The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.     

Interaction of hyperbaric oxygen, nitric oxide, and heme oxygenase on DNA strand breaks in vivo

Hyperbaric oxygen (HBO), e.g. pure oxygen breathing at supra-atmospheric pressures, represents a well-suited model for investigating oxidative stress-induced DNA damage as well as protective mechanisms. While the induction of heme oxygenase-1 (HO-1) seems to be crucial for this protection against this DNA damage, the role of nitric oxide (NO) remains unclear. HO-1 expression is a major regulator of the inducible NO synthase (iNOS), and therefore we investigated the effect of the interaction between HBO, NO, and HO-1 on DNA damage. Prior to exposure to HBO (3 h at 3 bar ambient pressure) rats randomly received vehicle (HBO alone, 1 mL 0.9% saline, n=8), the NO donor molsidomine (SIN-10, 40 mg/kg, n=8) or the HO-1 blocker tin-mesopophyrin (Sn-MP, 50 micromol/kg, n=8). Additional groups received SIN-10 without exposure to HBO, i.e. breathing air under normobaric conditions for 3h (SIN-10 alone, 40 mg/kg, n=6), vehicle without HBO (negative controls, n=6), and ethylmethanesulfonate without HBO (EMS, 200 mg/kg) (positive controls n=4). Immediately after the 3 h HBO or air breathing period blood was analysed for DNA strand breaks (tail moment in the alkaline comet assay) and nitrite+nitrate (chemoluminescence). Whereas the tail moment was ten-fold higher after EMS than in the negative controls, there was no effect of HBO nor SIN-10 alone. Together with HBO, pretreatment with SIN-10 doubled the tail moment, and Sn-MP increased it by 50%. In contrast to Sn-MP or HBO alone, SIN-10 resulted in a five-fold increase of nitrite+nitrate concentrations. We conclude that both HO-1 blockade and excess NO release promote DNA damage during HBO exposure in vivo. The effect of HO-1 inhibition is probably independent of the regulatory function of HO-1 for iNOS.     

Interaction of nitric oxide with human heme oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.     

Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages

Leishmania donovani promastigotes, the causative agent of visceral leishmaniasis, survive inside macrophages by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for parasite survival. LPG has several detrimental effects on macrophage function, including inhibition of periphagosomal filamentous actin (F-actin) breakdown during phagosomal maturation. However, in RAW 264.7 macrophages pre-stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNgamma), known to up-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, L. donovani promastigotes are unable to inhibit periphagosomal F-actin breakdown and phagosomal maturation proceeds normally. Moreover, the iNOS inhibitor aminoguanidine, blocked the positive effects of LPS/IFNgamma suggesting that NO is a key player in F-actin remodeling. In conclusion, production of NO by stimulated macrophages seems to allow phagosomal maturation following uptake of L. donovani promastigotes, suggesting a novel mechanism whereby NO facilitates killing of an intracellular pathogen.     

Erythrophagocytosis by macrophages: suppression of heme oxygenase by cyclic AMP.

On incubation of peritoneal macrophages with antibody-coated radiolabeled erythrocytes, a reproducible fraction of the erythrocytes was phagocytized and heme oxygenase was induced. Addition of cyclic AMP, dbcyclic AMP, or theophylline to the incubation medium suppressed the substrate-mediated induction of heme oxygenase in a dose-related manner but did not impair the rate or extent of erythrophagocytosis. A similar effect was produced by epinephrine, norepinephrine, isoproterenol, and prostaglandins, which generate endogenous cyclic AMP by stimulating the adenyl cyclase system. Propanolol completely blocked the suppressive effect of epinephrine, while phentolamine was ineffective. In contrast to the cyclic adenosine nucleotide, cyclic GMP probably slightly enhanced the substrate-mediated induction of heme oxygenase and partly reversed the suppressive effect of cyclic AMP. Cyclic adenosine nucleotides, prostaglandin, and theophylline significantly reduced the incorporation of labeled uridine or leucine into RNA and protein of erythrophagocytic macrophages, but failed to impair the uptake of these precursors by the phagocytizing cells. These compounds also reduced the conversion of [1-¹⁴C] glucose to ¹⁴CO₂ by the incubated macrophages, whereas ¹⁴CO₂ formation was enhanced by epinephrine. None of these effects was reversible by addition of insulin or by glucose supplementation, which is in sharp contrast to the suppressive effect of glucocorticoids on heme oxygenase induction.     

Anti-inflammatory effect and inhibition of nitric oxide production by targeting COXs and iNOS enzymes with the 1,2-diphenylbenzimidazole pharmacophore

Being the base of several non-communicable diseases, including cancer, inflammation is a complex process generated by tissue damage or change in the body homeostatic state. Currently, the therapeutic treatment for chronic inflammation related diseases is based on the use of selective cyclooxygenase II enzyme, COX-2, inhibitors or Coxibs, which have recently regained attention giving their preventive role in colon cancer. Thus, the discovery of new molecules that selectively inhibit COX-2 and other inflammatory mediators is a current challenge in the medicinal chemistry field. 1-Phenylbenzimidazoles have shown potential COX inhibitory activity, because they can reproduce the interaction profile of known COX inhibitors. Therefore, in the present investigation a series of 1,2-diphenylbenzimidazoles (DPBI) with different aromatic substitutions in the para position were synthesized and their interaction with COX-2 and nitric oxide synthase, iNOS, was determined in silico, in vitro and in vivo. Compound 2-(4-bromophenyl)-1-(4-nitrophenyl)-1H-benzo[d]imidazole showed the best inhibition towards COX-2, while compounds N-(4-(2-(4-bromophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide and N-(4-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide diminished the production of NO in vitro. Additionally, they had a significant anti-inflammatory activity in vivo when given orally.     

Baicalein inhibition of hydrogen peroxide-induced apoptosis via ROS-dependent heme oxygenase 1 gene expression

In the present study, baicalein (BE) but not its glycoside, baicalin (BI), induced heme oxygenase-1 (HO-1) gene expression at both the mRNA and protein levels, and the BE-induced HO-1 protein was blocked by adding cycloheximide (CHX) or actinomycin D (Act D). Activation of ERK, but not JNK or p38, proteins via induction of phosphorylation in accordance with increasing intracellular peroxide levels was detected in BE-treated RAW264.7 macrophages. The addition of the ERK inhibitor, PD98059, (but not the p38 inhibitor, SB203580, or the JNK inhibitor, SP600125) and the chemical antioxidant, N-acetyl cysteine (NAC), significantly reduced BE-induced HO-1 protein expression by respectively blocking ERK protein phosphorylation and intracellular peroxide production. Additionally, BE but not BI effectively protected RAW264.7 cells from hydrogen peroxide (H(2)O(2))-induced cytotoxicity, and the preventive effect was attenuated by the addition of the HO inhibitor, SnPP, and the ERK inhibitor, PD98059. H(2)O(2)-induced apoptotic events including hypodiploid cells, DNA fragmentation, activation of caspase 3 enzyme activity, and a loss in the mitochondrial membrane potential with the concomitant release of cytochrome c from mitochondria to the cytosol were suppressed by the addition of BE but not BI. Blocking HO-1 protein expression by the HO-1 antisense oligonucleotide attenuated the protective effect of BE against H(2)O(2)-induced apoptosis by suppressing HO-1 gene expression in macrophages. Overexpression of the HO-1 protein inhibited H(2)O(2)-induced apoptotic events such as DNA fragmentation and hypodiploid cells by reducing intracellular peroxide production induced by H(2)O(2), compared with those events in neo-control (neo-RAW264.7) cells. In addition, CO, but not bilirubin and biliverdin, addition inhibits H(2)O(2)-induced cytotoxicity in macrophages. It suggests that CO can be responsible for the protective effect associated with HO-1 overexpression. The notion of induction of HO-1 gene expression through a ROS-dependent manner suppressing H(2)O(2)-induced cell death is identified in the present study.     

Nitric oxide and prostaglandin E2 participate in lipopolysaccharide/interferon-gamma-induced heme oxygenase 1 and prevent RAW264.7 macrophages from UV-irradiation-induced cell death

Induction of heme oxygenase (HO)-1 during inflammation has been demonstrated in many cell types, but the contribution of inflammatory molecules nitric oxide (NO) and prostaglandin E(2) (PGE(2)) has remained unresolved. Here we show that NO donors including sodium nitroprusside (SNP) and spermine nonoate (SP-NO), and PGE(2) significantly stimulate HO-1 expression in RAW264.7 macrophages, associated with alternative induction on NO and PGE(2) in medium, respectively. NO donors also show the inductive effect on cyclo-oxygenase 2 protein and PGE(2) production. In the presence of lipopolysaccharide and interferon-gamma (LPS/IFN-gamma), HO-1 protein was induced slightly but significantly, and SNP, SP-NO, and PGE(2) enhanced HO-1 protein induced by LPS/IFN-gamma. L-Arginine analogs N-nitro-L-arginine methyl ester (L-NAME) and N-nitro-L-arginine (NLA) significantly block HO-1 protein induced by LPS/IFN-gamma associated with a decrease in NO (not PGE(2)) production. And, NSAIDs aspirin and diclofenase dose dependently inhibited LPS/IFN-gamma-induced HO-1 protein accompanied by suppression of PGE(2) (not NO) production. PD98059 (a specific inhibitor of MEKK), but not SB203580 (a specific inhibitor of p38 kinase), attenuated PGE(2) (not SP-NO) induced HO-1 protein. Under UVC (100 J/m(2)) and UVB (50 J/m(2)) irradiation, PGE(2) or SP-NO treatment prevents cells from UVC or UVB-induced cell death, and HO-1 inhibitor tin protoporphyrin (SnPP) reverses the preventive effects of PGE(2) and SP-NO. The protective activity induced by PGE(2) on UVC or UVB irradiation-induced cell death was blocked by MAPK inhibitor PD98059 (not SB203580). These results demonstrated that inflammatory molecules NO and PGE(2) were potent inducers of HO-1 gene, and protected cells from UV-irradiation-induced cell death through HO-1 induction.     

In vitro and in vivo induction of heme oxygenase 1 in mouse macrophages following melanocortin receptor activation

RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4-24 h) and concentration-related (1-100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (Mphi) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary Mphi. HO-1 induction in peritoneal Mphi was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the Mphi, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.     

Modified secoiridoid from Acicarpha tribuloides and inhibition of nitric oxide production in LPS-activated macrophages

Bioassay-guided fractionation of Acicarpha tribuloides Juss. resulted in the isolation of an uncommon non-glycosylated secoiridoid, tribulolide (1), two known secoiridoid glycosides named secologanic acid (2) and vogeloside (3) as well as two natural chromones, 6,7-dimethoxychromone (4) and 7-hydroxy-6-methoxy-chromone (5). Compounds 1-3 showed inhibition of nitric oxide production in lipopolysaccharide-activated macrophages; their activity is comparable to that of aminoguanidine, a classic inhibitor.     

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H₄B) and is the site for NO synthesis. The role of H₄B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H₄B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H₄B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H₄B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005) Supported by Calcutta University Research Grants.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

Protective effect of Hypericum calabricum Sprengel on oxidative damage and its inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages

The present study shows for the first time the phenolic composition and the in vitro properties (antioxidant and inhibition of nitric oxide production) of Hypericum calabricum Sprengel collected in Italy. The content of hypericins (hypericin and pseudohypericin), hyperforin, flavonoids (rutin, hyperoside, isoquercetrin, quercitrin, quercetin and biapigenin) and chlorogenic acid of H. calabricum, have been determined. The ethyl acetate fraction from the aerial parts of H. calabricum exhibited activity against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 value of 1.6 jig/ml. The test for inhibition of nitric oxide (NO) production was performed using the murine monocytic macrophage cell line RAW 264.7. The ethyl acetate fraction had significant activity with an IC50 value of 102 jig/ml and this might indicate that it would have an anti-inflammatory effect in vivo.