Modification of alpha1 -adrenoceptors by peroxynitrite as a possible mechanism of systemic hypotension in sepsis

OBJECTIVE: It is well known that nitric oxide synthase is induced by endotoxin or inflammatory cytokines, and consequently large amounts of nitric oxide cause vascular hyporeactivity to vasoconstrictor agents and myocardial dysfunction, hence hypotension. However, there is considerable controversy as to whether these pathologic cardiovascular features are mediated directly by nitric oxide or also through the formation of secondary reaction products such as peroxynitrite (ONOO-1). Our objective was to investigate inhibitory effects of ONOO-1 on alpha1-adrenoceptors. DESIGN: Prospective, controlled, in vitro, laboratory study. SETTING: Laboratory of a health sciences university. SUBJECTS: Chinese hamster ovary cells that expressed the human recombinant alpha1a-, alpha1b-, or alpha1d-adrenoceptors, rat aorta strips. INTERVENTIONS: Binding experiments of [3H]prazosin were done in the Chinese hamster ovary cell membranes pretreated with 100 microM to 3 mM ONOO-1. Displacement experiments with noradrenaline or 3-nitro-l-tyrosine also were conducted. Mobilization of intracellular Ca2+ evoked by 1 nM to 10 microM noradrenaline was monitored in a fluorescence spectrophotometer with dual excitation at 340 nm/380 nm and emission at 500 nm in fura-2/AM-loaded Chinese hamster ovary cells. Contractile force produced by noradrenaline was monitored in rat aorta strips that have alpha1a- and alpha1d-adrenoceptors, pretreated with 1 mM ONOO-1. Either 0.3 N NaOH or the decomposed ONOO-1 was used as the control. MEASUREMENTS AND MAIN RESULTS: The specific binding of [3H]prazosin to alpha1a- and alpha1d-adrenoceptor was inhibited by ONOO-1 in a concentration-dependent manner. We found that 3 mM ONOO-1 decreased maximum binding sites by 40% to 50% in alpha1a- and alpha1d-adrenoceptors. Binding affinities for prazosin and noradrenaline were not affected by 1 mM ONOO-1 in all subtypes. We found that 3-nitro-l-tyrosine did not affect the prazosin binding to three adrenoceptor subtypes. Noradrenaline increased intracellular Ca2+ concentration ([Ca2+]i) concentration-dependently, which was inhibited by ONOO-1 in alpha1a- and alpha1d-adrenoceptors. ONOO-1 had no effect on alpha1b-adrenoceptor. Contractile force produced by noradrenaline decreased significantly in aorta strips pretreated with ONOO-1. CONCLUSION: ONOO-1 reduces the binding capacity of alpha1a- and alpha1d- but not alpha1b-adrenoceptors without changing the affinities. Treatment with ONOO-1 attenuates noradrenaline-stimulated increase in [Ca2+]i in alpha1a- and alpha1d-adrenoceptors but not in alpha1b-adrenoceptor. ONOO-1 also weakens noradrenaline-induced contractions in rat aorta that has alpha1a- and alpha1d-adrenoceptors. Cardiovascular hyporeactivity to catecholamines in septic shock may be caused in part by the inactivation of alpha-adrenoceptors by ONOO-1.