Qualitative and quantitative aspects on nitric oxide metabolism. An experimental study in the rat and in man

Abstract

Background. Nitric oxide (NO) is a biomediator with significant physiological functions in the cardiovascular, nervous, and immune systems. NO is synthesized in the vascular endothelium by enzymatic conversion of L-arginine and O2 to L-citrulline in the presence of NO synthase. Nitrate is the main end-product of NO metabolism. NO plays an important role in a number of disease states. The aim of the present theses was therefore to develop and evaluate methods for assessment of formation of NO, qualitatively as well as quantitatively. Experimental Animals, Subjects and Methods. The study comprised anaesthetized and awake normotensive Wistar rats (WR), young and adult spontaneously hypertensive rats (SHR) and five healthy men. WR were injected s.c. equimolar amounts of 15NO gas or K15NO3. The appearance of 15N-labeled nitrate in plasma was followed for 450 min. In another study, WR were injected i.v. with 15N-L-arginine, and the appearance of 15N-nitrate was followed in plasma, urine, and faeces for up to 7 days. In other two studies, WR and SHR were placed in an air-tight cage and allowed to breathe 18-Oxygen containing mixture for 2 hours. The newly formed 18O-nitrate was measured in plasma with GC/MS, and the total formation of NO was calculated. WR and SHR were also treated with norepinephrine (NE) and adenosine, respectively, and the effects of blood pressure changes on NO formation were evaluated. Finally, healthy men were breathing 18O-containing mixture, and NO formation was estimated. The effect of NO synthase inhibitors (L-NAME and L-NMMA) on NO formation was determined in rats and in men. Results. Out of the subcutaneously administered 15NO gas in rats 89% was detected as 15N-nitrate in plasma. After the administration of 15N-L-arginine, during 7 days no 15N-nitrate was detected in faeces, while high concentrations of 15N-nitrate were measured in plasma and urine. The total formation of NO in young WR was estimated to 0.55±0.04 mmol/kg/h. L-NAME dose-dependently decreased the synthesis of NO. In young SHR the formation of NO was increased to 0.72±0.04 mmol/kg/h. It was somewhat lower in adult SHR, while renal clearance of nitrate was decreased significantly in this group. Lowering of blood pressure in SHR decreased the formation of NO. Treatment with NE did not evoke any difference in NO formation. The synthesis rate of NO in healthy men was estimated to 0.38±0.06 mmol/kg/h. It decreased to 0.17±0.03 mmol/kg/h after the same subjects were treated with L-NMMA. Conclusions. 15NO gas administered s.c. follows the same metabolic pathway as inhaled NO. Nitrate appears to be the main metabolite of endogenously formed NO. Nitrate excretion via faeces is not a significant route of elimination in healthy rats. Estimation of the formation of NO, both in laboratory animals and humans, was possible utilizing the 18-oxygen inhalation technique. This method may be useful in the assessment of various physiological and pathophysiological conditions affecting NO formation