 &mgr;mol is metabolized by DDAHs. Conclusions—This study defines the cardiovascular effects of a systemic increase in ADMA in humans. These are similar to changes seen in diseases associated with ADMA accumulation. Finally, our data also indicate that ADMA is metabolized by DDAHs extensively in humans in vivo.

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase

1 Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG‐monomethyl‐L‐arginine and NG, NG‐dimethy‐L‐arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2 S‐2‐amino‐4(3‐methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]‐NG‐monomethyl‐L‐arginine to [14C]‐citrulline by rat liver homogenates (IC50 416 ± 66μm; n = 9), human cultured endothelial cells (IC50 250 ± 34 μm; n = 9)and isolated purified dimethylarginine dimethylaminohydrolase. 3 Addition of 4124W to culture medium increased the accumulation of endogenously‐generated NG, NG‐dimethy‐L‐arginine in the supernatant of human cultured endothelial cells from 3.1 ± 0.3 to 5 ± 0.7 μm (n=15; P < 0.005). 4 4124W (1 μm‐1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium‐dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 ± 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L‐arginine (100 μm). 4124W reversed endothelium‐dependent relaxation of human saphenous vein (19.2 ± 6.7% reversal of bradykinin‐induced relaxation at 1 mM 4124W). 5 These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular concentration of NG, NG‐dimethyl‐L‐arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.

Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis

1 The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC‐7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]‐monomethyl‐l‐arginine ([14C]‐l‐NMMA) and the cytosolic extract analysed by high performance liquid chromatogaphy (h.p.l.c.) with on‐line radioisotope detection. 2 SGHEC‐7, HUVEC and human saphenous vein metabolized [14C]‐l‐NMMA to a compound which co‐eluted with [14C]‐citrulline. A second metabolite which co‐eluted with [14C]‐arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3 The intracellular levels of [14C]‐l‐NMMA and [14C]‐citrulline in SGHEC‐7 cells incubated with [14C]‐l‐NMMA (0.5 μCi ml−1: 8.9 μm) for 1 h were 113 ± 22 and 67.6 ± 6.2 pmol mg−1 cell protein respectively (n = 7). Co‐incubation with NGNGdimethyl‐l‐arginine (ADMA; 100 μm) but not NGN′Gdimethyl‐l‐arginine (SDMA; 100 μm) reduced the intracellular level of [14C]‐citrulline to 26.3 ± 3.7 pmol mg−1 cell protein (P < 0.01; n = 3) without reducing the intracellular level of [14C]‐l‐NMMA. 4 The intracellular levels of [14C]‐citrulline in SGHEC‐7 cells incubated with [14C]‐l‐NMMA for 1 h were reduced following co‐incubation with NGnitro‐l‐arginine methylester (l‐NAME; 1 mm), NGnitro‐l‐arginine (l‐NOARG; 1 mm) and l‐canavanine (1 mm) to 47.1 ± 6.2, 24.7 ± 3.6 and 12.5 ± 2.8% of control levels (P < 0.001; n = 9). ADMA (1 mm; n = 3) reduced intracellular [14C]‐citrulline levels to 4 ± 4% of control (P < 0.01) but SDMA (1 mm; n = 3) had no effect. 5 The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC‐7 cells was increased by co‐incubation with l‐NMMA (1 mm) from 1.98 ± 0.08 to 2.74 ± 0.36 nmol mg−1 cell protein, an increase of 40%. 6 These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize l‐NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium‐derived ADMA following co‐incubation with l‐NMMA is consistent with competition between ADMA and l‐NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.

Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia.

OBJECTIVE We investigated the change in the plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in early-, mid-, and late-gestation normotensive pregnancies and in gestational age-matched preeclamptic pregnancies and compared the observed changes with changes in blood pressure. STUDY DESIGN Blood pressure and peripheral plasma asymmetric dimethylarginine concentrations were measured in 20 nonpregnant and 145 pregnant women (33 first-trimester, 50 second-trimester, and 44 third-trimester normotensive pregnancies and 18 third-trimester pregnancies complicated by preeclampsia). In 23 normotensive pregnancies serial plasma asymmetric dimethylarginine concentrations were measured. Statistical analysis was by analysis of variance and linear regression. RESULTS The blood pressures recorded throughout normal pregnancy were significantly lower than in nonpregnant subjects (p < 0.0001). The mean systolic, diastolic, and average blood pressures were significantly higher in the second-trimester groups than in the first-trimester groups, whereas in the third trimester average and diastolic blood pressures were significantly higher than in the second trimester. The mean (+/-SD) systolic and diastolic blood pressures in third-trimester preeclamptic patients was 157.7 +/- 11.2 and 110.9 +/- 8.5 mm Hg. The mean plasma asymmetric dimethylarginine concentration in nonpregnant women was 0.82 +/- 0.31 micromol/L (significantly higher than in normotensive pregnancy, p < 0.0001). The plasma asymmetric dimethylarginine concentration was also significantly higher in second-trimester than in first-trimester normotensive groups (respectively, 0.52 +/- 0.20 micromol/L and 0.40 +/- 0.15 micromol/L, p = 0.001) and was higher in third-trimester normotensive pregnancy 0.56 +/- 0.23 micromol/L than it was in the second trimester. The asymmetric dimethylarginine concentration in third-trimester preeclamptic patients was 1.17 +/- 0.42 micromol/L (p < 0.0001 vs normotensive third-trimester subjects). CONCLUSIONS It is well recognized that blood pressure falls in early normal pregnancy and rises again toward term. These studies show that the early fall in blood pressure is accompanied by a significant fall in the plasma asymmetric dimethylarginine concentration. Later in pregnancy circulating concentrations increase and, when pregnancy is complicated by preeclampsia, concentrations are higher than in the nonpregnant state. Our data support a role for both asymmetric dimethylarginine and nitric oxide in the changes in blood pressure seen in both normal and preeclamptic pregnancy.

Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide

The expression of hepatocyte growth factor (HGF) is essential for normal placental development although its function is unknown. In this study we examined the effect of HGF on trophoblast cell motility and invasion of fibrin gels and investigated the possible role of nitric oxide (NO) in this process. The human extravillous trophoblast cell line SGHPL‐4 express both the constitutive and inducible isoforms of nitric oxide synthase (NOS). HGF significantly stimulates cell motility in monolayer culture, the invasion of fibrin gels and the production of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP). Invasion, motility and cyclic GMP production were inhibited by Ng‐monomethyl‐L‐arginine (L‐NMMA). Cell motility was also significantly inhibited by the inducible NOS specific inhibitor 1400 W. Neither 8 Br‐cyclic GMP nor the NO donor spermine‐NO had any significant effect on basal trophoblast cell motility. The data presented in this study demonstrate a direct effect of trophoblast‐derived NO synthesis on trophoblast cell function and support the idea that HGF is involved in the regulation of trophoblast invasion through mechanisms that involve the production of NO. However neither exogenous NO nor activation of cyclic GMP‐dependent pathways alone are sufficient to stimulate trophoblast cell motility.

Pre-eclampsia: fitting together the placental, immune and cardiovascular pieces†

The success of pregnancy is a result of countless ongoing interactions between the placenta and the maternal immune and cardiovascular systems. Pre‐eclampsia is a serious pregnancy complication that arises from multiple potential aberrations in these systems. The pathophysiology of pre‐eclampsia is established in the first trimester of pregnancy, when a range of deficiencies in placentation affect the key process of spiral artery remodelling. As pregnancy progresses to the third trimester, inadequate spiral artery remodelling along with multiple haemodynamic, placental and maternal factors converge to activate the maternal immune and cardiovascular systems, events which may in part result from increased shedding of placental debris. As we understand more about the pathophysiology of pre‐eclampsia, it is becoming clear that the development of early‐ and late‐onset pre‐eclampsia, as well as intrauterine growth restriction (IUGR), does not necessarily arise from the same underlying pathology. Copyright

Uterine Spiral Artery Remodeling Involves Endothelial Apoptosis Induced by Extravillous Trophoblasts Through Fas/FasL Interactions

Objective— Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. Methods and Results— Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. Conclusions— Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.

all-Trans-Retinoic Acid Increases Nitric Oxide Synthesis by Endothelial Cells. A Role for the Induction of Dimethylarginine Dimethylaminohydrolase

all-trans-Retinoic acid (atRA) has important effects on the developing and mature cardiovascular system. Nitric oxide (NO) production has been associated with the atRA-induced differentiation of neuronal cells, and we hypothesized that NO may also mediate certain actions of atRA in the cardiovascular system. We studied the effects of atRA on NO production by endothelial cells and determined whether regulation of enzymes responsible for metabolism of asymmetric dimethylarginine (ADMA) contributed to the effects seen. Murine endothelioma (sEnd.1) cells were incubated with or without atRA. Nitrite production was determined using the Griess reaction. The expression of NO synthase (NOS) and dimethylarginine dimethylaminohydrolase (DDAH) genes was determined by Northern blotting. A reporter gene assay was also used to study the effect of atRA on the DDAH II promoter. atRA significantly increased nitrite production by sEnd.1 cells despite no increase in eNOS expression. atRA also increased DDAH II gene expression and promoter activity and reduced the ratio of ADMA to symmetric dimethylarginine (SDMA) in culture medium. The DDAH inhibitor 4124W significantly reduced the induction of NO synthesis by atRA. The present study demonstrates that atRA increases NO synthesis in endothelial cells without increasing eNOS expression. atRA also increases the expression of DDAH II, the predominant DDAH isoform in endothelial cells. Our data suggests that the induction of NO synthesis by atRA may be facilitated by DDAH II. This pathway may help to explain some of the effects of atRA on the cardiovascular system.

Estrogen Stimulates Dimethylarginine Dimethylaminohydrolase Activity and the Metabolism of Asymmetric Dimethylarginine

Background—Experimental evidence suggests that estrogens stimulate the production of nitric oxide (NO) by vascular endothelial cells. This effect has been attributed to increased expression and enzymatic activity of both the constitutive and inducible isoforms of NO synthase. In this study, we have investigated whether estrogens regulate the metabolism or release of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase. Methods and Results—The concentration of ADMA in the plasma of 15 postmenopausal women was 0.722±0.04 &mgr;mol/L (mean±SEM). Two weeks after subcutaneous implantation with estradiol, there was an increase in plasma estradiol concentration from 0.693±0.075 to 0.81±87 nmol/L, which was accompanied by a significant fall in plasma ADMA concentration to 0.588±0.03 &mgr;mol/L (P =0.006). Human and murine endothelial cell lines previously cultured in estrogen-free medium and then exposed to 17&bgr;-estradiol showed a dose-dependent decrease in the release of ADMA. This reached statistical significance at 10−14 mol/L 17&bgr;-estradiol and was accompanied by a corresponding increase in the activity of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catalyzes the metabolism of ADMA. Conclusions—We have demonstrated that estrogens can alter the catabolism and release of ADMA in vitro and reduce the circulating concentration in vivo. We therefore propose that increased DDAH activity and the subsequent fall in ADMA could contribute to the positive effect of estrogen on NO synthesis.

Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension

Background—Chronic hypoxia–induced pulmonary hypertension is associated with increased pulmonary expression of nitric oxide synthase (NOS) enzymes. Nevertheless, some reports have indicated decreased pulmonary production of NO in the disease. To address this paradox, we determined pulmonary concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in the hypoxia-induced pulmonary hypertension rat model. In addition, we determined whether dysregulation of the ADMA-metabolizing enzyme dimethylarginine dimethylaminohydrolase I (DDAH I) plays a role in this disease. Methods and Results—Adult male rats were exposed for 1 week to either normoxia or hypoxia (10% oxygen). Lung tissues were used for Western blot analysis of endothelial NOS and DDAH I expression, measurement of lung NO and ADMA content, and in vitro assay of DDAH enzyme activity. Western blot analysis revealed a 1.9-fold increase in endothelial NOS protein and a 37% decrease in DDAH I protein in the lungs of hypoxia-exposed rats. Both pulmonary DDAH enzyme activity and NO content were significantly decreased in the hypoxic group (by 37% and 22%, respectively), but pulmonary ADMA concentrations were increased by 2.3-fold compared with the normoxic group. Conclusions—These data demonstrate that the rat chronic hypoxia–induced pulmonary hypertension model is associated with increased pulmonary concentrations of the NOS inhibitor ADMA. Moreover, pulmonary hypertensive rats exhibit reduced pulmonary expression and activity of the ADMA-metabolizing enzyme DDAH I. The decreased DDAH I and increased ADMA concentrations may therefore contribute to pulmonary hypertension via the competitive inhibition of pulmonary NOS enzymes.