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

Abstract

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. 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. 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 (EC(50) = 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 10(2) 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. 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.