Different Disappearance Rates of Plasma Nitrite (NO2-) Contribute to Apparent Steady-State Arterio-Venous Differences in Anesthetized Animals

Abstract

) is not only an oxidative product of nitric oxide (NO), but also serves as an indicator of endothelial nitric oxide synthase activity and as a circulating reservoir of NO bioactivity in the circulatory system. [1][2][3] The indicator status is supported by observations that approximately 70-90% of circulating plasma NO 2 Ϫ levels derive from NO produced by endothelial nitric oxide synthase. 4,5) The reservoir activity is widely accepted (see reviews [1][2][3] ) as is maintained when NO 2 Ϫ reacts with deoxygenated hemoglobin (Hb) in red blood cells (RBCs) to form NO and methemoglobin (metHb), leading to biological responses including vasodilation.2,6,7) Therefore, precise evaluation of NO 2 Ϫ plasma levels, including detailed kinetic knowledge, is required for further understanding of NO 2 Ϫ biology in the circulation. However, only limited kinetic data [8][9][10] are presently available, and many problems of NO 2 Ϫ biology await resolution.For example, it is still controversial whether arterio-venous (A-V) differences in plasma NO 2 Ϫ levels actually exist. 10-16)The A-V difference may reflect consumption of NO 2 Ϫ to form NO during peripheral circulation for preservation of blood flow at lower oxygen tension.12) Although we have previously reported a small but significant A-V difference under steadystate conditions, 10,14,17) these observations are complicated by a report that, in isolated blood, uptake of NO 2 Ϫ by RBCs is faster in venous blood than in arterial blood, 15) a scenario that may cause artificial apparent A-V differences when arterial and venous blood samples are treated after some delay. If this situation is indeed the case, our previous work reporting kinetic parameters 10) should also be re-evaluated. It is also unclear whether plasma NO 2 Ϫ levels reflect in vivo changes in NO 2 Ϫ levels. 10,12,18) Exogenously administered NO 2 Ϫ or NO resulted in a large A-V difference in plasma NO 2 Ϫ , with arterial blood preferentially reflecting the changes, 10,14,17) presumably due to greater contributions by RBCs or tissue as a buffering compartment under a state of lower oxygenation. A possible difference in the uptake rate of RBCs could not be large enough to explain such a large A-V difference, and contributions of tissue factors would be a great concern. 18,19) The emerging role of tissue as a reservoir of NO bioactivity via NO 2 Ϫ should be taken into consideration; some reports indicate that plasma NO 2 Ϫ levels may not accurately reflect increases in NO or NO 2 Ϫ levels in biological systems. 3,20,21) However, no alternative measures exist for assessing NO bioactivity in easily accessible biomaterials (such as blood). Therefore, the aim of this study was to estimate the steady-state concentration of plasma NO 2 Ϫ in order to evaluate whether the putative A-V difference actually exists. We also re-evaluated the pertinent kinetic features in vivo, considering uptake by RBC and tissue factors. MATERIALS AND METHODSJapanese white rabbits weighing 2.4-3.5 kg were anesthetized with intravenous sodium...

Role of Plasma S-Nitrosothiols in Regulation of Blood Pressure in Anesthetized Rabbits with Special References to Hypotensive Effects of Acetylcholine and Nitrovasodilators

“…In this study, no Cys-NO, G-SNO, or Alb-SNO were found in circulating blood under steady-state conditions as we have previously reported. 26) In addition, prevention of transnitrosation leading to the degradation of R-SNOs by NEM 19,20) failed to detect not only Alb-SNO, but also LMWR-SNOs. Although the expected concentration of NEM in circulating blood (2 mM in whole blood) is one quarter of that used ex vivo (8 mM in the sampling tube; see Materials and Methods), this dose is practically the maximum in vivo, because higher doses of NEM caused death in our preliminary study.…”

“…Quantification of Alb-SNO Alb-SNO was determined by HPLC-Saville's method 26) as was originally described by Akaike et al 27) with some modifications including an additional column to avoid interference by plasma proteins and composition of mobile phase ( Fig. 1A and its legend).…”

Effects of Cyanamide, an Aldehyde Dehydrogenase Type 2 Inhibitor, on Glyceryl Trinitrate- and Isosorbide Dinitrate-Induced Vasodilation in Rabbit Excised Aorta and in Anesthetized Whole Animal

“…The first control parameters and arterial blood sample were obtained after a stabilization period of 20-30 min and the latter was treated with special attention to NO x contamination as described previously. 10,12) The exact time period from blood sampling to plasma separation was measured for each sampling and was used for correction of plasma NO 2 − concentration in vivo (as described below). After intragastric administration of cyanamide (1 mL/kg of 100 mg/mL solution, i.e., 100 mg/kg) or vehicle (1 mL/kg of distilled water), another 60 min was allowed with additional pentobarbital for a steady-state followed by measurement of the second control parameters and sampling of the control blood specimen.…”

“…Because the disappearance rate of NO 2 − ex vivo (after sampling) is slower in arterial blood than in venous blood, error could be smaller in arterial blood when the measured NO 2 − concentration is corrected for time (from sampling to plasma separation) and the disappearance rate for estimating plasma NO 2 − concentration in vivo. 10) Furthermore, arterial plasma NO 2 − concentrations are considered to preferentially reflect changes (increases) in plasma NO 2 − concentration even by derangement with exogenous NO 2 − in vivo.…”

“…In addition, this study includes a special reference to changes in arterial plasma NO 2 − concentration in whole animal experiment, because it is considered to reflect NO status in whole body 8,9) and that measurement of these changes in concentration are feasible by our sophisticated method with high sensitivity and reliability. 10) The rationale for the study was that if bioactivation status or pathways of GTN and ISDN are differently affected by ALDH2 inhibitor, changes in plasma NO 2 − concentration should reflect changes in the active product (NO) or its oxidized product (NO 2 − ).…”