The role of heme in the regulation of tryptophan-2,3-dioxygenase activity and content of cytochrome P-450 in rat liver

The effects of exogenous heme on the activity of delta-aminolevulinate synthase, heme oxygenase, tryptophan-2.3-dioxygenase and microsomal cytochrome content in rat liver were studied. It was shown that hemin chloride diminishes the delta-aminolevulinate synthase activity and provokes heme oxygenase induction. This is paralleled with the induction of the tryptophan 2.3-dioxygenase apoenzyme and an increase in the saturation of the enzyme with heme. The cytochrome b5 content does not change thereby, whereas that of cytochrome P-450 shows a decrease. Upon combined administration of actinomycin D and hemin the cytochrome P-450 level is markedly increased. Actinomycin D by itself has no effect on the hemoprotein concentration. It is concluded that the increase in the cytochrome P-450 level results from the activation of heme-induced mRNA translation.     

Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis

Free radical-mediated mitochondrial dysfunction may play a role in the genesis of sepsis-induced multiorgan failure. Several cellular defenses protect against free radicals, including heme oxygenase. No previous study has determined if measures that increase heme oxygenase levels reduce mitochondrial dysfunction following endotoxin. The purpose of the present study was to determine if mitochondrial dysfunction following endotoxin (LPS) administration can be attenuated by administration of hemin, a pharmacological inducer of heme oxygenase. Blood pressure, heart rate, cardiac and diaphragm mitochondrial function, plasma nitrite/nitrate levels, and tissue markers of free radical generation were compared among rats given saline, LPS, hemin, or a combination of hemin and LPS. Endotoxin (LPS) administration produced large reductions in mitochondrial function (e.g., ATP production rate decreased in both tissues, P < 0.001). Administration of hemin increased tissue heme oxygenase levels, ablated LPS-induced alterations in mitochondrial function, attenuated LPS-induced increases in plasma nitrite/nitrate levels, and prevented LPS-mediated increases in tissue markers of free radical generation. These data indicate that tissue heme oxygenase levels modulate the degree of LPS-induced mitochondrial dysfunction. Measures that increase heme oxygenase levels may provide a means of reducing sepsis-induced mitochondrial dysfunction and tissue injury.     

Bilirubin and ferritin as protectors against hemin-induced oxidative stress in rat liver.

The in vivo effect of hemin on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 hr after hemin administration. Heme oxygenase-1 activity and expression appeared 6 hr after treatment, reaching a maximum between 12 and 15 hr after hemin administration. Such induction was preceded by a decrease in the soluble and enzymatic defenses, both effects taking place some hours before induction of heme oxygenase. Ferritin content began to increase 6 hr after heme oxygenase induction, and these increases were significantly higher 15 hr after treatment and remained high for at least 24 hr after hemin injection. Co-administration of tin protoporphyrin IX, a potent inhibitor of heme oxygenase, completely prevented the enzyme induction and the increase in ferritin levels, increasing the appearance of oxidative stress parameters. Administration of bilirubin, prevented the heme oxygenase induction as well as the decrease in hepatic GSH and the increase of lipid peroxidation when it was administered 2 hr before hemin treatment. These results indicate that the induction of heme oxygenase by hemin may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Heme oxygenase activity in rat organs during cadmium chloride administration

Heme oxygenase activity, the level of spontaneous and ascorbat-induced LPO in the liver, kidney and spleen homogenates of rats and blood serum absorption spectrum in the Soret region in different periods both after CdCl2 and prior alpha-tocopherol administration were studied. The increase in the hemolysis products content in the serum was observed in 15 min after CdCl2 injection and remained during 24 h. Heme oxygenase activity in the liver and kidney increased after 6 h and stayed at the same level 24 h after CdCl2 administration. The level of spontaneous LPO in the spleen increased after 6 h, and in the liver and kidney the level of spontaneous and ascorbat-induced LPO increased in 24 h after CdCl2 injection. The preliminary alpha-tocopherol administration did not prevent the accumulation of hemolysis products in the serum and the increase of heme oxygenase activity in the liver and kidney caused by CdCl2 administration. However, the increase in the ascorbat-induced LPO in these organs was completely blocked. The role of heme and LPO in the heme oxygenase induction by CdCl2 are discussed.     

Activity of key enzymes of heme metabolism and cytochrome P-450 content in the rat liver in experimental rhabdomyolysis and hemolytic anemia

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: evidence for oxidative stress involvement.

The induction of heme oxygenase in rat liver by cobaltous chloride (CoCl2) and Co-protoporphyrin IX is entirely prevented by the administration of alpha-tocopherol and allopurinol. CoCl2 was converted in the liver into Co-protoporphyrin IX before it induced heme oxygenase activity. Actinomycin and cycloheximide affected to a similar degree the induction of heme oxygenase by both CoCl2 and Co-protoporphyrin IX. Administration of either CoCl2 or Co-protoporphyrin strongly decreased the intrahepatic GSH pool, a decrease which was completely prevented by the administration of either alpha-tocopherol or allopurinol. The latter compounds prevented heme oxygenase induction as well as the decrease in hepatic GSH when administered 2 h before, together with, or 2 h after CoCl2. However, when given 5 h after administration of CoCl2, alpha-tocopherol and allopurinol showed no preventive effect. Similar results were obtained when Co-protoporphyrin IX was used, with the difference that when alpha-tocopherol and allopurinol were given 2 h after administration of the inducer, they showed no protective effect. Phenylhydrazine and diamide also induced heme oxygenase activity in rat liver. This inductive effect was preceded by a decrease in the intrahepatic GSH pool, which took place several hours before induction of the oxygenase. Administration of alpha-tocopherol and allopurinol prevented induction of the oxygenase but had no effect on the decrease in GSH levels. These results suggest that the induction of heme oxygenase by phenylhydrazine and the diamide is preceded by an oxidative stress which very likely originates in the depletion of GSH. The induction of heme oxygenase by hemin was not prevented by administration of alpha-tocopherol or allopurinol. Coprotoporphyrin IX did not affect the pattern of the molecular forms of hepatic biliverdin reductase, at variance with CoCl2, which is known to convert molecular form 1 of the enzyme into molecular form 3.     

Metabolism of heme and hemoproteins in rat liver upon administration of mercuric chloride

Rat liver delta-aminolevulinate synthase (delta-ALAS) activity in the early period after mercury chloride administration (0.7 mg per 100 g body weight) was found to be followed by free heme level increase, which was registered by the increase of heme saturation of the heme-binding protein tryptophan-2,3-dioxygenase (T-2,3-DO). delta-ALAS and heme oxygenase activity increase was observed 24 h after action. Microsomal cytochromes P450 and b5 levels decrease. Heme saturation of the T-2,3-DO returned to control level. Heme oxygenase and T-2,3-DO induction promoted hepatocytes free heme level normalization. Heme oxygenase and delta-ALAS induction role in the liver cells defense from the oxidative damage is discussed.     

Hemopexin decreases hemin accumulation and catabolism by neural cells

Hemopexin is a serum, CSF, and neuronal protein that is protective after experimental stroke. Its efficacy in the latter has been linked to increased expression and activity of heme oxygenase (HO)-1, suggesting that it facilitates heme degradation and subsequent release of cytoprotective biliverdin and carbon monoxide. In this study, the effect of hemopexin on the rate of hemin breakdown by CNS cells was investigated in established in vitro models. Equimolar hemopexin decreased hemin breakdown, as assessed by gas chromatography, by 60–75% in primary cultures of murine neurons and glia. Extracellular hemopexin reduced cell accumulation of ⁵⁵Fe-hemin by over 90%, while increasing hemin export or extraction from membranes by fourfold. This was associated with significant reduction in HO-1 expression and neuroprotection. In a cell-free system, hemin breakdown by recombinant HO-1 was reduced over 80% by hemopexin; in contrast, albumin and two other heme-binding proteins had no effect. Although hemopexin was detected on immunoblots of cortical lysates from adult mice, hemopexin knockout per se did not alter HO activity in cortical cells treated with hemin. These results demonstrate that hemopexin decreases the accumulation and catabolism of exogenous hemin by neural cells. Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products.     

Cyclic oscillations in rat hepatic heme oxygenase and delta-aminolevulinic acid synthetase following intravenous heme administration.

Heme administration in vivo results in the suppression of synthesis of rat hepatic δ-aminolevulinic acid (ALA) synthetase and induction of rat hepatic heme oxygenase. Intravenous heme administration in vivo results in the appearance of cyclic progressively damped oscillations of both hepatic ALA synthetase activity and hepatic heme oxygenase activity. Heme oxygenase induction precedes in time the induction of ALA synthetase. ALA synthetase oscillations are observed in hepatic cell cytosol and mitochondrial fractions as well as in the total homogenate. Cycloheximide pretreatment abolishes both the ALA synthetase and heme oxygenase oscillations, while actinomycin D pretreatment has only a minimal effect on the induction of heme oxygenase. These results suggest that hepatic heme metabolism is closely regulated by rapid changes in the capacity to synthesize and catabolize heme, and the cyclic oscillations following intravenous heme may be a manifestation of the feedback regulation processes involved. This regulatory capacity is dependent on protein synthesis, and the primary site of regulation may be at the translational level on the endoplasmic reticulum.     

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12-24 h. Testicular mitochondrial delta-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.     

Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues as well as the induced enzymes

A specific antibody was prepared against rat liver heme oxygenase which had been induced by bromobenzene treatment. Immunochemical studies with this antibody (IgG) revealed that heme oxygenases from livers of rats treated with hemin, Cd2+, Co2+, or bromobenzene from rat spleen and also from kidney of Sn2+-treated rats were all immunochemically identical. Cell-free synthesis of heme oxygenase was performed in a rabbit reticulocyte lysate system using polysomes isolated from livers of rats treated with either hemin, Cd2+, or bromobenzene, and it was found that translatable mRNA specific for heme oxygenase was actually increased in the liver of rats treated with any of those inducers. Also, the ability of liver polysomes to direct cell-free synthesis of heme oxygenase was apparently proportional to the activity of heme oxygenase in the liver from which polysomes were prepared. The heme oxygenase protein synthesized either in vivo or in vitro showed a molecular weight of 31,000 when examined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. This value is essentially identical with the molecular weight of heme oxygenase purified from rat liver and indicates that a precursor form of heme oxygenase may not be involved in the heme oxygenase synthesis.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Human heme oxygenase cDNA and induction of its mRNA by hemin

Hemin treatment increased both activity and mRNA level of heme oxygenase in human macrophages. Using poly(A)-rich RNA prepared from human macrophages treated with hemin, we have constructed a cDNA library in the Okayama-Berg vector. The human heme oxygenase cDNA was isolated by screening this library with a rat cDNA and was subjected to nucleotide sequence analysis. The deduced human heme oxygenase is composed of 288 amino acids with a molecular mass of 32,800 Da. The homology in amino acid sequences between rat and human heme oxygenase is 80%. Like rat heme oxygenase, human enzyme has a putative membrane segment at its carboxyl terminus, which is probably essential for the insertion of heme oxygenase into endoplasmic reticulum. Both rat and human heme oxygenase have no cysteine residues. Recently we have shown that rat heme oxygenase is a heat-shock protein [J. Biol. Chem. 262, 12889-12892 (1987)], and therefore we examined the effects of heat treatment on the induction of heme oxygenase in human macrophages and glioma cells. In contrast to hemin treatment, heat treatment had no apparent effects in either human cell line on the activity of heme oxygenase and its mRNA levels. These results suggest that human heme oxygenase may not be a heat-shock protein.     

Differential heme oxygenase induction by stannous and stannic ions in the heart

Heme oxygenase is the rate-limiting enzyme in heme catabolism, and is induced by oxidative stress, foreign and endogenous chemicals, and many trace elements and heavy metals. This study examined the effect of the oxidative state of the heavy metal tin, on heme oxygenase-1 induction in cardiac tissue. Subcutaneous administration of stannous and stannic chloride failed to induce the enzyme in this tissue. Atomic absorption spectroscopy revealed the absence of tin in the heart cells. Investigation of several metal formulations showed that both stannous and stannic citrate were able to enter the bloodstream from the injection site and into heart tissue. Northern blot analysis revealed that heme oxygenase-1 mRNA was elevated several-fold in rat hearts from animals which received either stannous or stannic citrate, and that mRNA levels corresponded with the increase in enzyme activity. The presence of citrate facilitated the transport of the tin ion into the blood stream and possibly across cardiac cell membrane. The stannous ion was more potent as an inducer of heme oxygenase than was the stannic ion.     

Acute antihypertensive action of Tempol in the spontaneously hypertensive rat

Acute intravenous Tempol reduces mean arterial pressure (MAP) and heart rate (HR) in spontaneously hypertensive rats. We investigated the hypothesis that the antihypertensive action depends on generation of hydrogen peroxide, activation of heme oxygenase, glutathione peroxidase or potassium conductances, nitric oxide synthase, and/or the peripheral or central sympathetic nervous systems (SNSs). Tempol caused dose-dependent reductions in MAP and HR (at 174 micromol/kg; DeltaMAP, -57+/- 3 mmHg; and DeltaHR, -50 +/- 4 beats/min). The antihypertensive response was unaffected by the infusion of a pegylated catalase or by the inhibition of catalase with 3-aminotriazole, inhibition of glutathione peroxidase with buthionine sulfoximine, inhibition of heme oxygenase with tin mesoporphyrin, or inhibition of large-conductance Ca(2+)-activated potassium channels with iberiotoxin. However, the antihypertensive response was significantly (P < 0.01) blunted by 48% by the activation of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channels with cromakalim during maintenance of blood pressure with norepinephrine and by 31% by the blockade of these channels with glibenclamide, by 40% by the blockade of nitric oxide synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME), and by 40% by the blockade of ganglionic autonomic neurotransmission with hexamethonium. L-NAME and hexamethonium were additive, but glibenclamide and hexamethonium were less than additive. The central administration of Tempol was ineffective. The acute antihypertensive action of Tempol depends on the independent effects of potentiation of nitric oxide and inhibition of the peripheral SNS that involves the activation of K(ATP) channels.     

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

The dual oxygenase and peroxidase activities of porphobilinogen oxygenase and horseradish peroxidase: a study using the reaction with phenylhydrazine.

Porphobilinogen oxygenase and horseradish peroxidase show dual oxygenase and peroxidase activities. By treating porphobilinogen oxygenase with phenylhydrazine in the presence of H2O2 both activities were inhibited. When horseradish peroxidase was treated in the same manner only the peroxidase activity was lost while its oxygenase activity toward porphobilinogen remained unchanged. The phenylhydrazine treatment alkylated the prosthetic heme group of porphobilinogen oxygenase and N-phenylheme as well as N-phenylprotoporphyrin IX were isolated from the treated hemoprotein. In horseradish peroxidase the modified heme was mainly 8-hydroxymethylheme. The apoproteins of the alkylated enzymes were isolated and recombined with hemin IX. The oxygenase and peroxidase activities of porphobilinogen oxygenase were entirely recovered in the reconstituted enzyme, while the reconstituted horseradish peroxidase regained 75% of its peroxidase activity.