Elevation of intracellular calcium levels contributes to the inhibition ofnitric oxide production by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) attenuates LPS-induced inducible nitric ox ide synthase (iNOS) expression in murine macrophages by destabilizing iNOS mRNA. Because elevated intracellular free Ca2+ levels [Ca2+](i) reduce iNOS mRNA stability, the aim of the present study was to determine whether inhi bition of iNOS by ANP is due to alterations in intracellular calcium. As de termined by fluorescence photometry, ANP (10(-7) and 10(-6) mol/L) was show n to elevate intracellular calcium levels in bone marrow-derived macrophage s. This effect seemed to be mediated via the guanylate cyclase-coupled A re ceptor, because dibutyryl-cGMP mimicked and the A-receptor antagonist HS-14 2-1 partially abrogated the effect of ANP. Because the Ca2+ increase was al so observed in Ca2+-free buffer, it is suggested that the liberation of int racellular calcium pools contributes to the elevation of [Ca2+](i) by ANP. The B-receptor ligand C-type natriuretic peptide (CNP), which does not alte r iNOS expression, had no effect on [Ca2+](i). The Ca2+-ionophore 4-Br-A23 187 and thapsigargin, a compound known to liberate Ca2+ from intracellular stores, were further demonstrated to reduce LPS-induced NO production in ma crophages (Griess assay), confirming a functional link for elevated [Ca2+]( i) and iNOS inhibition. These effects were abrogated by coincubation with e xtra- as well as intracellular Ca2+ chelators (EGTA, 1,2-bis(o-aminophenoxy )ethane-N,N,N',N'-tetraacetic acid (BAPTA)). The inhibitory effect of ANP o n NO production was also abrogated by Ca2+ chelation. These findings suppor t a causal relationship between reduced iNOS induction and elevation of [Ca 2+](i). Taken together, the data indicate that intracellular Ca2+ elevation by ANP is involved in the inhibition of LPS-induced nitric oxide productio n in macrophages.