The l-Arginine-Nitric Oxide Pathway

The demonstration of the synthesis of nitric oxide fi-om the amino acid L-arginine by vascular endo-thehal cells has led to the elucidation of the importance of the L-arginine-nitric oxide pathway as a regulator of cell function in a number of tissues (Moncada et al. 1989). The reaction involves a 5- 

NOS-positive neurones and activated neuroglial cells were the most prominent citruUine-positive structures (Keilhoff et al. 2000). Lack of citruUine-immunoreaction in neurones of nNOS knockout mice emphasised the dependency of cit-ruUine positivity on NOS activity, and likewise there was no citrulline staining after application of the NOS inhibitors 7-nitroindazole and l-N -(1-iminoethyl)lysine. The inhibition of argininosuc-cinate synthetase by a-methyl-DL-aspartate increased the number of citrulline-positive cells, apparently due to the reduction of the turnover rate of citrulline. Cells positive for NOS but negative for citrulline may indicate that the enzyme is either not activated or inhibited by cellular control mechanisms. The fact that not all citrulline-positive cells were NOS positive may be explained by an insufficient detection sensitivity or by disparate sites of citrulline production and recycling. 

Using chemiluminescence resulting from the reaction of NO and O3, Maurer and Fung (2000) characterised enzyme activity for purified murine macrophage NOS n. They also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS n. The estimated parameters were in agreement with those reported using other methods. 

The most promising of all methods proposed thus far for direct measurement of NO appears to be the use of spin traps from stable paramagnetic adducts detectable by the electron paramagnetic resonance (EPR) method (Vanin 1999). Such traps were found to be complexes of bivalent iron with hydrophobic and hydrophihc derivatives of dithio- 

To a culture of NO-producing macrophages from murine bone marrow (5 x 10 cells in 2 ml of cultural medium) superoxide dismutase (10 M), Na-DETC (Img/ml) and FeS04 7H2O (10 M) are added successively (Vanin et al. 1991,1993). In 2 h, the cells harvested with the medium are centrifuged for 10 min at l,500xg, reconstituted in 0.3 ml of supernatant, and frozen in liquid nitrogen for the EPR analysis. The formed hydrophobic mononitrosyl iron complexes with DETC are located in membranous compartments of all cells. 

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East, S. J., and Garthwaite, J. (1991). NMDA receptor activation in the rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide pathway. Neurosci. Lett. 123, 17-19. 

Matheis, G., Sherman, M. P., Buckberg, G. D., Haybron, D. M., Young, H. H., and Ignarro, L. J. (1992). Role of the L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am. J. Physiol. 262, H616-H620. 

Fleming, I., Gray, G., and Stoclet, J. C. (1993). Influence of endothelium on induction of the L-arginine Nitric oxide pathway in rat aortas. Am. J. Physiol. 264, H1200-H1207. 

Thiemermann, C. (1994). Role of the L-arginine-nitric oxide pathway in circulatory shock. Adv. Pharmacol. 28, 45-79. 

As yet, it has not been possible to pinpoint the exact site of injury to the L-arginine nitric oxide pathway. The defect may lie with the surface receptors, signal transduction, L-arginine deficiency, nitric oxide synthase, tolerance of smooth muscle, physical obstruction to the diffusion of nitric oxide by a thickened intima, or impaired guanylate cyclase activation. However, endogenous guanylate cyclase can be activated by exogenously administered nitric oxide to relax smooth muscle, even when unresponsive to acetyl-... 

Boger, R.H., Bode-Boger, S.M., and Frdlich, J.C. (1996) The L-Arginine-Nitric Oxide Pathway Role in Atherosclerosis and Therapeutic Implications, Atherosclerosis 127,1 11. 

Moncada, S. (1993). The L-arginine nitric oxide pathway, cellular transduction and immunological roles. Adv. Second Messenger Phosphoprotein Res. 28, 97-99. 

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