Inhaled isobutyl nitrite inhibited macrophage inducible nitric oxide by blocking NF$kappa;B signaling and promoting degradation of inducible nitric oxide synthase-2

Abstract

We previously reported that inhaled isobutyl nitrite inhibited macrophage tumoricidal activity by inhibiting inducible nitric oxide (NO) production. In the present study, a much shorter inhalant exposure regimen (five daily exposures) inhibited inducible NO and the NO synthase (NOS2). One of the ways in which NO and NOS2 are regulated is by ubiquitin-dependent NOS2 degradation. Immunoprecipitated NOS2 showed increased poly-ubiquitination, following exposure to the inhalant. In addition, Western blots of macrophage nuclear extracts for the NFκB subunit, p65, showed that exposure to the inhalant inhibited NFκB signaling, necessary for induction of NOS2. The inhalant blocked phosphorylation of the NFκB inhibitor, IκBα. The inhibition of NFκB signaling following inhalant exposure was confirmed using mice transgenic for the κB-dependent promoter of the HIV 5′ LTR linked to luciferase. The data suggested that inhalant exposure likely inhibited macrophage NO production by blocking NFκB-mediated activation signaling and promoting poly ubiquitination of NOS2.