Induction of Nitric Oxide Synthase Gene by Interleukin-1f in Cultured Rat Cardiocytes

Background Impaired myocardial contractility in septic shock is protracting, which may be caused by cytokine-induced nitric oxide (NO) synthesis in the heart. However, the cellular mechanism by which cytokines induce nitric oxide synthase (NOS) in cardiocytes remains obscure. Methods and Results We studied the effect of human recombinant interleukin-l(3 (IL-1a8) on synthesis of N02-NO3(NOj) and the expression of NOS mRNA and protein in cultured neonatal rat cardiocytes. IL-1f3 dose-dependently (0.1 to 10 ng/mL) stimulated NOQ production as a function of time (6 to 48 hours). Northern blot analysis using complementary DNAs for rat brain-type constitutive (c) NOS and mouse macrophage-type inducible (i) NOS as probes showed that IL-1l3 induced expression of mRNA for iNOS but not for cNOS, starting after 6 hours and reaching a maximum after 48 hours in cardiocytes. IL-lp similarly induced iNOS mRNA expression in cultured adult rat cardiocytes in a time-dependent manner. Western blot analysis using specific antibody against the N-terminal fragment of mouse iNOS revealed the expression of 130-kD iNOS-like protein in IL-113-treated cardiocytes. Northern blotting and immunocytochemical study revealed that IL-1,3-induced iNOS mRNA and iNOS-like immunoreactivity were exclusively localized to cardiac myocytes but also to nonmyocytes, to a lesser extent. N0-monomethyl-L-arginine, an NOS inhibitor, completely blocked the IL-1p3-induced NO1 production, whose effect was reversed by L-arginine but not by D-arginine. Dexamethasone inhibited the IL-1X3-induced NO, production as well as iNOS mRNA expression. Cycloheximide and actinomycin D completely inhibited the IL-1,8-induced NO, production and iNOS mRNA expression. Neither a calmodulin inhibitor (W-7), a protein kinase C inhibitor (calphostin C), nor a Ca2' channel antagonist (nicardipine) showed any effect on the IL-13-induced NO, production. Conclusions These data demonstrate that IL-1,3 induces macrophage-type iNOS mRNA expression mainly by cardiac myocytes but also by nonmyocytes to a lesser extent, and subsequent de novo protein synthesis of iNOS leads to excessive local production of NO by cardiocytes. (Circulation. 1994;90:375-383.)