INVOLVEMENT OF NITRIC OXIDE IN THE INHIBITION OF HUMAN P45OS - POSSIBLE CLINICAL RELEVANCE R. Pave1 Marbsek, and Bettie Sue Masters. U. Texas Health Science Center, San Antonia, Texas, 78229.

Overproduction of nitric oxide (NO) by the inducible form of nitric oxide synthase (NOS) has been implicated in the decreased cytochrome P450 activity which occurs during sepsis. Suppression of P450-mediated drug metabolism is important in that it prolongs the duration and intensity of action of drugs and endogenously produced substances. Since septic patients are often given therapeutic drugs that require I’450-mediated metabolism, adverse effects of these drugs may be a common but unrecognized problem. Interestingly, the mechanism of P450 inhibition during sepsis remains largely unknown. Since levels of inducible NOS, and thus NO, are increased during sepsis, we have begun examining the ability of NOS isoforms and human P450 isoforms to bind NO and stabilize an inhibitory iron-nitrosyl complex. E. c&-expressed nNOS forms a stable ferrous nitrosyl complexe during enzymatic turnover, which is believed to be a form of autoregulation. In contrast, the ferric nitrosyl complex of nNOS is unstable. We have discovered that the ability of E. c&-expressed human P450 isoforms to form and/or stabilize either a ferrous or ferric nitrosyl complex is isoform-specific. In activity assays, using baculovirus-expressed microsomes, initial results suggest that P450 isoforms are differentially susceptible to inhibition during exposure to fluxes of NO. The rank order for inhibition of NADPH oxidation by NO was CYP 2C9 > CYP 2D6 > CYP 3A4. These data imply a role for NO in the inhibition of P450-mediated drug metabolism, and it is our belief that studies of this type will provide information as to the significance of altered drug metabolism in critically ill septic patients.(Supported by NIH Grants GM52419, HL30050 and Welch Foundation Grant AQ-1192 to BSSM).

FORMATION OF S-NITROSOALBUMIN IN HUMAN PLASMA BY NITRIC OXIDE. ~Richard Marleyl, Rakesh Patel2, and Victor Dar1 -Usmar Royal Free and University College Medical Schml, London NW3 r QG UK.

S-nitrosothiols are potentially important modulators of biological processes such as vasodiltatation, apoptosis and thrombosis. We have recently shown that the plasma concentrations of S-nihosothiols in humans is -25nM. The mechanisms of formation and metabolism of the low nM concentrations required to exert biological effects remains unknown. An important issue that remains unresolved is the significance of the reactions of NO with oxygen to form S-nitrosothiols in a complex biological medium such as plasma. In the present study a sensitive assay for S-nitrosothiol formation in plasma exposed to varying fluxes of NO has been examined. In the presence of physiological concentrations of oxygen and nitric oxide, plasma thiols (predominantly as albumin) are S-nitrosated yielding S-nitrosothiols in the low r&i concentration range. The stability of S-nitrosoalbumin in plasma largely depends on the availability of free low molecular weight thiols. Using the rate constants derived from these experiments simulation of S-nitrosothiol formation as a function of oxygen and nitric oxide tension was determined. From these data it is concluded that despite the low rates of reaction of oxygen with NO biologically relevant concentrations of S-nitrosothiols can be formed from this reaction in plasma.

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INCREASED FORMATION OF S-NITROSOTHIOLS IN THE ACUTE BILE DUCT LIGATED RAT KevinSteven Halt and Richard Marl

8 Collqe Medical School, London NW3 2QG K. Royal Free and University

Nitrosothiols are formed by the reaction of nitric oxide and a thiol residue under aerobic conditions and S-nitrosoalbumin is the major circulating RSNO. S-Nitrosothiols can then circulate and release nitric oxide distal from their site of synthesis. We have recently developed an assay for plasma S-nitrosothiols using chemiluminescence. This assay is highly reproducible with an inter-assay variation of 4%, and a detection limit of 5nM. The development of vasodilatation is a well recognised complication of liver disease. Whilst many have attributed vasodilatation to the increased formation of nitric oxide acting locally in the vasculature, there have been no studies evaluating the formation of S-nitrosothiols in models of liver disease. In this study we measured plasma S-nitrosothiols in both sham controls and rats that had undergone complete bile duct ligation to cause acute biliary obstruction. Twenty four hours following bile duct ligation or sham procedure, animals were sacrificed under anesthesia by exsanguination. Plasma S-nitrosothiols were then measured by chemiluminescence. Plasma S-nitrosothiols increased from a basal level of 20 !r 1 nM to 37 It 3 nM following bile duct ligation (p<O.O5). Plasma total nitrite and nitrate were also measured by chemihuninescence, and increased from 12 + 2 mM to 28 f 4 mM (~~0.05). We conclude that there is a rapid up-regulation of nitric oxide synthesis following bile duct ligation in our model, and this is accompanied by increased formation of S-nitrosothiols. Further studies on the mechanism of formation of these compounds under pathological conditions will be undertaken. This work was funded by the Medical Research Council, UK.

INVOLVEMENT OF MAMMALIAN MITOCHONDRIA IN RECYCLING OF THE NO-METABOLITE NITRITE TO NITRIC MONOXIDE. Hans Katrin Staniek, and Andr V. Kozlov. Inst. Pharmacolo & Toxicolog!+ Univ. of Veterinmy Me mm, Vmnn A-1210 AUST 2. B A.

Various reports exist on elevated nitrite levels in a variety of diseases including HIV, reperfusion injuries and hypovolemic shock. Nitrite is a metabolite of nitric monoxide which is increasingly generated under many metabolic stress conditions. Nitrite is also augmented in patients supplemented with NO-donors to cope with angina pectoris. The low frequency of hypertension in vegetarians was discussed with respect to high dietary nitrite/nitrate uptake indicating the biological transformation to the vasodilatator nitric monoxide. Similarities between cytochromes involved in mitochondrial respiration of mammalians and cytochromes involved in nitrite-reduction of denitrifying bacteria led us to study the role of mitochondrial cytochromes in nitrite reduction. Our results which were based on EPR-studies demonstrate that the ubiquinone cytbcl-redox couple of mitochondria recycles nitrite to the bioregulator NO. This is the first observation of the existence of nitrite reductase activity in mammalians.

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