Deactivation of norepinephrine by peroxynitrite as a new pathogenesis in the hypotension of septic shock

Background: Vascular hyporeactivity to catecholamines limits successful treatment of hypotension in septic shock. Large amounts of nitric oxide (NO) and superoxide anion (O-2(-1.)) are produced in response to bacterial endotoxins and/or inflammatory cytokines. NO reacts with O-2(-1). to form the potentially toxic NO metabolite, peroxynitrite (ONOO-1). The purpose of this study was to investigate whether ONOO-1 decreases the vasocontractile activity of norepinephrine. Methods: Norepinephrine was treated with ONOO-1 or 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1; an ONOO-1 producer) in a 5 X 10(-2) m sodium phosphate buffer solution at pH 7.4, and absorbance of the product was measured spectrophotometrically at 295 and 370 nm. Norepinephrine pretreated with ONOO-1 was administered to isolated rat thoracic aortas to observe contractions in functional experiments. The rate constant between norepinephrine and ONOO-1 was determined via a competition assay with cysteine in functional experiments. Norepinephrine pretreated with ONOO-1 was injected intravenously into anesthetized rats to measure blood pressure. Results: Norepinephrine pretreated with ONOO-1 was confirmed spectrally as oxidized norepinephrine. Norepinephrine pretreated with ONOO-1 decreased its vasocontractile force in an ONOO-1 (10(-6), up to 3 X 10(-4) M) concentration-dependent manner (EC50 = 5.1 X 10(-5) m). The decrease in its force was lower at pretreatment with ONOO-1 in a lower pH buffer. A rate constant for the ONOO-1-norepinephrine reaction was 6 X 102 m/s. Norepinephrine (10(-7) m) incubated with SIN-1 (10(-3) M) decreased its vasocontractile force in an incubation time-dependent manner. Administration of norepinephrine pretreated with ONOO-1 to anesthetized rats caused no significant change in arterial blood pressure. Conclusions. These results indicate that norepinephrine was oxidized and deactivated by ONOO-1. This deactivation may, at least in part, account for the hyporeactivities of vasocontraction to norepinephrine in septic shock.