N-Methyl-D-arginine

Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 +/- 0.7 nmol/l vs baseline (28.7 +/- 1.4 nmol/l, P < 0.001) in response to papaverine-induced flow increases of 121 +/- 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap alpha-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

The potential role of nitric oxide (NO) production in secondary pathologic processes that follow spinal cord injury was examined in a guinea pig model that shows secondary loss of function for at least 3 days after trauma. Lateral compression injury of the lower thoracic cord was performed under ketamine/xylazine/acepromazine anesthesia. A fine polyethylene cannula was inserted through an incision in the dura rostral to the injury and run along the dorsal subdural space to the lesion level. The tube was connected to an osmotic pump delivering 1 microL/h of a 10 mM solution of either N-methyl-L-arginine or N-methyl-D-arginine in normal saline (pH 7.2). N-Methyl-L-arginine blocks both constitutive and inducible forms of NO synthase (NOS), present in neurons and inflammatory cells, respectively: N-methyl-D-arginine is the inactive stereoisomer. Two groups of 10 animals were used. Behavioral analysis and somatosensory evoked potential measurements were performed daily for 3 days, then the animals were fixed and survival of white matter at the center of the injury was evaluated, using toluidine-blue stained, 1 microns plastic sections. No significant difference was found between treated and control groups in degree or rate of secondary loss of spinal cord function or in the cross-sectional area of surviving white matter. These data do not support the hypothesis that local NO production by phagocytes, neurons, or other cells plays a significant role in secondary pathology of injury in this model.