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

Abstract

Atrial natriuretic peptide (ANP) attenuates LPS‐induced inducible nitric oxide 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 inhibition of iNOS by ANP is due to alterations in intracellular calcium. As determined by fluorescence photometry, ANP (10–7 and 10–6 mol/L) was shown to elevate intracellular calcium levels in bone marrow‐derived macrophages. This effect seemed to be mediated via the guanylate cyclase‐coupled A receptor, because dibutyryl‐cGMP mimicked and the A‐receptor antagonist HS‐142‐1 partially abrogated the effect of ANP. Because the Ca2+ increase was also observed in Ca2+‐free buffer, it is suggested that the liberation of intracellular calcium pools contributes to the elevation of [Ca2+]i by ANP. The B‐receptor ligand C‐type natriuretic peptide (CNP), which does not alter iNOS expression, had no effect on [Ca2+]i. The Ca2+‐ionophore 4‐Br‐A23187 and thapsigargin, a compound known to liberate Ca2+ from intracellular stores, were further demonstrated to reduce LPS‐induced NO production in macrophages (Griess assay), confirming a functional link for elevated [Ca2+]i and iNOS inhibition. These effects were abrogated by coincubation with extra‐ 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 on NO production was also abrogated by Ca2+ chelation. These findings support a causal relationship between reduced iNOS induction and elevation of [Ca2+]i. Taken together, the data indicate that intracellular Ca2+ elevation by ANP is involved in the inhibition of LPS‐induced nitric oxide production in macrophages.

Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture

“…It was reported that application of TG is also associated with activation of programmed cell death in various liver epithelial cells: human liver-derived cell line, Huh7 cells (Xie et al, 2002;Chae et al, 2004), different hepatoma cell lines (Sohn et al 2003), and primary rodent hepatocytes (Li and Holbrook, 2004;. Moreover, TG can modulate NO production in macrophages as previously reported (Jordan et al, 1995;Jun et al, 1996;Park et al, 1996;Chen BC et al, 2001;Kiemer and Vollmar, 2001;Chen YJ et al, 2005;Kmonickova et al, 2005). TG can stimulate and/or inhibit NO production and iNOS expression mostly depending on an increase in [Ca 2+ ] i , immunological stimulation, and protein kinase C status of macrophages.…”

“…We have previously observed (Kmonickova et al, 2005) that TG suppressed immunologically induced high-output NO production that was accompanied by decreased number of viable mouse peritoneal macrophages in culture. Although iNOS is traditionally considered as Ca 2+ independent, it was shown that an increase in [Ca 2+ ] i caused by TG and other ionophores downregulates iNOS mRNA induction and NO synthesis in lipopolysaccharide-stimulated macrophages (Jordan et al, 1995;Kiemer and Vollmar, 2001). The differential effects of TG in the 0-24 h and 24-48 h periods are noteworthy and may be correlated with different degrees of activation of the basal iNOS pathway observed immediately after collagenase method isolation or 24 h after cell isolation.…”

Endothelial Dysfunction and Damage in Congestive Heart Failure

“…Thus far, there is only conflicting evidence from a few studies of such a possible relation, some showing upregulation of eNOS and iNOS by ANP and BNP 23,24 and others downregulation of tumor necrosis factor-␣ (TNF-␣) and, indirectly, iNOS by ANP. [25][26][27] Although strictly speaking, the latter studies were performed using ANP, both ANP and BNP appear to have identical functions. 28 Therefore, whereas upregulation of eNOS and iNOS should improve FMD as shown by exogenous administration of BNP, albeit in healthy individuals, 11,12 downregulation of TNF-␣ and consequently iNOS may impair FMD further.…”

Induction of Glucocorticoid-induced Leucine Zipper (GILZ) Contributes to Anti-inflammatory Effects of the Natural Product Curcumin in Macrophages

“…After 20 h, the concentration of nitrite, a stable metabolite of NO, was measured in the supernatants by Griess assay as described previously (58). Briefly, 90 l of 1% sulfanilamide in 5% H 3 PO 4 and 90 l of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in H 2 O were added to 100 l of the cell culture supernatant followed by absorbance measurement at 550 nm and the reference wavelength 630 nm using a Tecan Sunrise microplate reader.…”