β-Adrenergic stimulation of rat cardiac fibroblasts enhances induction of nitric-oxide synthase by interleukin-1β via message stabilization

We have investigated factors modulating expression of inducible NO synthase (iNOS) in isolated adult rat cardiac fibroblasts. Treatment of cardiac fibroblasts with interleukin-1 beta (IL-1 beta) promotes induction of iNOS mRNA and protein and production of NO. Simultaneous incubation of cells with isoproterenol enhances the response to IL-1 beta, even though isoproterenol alone is without effect. N-G-nitro-L-arginine methyl ester inhibits the effect of isoproterenol + IL-1 beta on NO production. beta (2)-Adrenergic receptors appear to mediate this effect of isoproterenol. Reverse transcriptase-polymerase chain reaction analyses show that beta (2)-receptor mRNA is the predominant beta -receptor message; in pharmacologic studies, ICI-118,551 significantly antagonizes isoproterenol-stimulated cyclic AMP production whereas CGP20712A does not. Dibutyryl-cyclic AMP and forskolin mimic the synergistic effect of isoproterenol on IL-1 beta -induced NO production; H-89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, antagonizes the enhancing effect of isoproterenol. Nuclear run-off experiments indicate that enhancement of iNOS by isoproterenol does not occur at the level of transcription. Message stability studies demonstrate that isoproterenol increases the half-life of iNOS mRNA from 1.0 to 1.9 h; this change is sufficient to account for the observed augmentation of iNOS mRNA and protein. Thus, cardiac fibroblasts produce significant amounts of NO in response to IL-1 beta via induction of iNOS; beta -adrenergic stimulation enhances the IL-1 beta effect by stabilizing the iNOS message. These data suggest that cardiac fibroblasts could participate in a paracrine mechanism whereby the direct positive inotropic effect of beta (1)-adrenergic stimulation of myocytes is opposed by beta (2)-adrenergic enhancement of NO production, a negative inotropic event, in neighboring fibroblasts.