RESUMEN
Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Arginina/análogos & derivados , Fenómenos Fisiológicos Cardiovasculares , Homeostasis/genética , Modelos Moleculares , omega-N-Metilarginina/metabolismo , Acetilcolina/farmacología , Amidohidrolasas/antagonistas & inhibidores , Animales , Arginina/metabolismo , Presión Sanguínea/genética , Vasos Sanguíneos/efectos de los fármacos , Northern Blotting , Western Blotting , Calcimicina/farmacología , Cromatografía Líquida de Alta Presión , Cristalografía , Relación Dosis-Respuesta a Droga , Ecocardiografía , Endotelio/metabolismo , Eliminación de Gen , Humanos , Ratones , Contracción Muscular/efectos de los fármacos , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genética , Resistencia Vascular/genéticaRESUMEN
The enzyme DDAH metabolizes methylarginines that are inhibitors of nitric oxide synthase (NOS). Substrate-based inhibitors of mammalian DDAH have been synthesized, with optimization to give selective inhibition of DDAH with no significant direct effect on NOSs. These are the first examples of reversible DDAH inhibitors with significant activity and selectivity. In vivo administration increases plasma ADMA levels, giving proof of concept that these inhibitors can be used to probe the physiological effects of DDAH inhibition, with potential for pharmaceutical use of DDAH inhibitors in diseases where excess NO production is implicated.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Arginina/análogos & derivados , Arginina/síntesis química , Animales , Arginina/sangre , Arginina/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/enzimología , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Endogámicas WKY , Relación Estructura-ActividadRESUMEN
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 micromol of ADMA per day, of which approximately 250 micromol 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.
Asunto(s)
Amidohidrolasas/metabolismo , Arginina/análogos & derivados , Arginina/administración & dosificación , Arginina/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Adulto , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Método Doble Ciego , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Inyecciones Intravenosas , Masculino , Resistencia Vascular/efectos de los fármacosRESUMEN
Asymmetric dimethylarginine (ADMA) and N(G) monomethyl-L-arginine (L-NMMA) are endogenous inhibitors of nitric oxide synthases (NOS) and their local concentration is determined by the activity of dimethylarginine dimethylaminohydrolases (DDAHs). The current study in male Wistar rats was designed to immunolocalise DDAH I and II in relation to NOS and to investigate changes in distribution, activity and ADMA content in the acute period following myocardial infarction (MI) resulting from coronary artery ligation. Seven days after the coronary artery ligation, L-Arg and methylated arginine content, as well as DDAH activity were determined in homogenates of left ventricular (LV) infarct and border. The distribution of immunoreactive DDAH I, DDAH II, eNOS and iNOS were determined in sections of LV. In healthy hearts, DDAH I was absent, however, DDAH II was localized to endothelium and endocardium with a similar distribution to that of eNOS. Following MI, LV DDAH activity was increased (to 210+/-19% of control, P<0.05). Both DDAH I and DDAH II proteins were detected in peri-infarct cardiomyocytes, while DDAH II immunoreactivity was additionally localized to infiltrating inflammatory cells and blood vessels in the healing infarct. Both plasma and LV concentrations of the DDAH substrate, ADMA, were increased post-MI, although the ratio of Arg:ADMA was retained in the LV post-MI relative to sham operated controls. In conclusion, DDAH II has a distribution similar to eNOS in healthy myocardium. The increased levels and activity of DDAH I and DDAH II enzymes following myocardial infarction suggest a potential role for them in local protection of NOS enzymes from inhibition by methylated arginines during infarct healing.
Asunto(s)
Amidohidrolasas/metabolismo , Infarto del Miocardio/enzimología , Miocardio/enzimología , Animales , Aorta/enzimología , Arginina/análogos & derivados , Arginina/sangre , Arginina/metabolismo , Modelos Animales de Enfermedad , Endocardio/enzimología , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Masculino , Infarto del Miocardio/sangre , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Ratas , Ratas WistarRESUMEN
UNLABELLED: Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function. CONCLUSION: Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis.