Patrick Kearns

Evidence for the Pathophysiological Role of Endogenous Methylarginines in Regulation of Endothelial NO Production and Vascular Function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated l-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-l-arginine (l-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and l-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for l-arginine is 3.14 μm with a Vmax of 0.14 μmol mg–1 min–1, whereas Ki values of 0.9 μm and 1.1 μm were determined for ADMA and l-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 μm level of l-arginine, MA levels of >10μm were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 μm. Similar effects were observed in isolated vessels where 5 μm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and l-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. l-Arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, ∼4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.

NON-INVASIVE IMAGING OF THE LOCALIZATION AND TIME COURSE OF CELL DEATH IN A CANINE MODEL OF ACUTE MYOCARDIAL INFARCTION AND REPERFUSION: DEMONSTRATION OF EARLY AND LATE PHASES OF REPERFUSION CELL DEATH

Methods: The left anterior descending artery (LAD) was occluded using a 3 mm PTCA balloon catheter (n = 12). A constant gadolinium (Gd) infusion was administered during MRI to assess MI. Regional myocardial signal enhancement and function were recorded every 10 min during 90 min of ischemia followed by 120 min of R, and again at 24 and 48 hr post R. A catheter was inserted into the coronary sinus to measure creatine kinase (CK) leak across the coronary circulation.