Induction of liver cell haem oxygenase in iron-overloaded rats.

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.     

Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.     

The role of haem in the regulation of rat liver tryptophan metabolism.

Anion-exchange h.p.l.c. analysis of [3H]inositol phosphates derived from glucose-stimulated isolated pancreatic islets that had been prelabelled with myo-[3H]inositol revealed that the predominant inositol trisphosphate was the 1,3,4-isomer [Ins(1,3,4)P3]. The 1,4,5-isomer [Ins(1,4,5)P3] was also detectable, as was a more polar inositol phosphate with the chromatographic properties of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Glucose-induced accumulation of Ins(1,3,4)P3 was augmented by Li+ and occurred after maximal accumulation of Ins(1,4,5)P3. These findings suggest a possible role for Ins(1,3,4)P3 or its probable precursor Ins(1,3,4,5)P4 in stimulus-secretion coupling in pancreatic islets.     

Differential responses to inducers of delta-aminolaevulinate synthase and haem oxygenase during pregnancy.

The responses of hepatic delta-aminolaevulinate synthase and microsomal haem oxygenase to inducers were examined in pregnant rats. 2-Allyl-2-isopropylacetamide-mediated induction of delta-aminolaevulinate synthase was greatly decreased during pregnancy and in the early post-partum period. Administration of allylisopropylacetamide to pseudopregnant rats induced delta-aminolaevulinate synthase normally. Treatment of pregnant rats with cortisol failed to restore the drug-mediated induction of delta-aminolaevulinate synthase. Microsomal cytochrome P-450 content and the activities of drug-metabolizing enzymes such as aniline hydroxylase and ethylmorphine. N-demethylase were significantly lowered during pregnancy. In contrast with the greatly impaired induction of delta-aminolaevulinate synthase, the induction of haem oxygenase in response to CoCl2 remained unaltered in pregnant rats. The normal perturbations of delta-aminolaevulinate synthase, consisting of an initial inhibition followed by a rebound increase in the enzyme activity associated with CoCL2 treatment, were observed during pregnancy. These findings indicate that hormones and metabolic factors associated with gestation exert significant but differential controls on the induction patterns of delta-aminolaevulinate synthase and haem oxygenase.     

Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride.

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.     

Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin.

Phenylhydrazine was found to be a potent inducer of microsomal haem oxygenase activity in rat liver and kidney, but not in spleen. The phenylhydrazine-mediated increase in haem oxygenase activity was time-dependent. Maximum activity was attained 12h after treatment in the liver, and 24h after treatment in the kidney. The increases in the activity of haem oxygenase in the liver and the kidney could be inhibited by cycloheximide. Furthermore, the increases could not be elicited by the treatment of microsomal preparations in vitro with phenylhydrazine. In consonance with the increased haem oxygenase activity, a marked increase (16-fold) was observed in the serum total bilirubin concentration in phenylhydrazine-treated rats. The mechanism of haem degradation promoted by phenylhydrazine in vivo appears to differ from that in vitro; only in the former case is bilirubin formed as the end-product of haem degradation. When rats were given zinc-protoporphyrin (40 mumol/kg) 12h before and after phenylhydrazine treatment, the phenylhydrazine-mediated increases in haem oxygenase activity in the liver and the kidney were effectively blocked. Treatment of rats in vivo with the metalloporphyrin also inhibited the activity of splenic haem oxygenase, and promoted a major decrease in the serum bilirubin levels. In phenylhydrazine-treated animals, the microsomal content of cytochrome P-450 was significantly decreased in the absence of a decrease in the microsomal haem concentration. The decrease in cytochrome P-450 content was accompanied by an increased absorption in the 420nm region of the reduced CO-difference spectrum, suggesting the conversion of the cytochrome to an inactive form. The marked depletion of cellular glutathione levels suggests that this conversion may be related to the action of active intermediates and free radicals formed in the course of the interaction of phenylhydrazine with the haem moiety of cytochrome P-450.     

Induction of heme oxygenase 1 in radiation nephropathy: role of angiotensin II

Datta, P. K., Moulder, J. E., Fish, B. L., Cohen, E. P. and Lianos, E. A. Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II. Radiat. Res. 155, 734-739 (2001). In a rat model of radiation-induced nephropathy, we investigated changes in expression of heme oxygenase 1 (Hmox1, also known as HO-1), an enzyme that catalyzes conversion of heme into biliverdin, carbon monoxide and iron. The study explored whether radiation induces Hmox1 expression in the irradiated kidney and whether angiotensin II (AII) mediates Hmox1 expression in glomeruli isolated from irradiated kidneys. To assess the effects of radiation on Hmox1 expression, rats received 20 Gy bilateral renal irradiation and were randomized to groups receiving an AII type 1 (AT(1)) receptor antagonist (L-158,809) or no treatment. Drug treatment began 9 days prior to bilateral renal irradiation and continued for the duration of the study. Estimation of Hmox1 levels in glomerular protein lysates assessed by Western blot analysis revealed a significant increase in Hmox1 protein at 50 and 65 days postirradiation. In animals treated with the AT(1) receptor antagonist, there was no induction of Hmox1, suggesting that AII may be a mediator of Hmox1 induction. To confirm that AII stimulates Hmox1 expression, animals were infused with 200, 400 or 800 ng/kg min(-1) of AII for 18-19 days, and Hmox1 protein levels in glomeruli were assessed. There was a significant induction of Hmox1 in glomeruli of animals infused with 800 ng/kg min(-1) of AII. These studies demonstrate that glomerular Hmox1 expression is elevated in the middle phase of radiation nephropathy and that AII can increase glomerular Hmox1 levels.     

The structural organization of haem synthesis in rat liver mitochondria

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe(3+) is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the K(m) for Co(2+) is 11x10(-6)m and that for mesoporphyrin is 5x10(-6)m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.     

Metal ion interactions in the control of haem oxygenase induction in liver and kidney.

Mn2+ and Zn2+ exhibit a striking ability to block the induction by Sn2+ and Ni2+ of haem oxygenase (EC 1.14.99.3) in kidney. The blocking effects of Mn2+ and Zn2+ were found to be greatest on simultaneous administration, time-dependent when administered up to 8 h before the inducing metal ions, and ineffective when administered as little as 10 min after the inducing metal ions. The decreases in cytochrome P-450 and haem contents and the sequential changes in delta-aminolaevulinate synthase (EC 2.3.1.37) activity that occur concomitant with haem oxygenase induction were largely eliminated with simultaneous or prior treatment with Mn2+ or Zn2+, but not when Mn2+ or Zn2+ was administered after Sn2+ or Ni2+. Mn2+ and Zn2+ did not increase the catabolism of the enzyme in vivo. Zn2+ on simultaneous administration was also able substantially to block the induction of haem oxygenase by Co2+, Cd2+ and Ni2+ in liver. The Zn2+ blockade of Cd2+ induction was examined in detail, and prior or simultaneous administration of Zn2+ was found to be effective in blocking the induction of haem oxygenase and the concomitant decreases in cytochrome P-450 and haem contents, ethylmorphine demethylase activity and the sequential changes in delta-aminolaevulinate synthase activity. Zn2+ administration 10 min or more after Cd2+ was ineffective in preventing the occurrence of these perturbations in haem metabolism. These findings describe a new and striking biological property of Mn2+ and Zn2+, and indicate the existence of significant metal ion interactions in the control of haem metabolism.     

Making light of it: the role of plant haem oxygenases in phytochrome chromophore synthesis

The haem oxygenase (HO) enzyme catalyses the oxidation of haem to biliverdin IX alpha, CO and Fe(2+), and performs a wide variety of roles in Nature, including degradation of haem from haemoglobin, iron acquisition and phycobilin biosynthesis. In plants, HOs are required for the synthesis of the chromophore of the phytochrome family of photoreceptors. There are four HO genes in the Arabidopsis genome. Analysis of a mutant deficient in HO1 (the hy1 mutant) has demonstrated that this plastid-localized protein is the major HO in the phytochrome chromophore synthesis pathway. HO2 may also have a minor role in this pathway, but our understanding of the divergent roles of this small gene family is still far from complete.     

Apoprotein E isoform-dependent expression and secretion of pro-inflammatory cytokines TNF-alpha and IL-6 in macrophages

The anti-atherogenic properties of human apoprotein E-associated lipoproteins have been partially attributed to its anti-inflammatory properties. We studied if endogenously expressed apoprotein E (apoE) elicits isoform-dependent effects on pro-inflammatory cytokine expression and secretion. Mouse J774A.1 peritoneal macrophages without native expression of apoE were used to establish cell lines with stable expression of the three human apoE isoforms, apoE2, apoE3 and apoE4. In the presence of lipopolysaccharide (LPS), expression and secretion of TNF-alpha and IL-6 in cells expressing different apoE isoforms were determined by RT-PCR, immunoblotting and ELISA assays. ApoE3-expressing cells have significantly lower expression and secretion levels of the two cytokines as compared to cells with apoE2 and apoE4 expression. Such observations were accompanied with the lowest ERK1/2 activity in apoE3-expressing cells. Further study shows that the apoE isoform-dependent variations of TNF-alpha and IL-6 expression/secretion in macrophages are diminished in the presence of ERK1/2 inhibitor U0126. In conclusion, apoE elicits isoform-dependent effects on macrophage TNF-alpha and IL-6 expression as well as secretion. The ERK1/2 signaling pathways are involved in mediating such apoE isoform-dependent effects.     

Induction of heme oxygenase mRNA by cobalt protoporphyrin in rat liver

The effect of cobaltic(III)-protoporphyrin on heme oxygenase activity and mRNA content was examined in vivo in the adult male rat liver. The activity of heme oxygenase, the rate-limiting enzyme in the degradation of heme, was enhanced, as expected, by cobalt protoporphyrin (25 mumol/kg body weight) in a time-dependent manner. Levels of enzyme activity were increased 2-fold by 8-16 h following treatment and were 6-fold higher than baseline values by 48 h. Administration of cobalt protoporphyrin resulted in a marked increase in heme oxygenase mRNA in the liver. Within 2 h of treatment, mRNA levels had increased 7.9-fold. The induction of heme oxygenase mRNA was maximal at 8 h when the levels were 58.5-fold above baseline. At every time point tested, the increase in heme oxygenase mRNA was several fold greater than that of enzyme activity.     

Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride.

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.     

Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells

Visfatin is a novel multifunctional adipocytokine with inflammatory properties. Although a link between visfatin and atherosclerosis has recently been suggested, its actions in the development of atherosclerosis remain unknown. Therefore, we investigated a potential role and underlying mechanism(s) of visfatin in monocytes/macrophages differentiation, a critical early step in atherogenesis, using phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell models. The co-incubation of PMA with visfatin-induced CD36 expression with a concomitant increase in the phagocytosis of latex beads compared with PMA alone treatment. Moreover, visfatin markedly increased interleukin (IL)-1β secretion by enhancing IL-1β mRNA stability in a short-term incubation. Visfatin also significantly elevated the secretion of IL-6 as well as IL-1β in a longer incubation period, which was partially suppressed by nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, and c-Jun-N-terminal kinase (JNK) inhibitor, SP600125. Furthermore, silencing IL-1β successfully blocked IL-6 secretion, CD36 expression, and NF-κB activation in response to visfatin. Collectively, these results suggest that visfatin enhances the IL-1β-dependent induction of IL-6 and CD36 via distinct signaling pathways mediated by JNK and NF-κB, respectively, and consequently, leading to the acceleration of monocytes/macrophages differentiation.     

Accelerated hepatic haem catabolism in the selenium-deficient rat.

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.     

Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.