Impact of silencing HO-2 on EC-SOD and the mitochondrial signaling pathway

The contribution of heme oxygenase HO-2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO-2, we examined the effect of suppressed levels of HO-2 on vascular antioxidant and survival proteins. In vivo HO-2 siRNA treatment decreased the basal levels of EC-SOD, pAKT proteins (serine-473 and threonine-308), without changing Akt protein expression. HO-2 siRNA treatment increased 3-nitrotyrosine (3-NT) and apoptotic signaling kinase-1 (ASK-1) (P < 0.01). HO activity was decreased by the use of siRNA HO-2. We extended these studies to the mitochondria, examining for the presence of HO-1 and its role in the regulation of pro- and anti-apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO-1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO-2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC-SOD, Akt, 3-NT, and ASK-1. In addition, localization of HO-1 in the mitochondrial compartment plays a critical role in mitochondria-mediated apoptosis.     

Catalytically inactive heme oxygenase-2 mutant is cytoprotective

Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself.     

Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.

It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.     

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.     

Heme oxygenase expression in selected regions of term human placenta

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase     

BMP6 attenuates oxidant injury in HK-2 cells via Smad-dependent HO-1 induction

Oxidative stress is involved in a variety of kidney diseases, and heme oxygenase 1 (HO-1) induction is a protective response to oxidative stress. Downregulation of bone morphogenetic protein 6 (BMP6) is associated with renal damage in intrauterine growth-restricted newborns. However, it is unknown whether BMP6 has a renoprotective effect or HO-1 induction property. In this study, we demonstrate that BMP6 effectively protects renal proximal tubule cells (HK-2) against hydrogen peroxide (H₂O₂)-induced cell injury. BMP6 also increased HO-1 gene expression and activity of HO. Inhibition of de novo gene expression, the HO inhibitor ZnPPIX, HO-1 knockdown, or the carbon monoxide (CO) scavenger hemoglobin attenuated the cytoprotective effect of BMP6, whereas HO-1 constitutive expression, the HO-1 inducer hemin, or the hemin metabolites bilirubin and CO ameliorated H₂O₂-induced cell injury. Stimulation of HK-2 cells with BMP6 activated Smad signaling but not mitogen-activated protein kinases. In addition, BMP6-mediated induction of HO-1 expression and increase in HO activity were inhibited by Smad5 knockdown. Furthermore, deletion or mutation of the Smad-binding element in the HO-1 promoter also inhibited BMP6-induced luciferase activity. In summary, these findings suggest that induction of HO-1 through a Smad-dependent mechanism is responsible for the cytoprotective effect of BMP6 in H₂O₂-mediated renal cell injury.     

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway: HEME OXYGENASE-2, CYTOCHROME P450 REDUCTASE,AND BILIVERDIN REDUCTASE*

Heme oxygenase (HO) catalyzes the rate-limiting step in the O₂-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The ¹H-¹⁵N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in K_m values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (K_d = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the ¹H-¹⁵N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence.     

Differential effect of cobalt protoporphyrin on distributions of heme oxygenase in renal structure and on blood pressure in SHR.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, releasing iron and carbon monoxide (CO). Thus, HO not only controls the availability of heme for the synthesis of hemeproteins but also generates CO, which binds to the heme moiety of hemoproteins, thereby affecting their enzymatic activity. The present study was undertaken to explore changes in the relative expression of renal HO-1 and HO-2 in response to modulators and the effect on blood pressure regulation in spontaneously hypertensive rats (SHR). Immunohistochemistry confirmed a cobalt protoporphyrin (CoPP)-mediated increase in HO-1 protein. After a single injection of CoPP (5 mg/100 gram body weight) in 7-week-old SHR, blood pressure significantly decreased (p<0.01) while renal HO activity increased 6-fold over controls. CoPP pretreatment deceased the levels of the renal cytochrome P450-derived arachidonic acid metabolite, 20-HETE, a powerful vasoconstrictor, by 65% in renal tissue. Western blot analysis demonstrated that CoPP significantly increased HO-1 protein expression in the cortex and outer medulla and, to a lesser degree, in the inner medulla of the rat kidney. HO-2 was constitutively expressed in all parts of the kidney, and did not significantly change after treatment with CoPP. These results indicate that selective induction of cortical and outer medullary HO-1 is associated with a decrease in 20-HETE and blood pressure, suggesting an important role for HO-1 activity in the regulation of urine volume, electrolyte excretion and blood pressure.     

Subcellular localization of iron and heme metabolism related proteins at early stages of erythrophagocytosis

Background

Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined.

Methodology/Principal Findings

We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes.

Conclusions/Significance

Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.     

Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.     

Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression

The extended postictal state is characterized by neurological problems in patients. Inadequate blood supply to the brain and impaired cerebral autoregulation may contribute to seizure-induced neuronal damage. Recent evidence in newborn pigs indicates that activation of the antioxidative enzyme heme oxygenase (HO) at the onset of seizures is necessary for increased cerebral blood flow during the ictal episode and for normal cerebral vascular functioning during the immediate postictal period. We hypothesized that seizures cause prolonged postictal cerebral vascular dysfunction that can be accentuated by HO inhibition and rescued by HO overexpression. Cerebral vascular responses to endothelium-dependent (hypercapnia, bradykinin) and -independent (isoproterenol, sodium nitroprusside) stimuli were assessed 48 h after bicuculline-induced seizures in: 1) saline-control newborn piglets, 2) HO-inhibited animals (HO was inhibited by tin protoporphyrin, SnPP, 3 mg/kg iv), and 3) HO-overexpressing piglets (HO-1 was upregulated by cobalt protoporphyrin, CoPP, 50 mg/kg ip). Extended alterations of HO expression in cerebral microvessels were confirmed by measuring CO production and inducible HO (HO-1) and constitutive HO (HO-2) proteins. Our data provide evidence that seizures cause a severe, sustained, postictal cerebral vascular dysfunction as reflected by impaired vascular reactivity to physiologically relevant dilators. During the delayed postictal state, vascular reactivity to all dilator stimuli was reduced in saline control and, to a greater extent, in HO-inhibited animals. In CoPP-treated piglets, no reduction in postictal cerebral vascular reactivity was observed. These findings may indicate that CoPP prevents postictal cerebral vascular dysfunction by upregulating HO-1, a finding that might have implications for preventing postictal neurological complications.     

A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes

Ultraviolet A (UVA) radiation is an oxidizing agent that strongly induces the heme oxygenase 1 (HO-1) gene and expression of the protein in cultured human skin fibroblasts but weakly induces it in skin keratinocytes. Lower basal levels of HO-1 and much higher basal levels of HO-2 protein are observed in keratinocytes compared with fibroblasts. Using both overexpression and knockdown approaches, we demonstrate that HO-2 modulates basal and UVA-induced HO-1 protein levels, whereas HO-1 levels do not affect HO-2 levels in skin fibroblasts and keratinocytes. Silencing of Bach1 strongly increases HO-1 levels in transformed HaCaT keratinocytes and these HO-1 levels are not further increased by either UVA irradiation or silencing of HO-2. This is consistent with the conclusion that high constitutive levels of HO-2 expression in keratinocytes are responsible for the resistance of these cells to HO-1 induction by UVA radiation and that Bach1 plays a predominant role in influencing the lack of HO-1 expression in keratinocytes. Bach1 inhibition leading to HO-1 induction reduced UVA-irradiation-induced damage as monitored both by the extent of LDH release and by nuclear condensation, so that Bach1 inhibition seems to protect against UVA-irradiation-induced damage in keratinocytes.     

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.     

Heme oxygenase activity in placenta: direct dependence on oxygen availability

Carbon monoxide (CO), which is formed endogenously from heme catalyzed by heme oxygenase (HO), is proposed to play a role in vascular control. The mRNA and protein expression of the inducible isoform of HO (HO-1) increases in response to hypoxia, and it has been assumed that HO activity also increases. This assumption requires evaluation because the catalytic activity of HO requires three molecules of O(2) for each molecule of CO formed from heme, and HO activity may be limited by O(2) availability. To test the hypothesis that low physiological O(2) concentrations limit HO activity, heme-derived CO formation by microsomal fractions of homogenates of chorionic villi of human placentas was determined after exposure to 0, 1, 5, or 21% O(2). Results revealed that HO activity was directly dependent on O(2) concentration. Thus, although hypoxia may increase HO protein and mRNA expression, there is a progressive decrease in HO activity with decreasing O(2) concentration and the dependence of HO activity on O(2) concentration is similar in chorionic villi from noninfarcted areas of preeclamptic and normotensive placenta.     

Carbon monoxide actuates O(2)-limited heme degradation in the rat brain

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.