Iron Released by Sodium Nitroprusside Contributes to Heme Oxygenase-1 Induction via the cAMP-Protein Kinase A-Mitogen-Activated Protein Kinase Pathway in RAW 264.7 Cells

Abstract

Nitric oxide (NO) is a potent inducer of heme oxygenase (HO)-1, and NO-induced HO-1 expression is dependent on the cGMPsignaling pathway. Sodium nitroprusside (SNP) produces NO and iron. However, it is unclear whether NO is exclusively responsible for induction of HO-1 by SNP in RAW 264.7 cells. We tested our hypothesis that iron may contribute more to the SNP induction of HO-1 than does NO by comparing the HO-1 protein level and the production of NO in RAW 264.7 cells treated with SNP and S-nitroso-N-acetyl-DL-penicillamine (SNAP). Although SNP induced less NO production than SNAP, SNP induced the production of more HO-1 protein than did SNAP. Deferoxamine (DFO) decreased SNP-but not SNAPinduced HO-1 expression but did not decrease the production of NO. SNP-induced HO-1 was significantly inhibited by specific protein kinase A (PKA) inhibitors or an antagonist of cAMP but not by guanylyl cyclase inhibitors. Exogenous iron (ferric ammonium citrate or ferricyanide) and forskolin increased the level of HO-1, which was inhibited by PKA inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). These results indicate that iron and cAMP, but not cGMP, play crucial roles in the induction of HO-1 in RAW 264.7 cells. Moreover, DFO and inhibitors of extracellular signal-related kinases 1/2 or c-Jun NH 2 -terminal kinase inhibited HO-1 production induced by SNP. This study illustrates that iron rather than NO from SNP contributes to HO-1 induction. Therefore, studies on the effects of SNP should consider the role of iron in some biological functions. We concluded that iron released by SNP contributes to HO-1 induction via the cAMP-PKA-mitogen-activated protein kinase pathway.Heme oxygenase (HO) is an essential enzyme in heme catabolism that cleaves heme to release carbon monoxide, iron, and biliverdin (Tenhunen et al., 1968(Tenhunen et al., , 1969. HO-1 is induced by a variety of physiological stimuli, including heme, heavy metals, inflammatory cytokines, endotoxins, and nitric oxide (NO) (Durante et al., 1997b;Maines, 1997;Yet et al., 1997). Recent studies have shown that HO-1 expression plays a critical role in mediating antioxidant, anti-inflammatory, and antiapoptotic effects (Brouard et al., 2000;Otterbein et al., 2000). The beneficial effects of HO-1 induction might occur via several postulated mechanisms. Increased HO-1 activity results in the degradation of the heme moiety, a potentially toxic prooxidant, and generates bilirubins, an antioxidant capable of scavenging peroxy radicals and inhibiting lipid peroxidation (Stocker et al., 1987;Llesuy and Tomaro, 1994;Nath et al., 1998).NO is a free radical involved in the regulation of many physiological functions, including endothelium-dependent vasodilation, neurotransmission, and the cell-mediated im-

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“…3B). A NO donor SNP releases iron along with NO (50). A small amount of iron may affect microglial viability.…”

No Nitric Oxide for HO-1 from Sodium Nitroprusside: Fig. 1.

“…In addition, scavengers of NO as well as inhibitors of its downstream signaling target, soluble guanylyl cyclase, are without effect on HO-1 induction by SNP in RAW macrophages. Kim et al (2006) conclude that free iron and not NO is the driving force behind the observed increases in HO-1 protein after exposure to SNP. Induction of HO-1 by SNP but not SNAP is blocked upon coincubation with the iron binding compound deferoxamine, and the authors are able to mimic the effect of SNP on HO-1 by use of exogenous iron (ferricyanide or ferric ammonium citrate).…”

“…Using RAW 264.7 murine macrophages, Kim et al (2006) clearly demonstrate that, in the case of HO-1 induction by SNP, NO has, at best, a negligible role as a mediator of this action of SNP. In a number of well designed experiments and by using appropriate controls such as the "true" donor of NO, SNAP, Kim et al (2006) provide compelling evidence that SNP's NO or NO ϩ contribute minimally to HO-1 induction.…”

“…Downstream effects of iron, after being dissociated from SNP, include increases in intracellular cAMP that are causally linked to subsequent phosphorylation of specific MAPK targets and enhanced HO-1 protein levels. Based on the data reported by Kim et al (2006), the use of SNP as an experimental tool to mimic intracellular effects of NO should be avoided in the future. This work not only helps revise concepts in NO and HO-1 research but also may direct future efforts to the role of iron and reactive oxygen species in the regulation of adenylyl cyclase.…”

“…The answer given by Kim et al (2006) is a very simple and unexpected one, based on the generally accepted concept of iron working through iron regulatory and binding proteins such IRP1 and IRP2. Using an inhibitor approach, Kim et al (2006) demonstrate that iron released from SNP induces HO-1 via adenylyl cyclase activation and enhanced formation of cAMP; exogenous iron as well as SNP increase both intracellular cAMP and HO-1 levels. HO-1 induction by both SNP and iron is almost completely blocked by inhibitors of protein kinase A (PKA).…”