Atrial Natriuretic Peptide Enhances IL-1β-Stimulated Nitric Oxide Production in Cultured Rat Vascular Smooth Muscle Cells

Abstract

Objective: To elucidate the effect of atrial natriuretic peptide (ANP) on cytokine-induced nitric oxide (NO) production, we examined whether ANP affects IL-1β-stimulated NO production and inducible NO synthase (iNOS) mRNA expression in cultured rat vascular smooth muscle cells (VSMC). Methods: VSMC were incubated with test agents for 24 h. The nitrite concentration of the culture medium, a stable-end product of NO, was measured by the column reduction method. NOS mRNA expression was analyzed by Northern blotting. The intracellular cAMP content was measured by enzyme immunoassay. cAMP-dependent kinase (PKA) activity was determined by a PKA assay system. Results: IL-1β stimulated nitrite production in VSMC. ANP (10 nM to 1 mM) enhanced the nitrite production and iNOS mRNA expression in the presence of IL-1β. Both 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-br-cGMP) and dibutylyl adenosine 3′,5′-cyclic monophate enhanced IL-1β-induced nitrite production. The effects of these two nucleotide donors on the nitrite production were not additive, suggesting a common pathway. KT 5720, an inhibitor of PKA, abolished the enhancement of nitrite production by ANP and 8-br-cGMP, while KT 5823, an inhibitor of cGMP-dependent protein kinase, did not inhibit the enhancement. In the in vitro assay 8-br-cGMP increased PKA activity. Conclusions: ANP and cGMP enhanced IL-1β-induced NO production in VSMC. PKA rather than PKG may be involved in the enhancement.