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PubMed2006

NO modulates NADPH oxidase function via heme oxygenase-1 in human endothelial cells.

NO is known to induce expression of heme oxygenase-1, an antioxidant enzyme in blood vessels. We tested whether NO might modulate the endothelial NADPH oxidase function via heme oxygenase-1. In human microvascular endothelial cells, the NO donor DETA-NONOate (0.1 to 1 mmol/L) strongly induced expression of heme oxygenase-1 but not Cu/Zn superoxide dismutase. This was associated with a reduction of the superoxide-generating capacity of NADPH oxidase, an effect that depended on de novo gene transcription and heme oxygenase-1 activity. Activation of NADPH oxidase by tumor necrosis factor (TNF) alpha increased generation of reactive oxygen species. DETA-NONOate alone had little effect on TNF-stimulated reactive oxygen species, but it enhanced the TNF response when: (1) heme oxygenase-1 expression was blocked with specific small-interfering RNA; (2) heme oxygenase-1 activity was blocked by zinc-protoporphyrin; or (3) NADPH oxidase activity was blocked by diphenyleneiodonium. Moreover, the heme oxygenase-1 end product bilirubin directly inhibited fully functional NADPH oxidase and seemed to interrupt the assembly and activation of the oxidase. In conclusion, NO may modulate superoxide production by NADPH oxidase in human vascular endothelial cells, at least partly by inducing heme oxygenase-1. Our results indicate that suppression of NADPH oxidase-dependent reactive oxygen species formation may represent a novel mechanism underlying the cardiovascular protective actions of heme oxygenase-1 and bilirubin.

Cells, Cultured↗

PubMed2006

Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death.

Cigarette smoke-induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.

Adenosine Triphosphate↗

PubMed1978

Reaction of the microsomal heme oxygenase with cobaltic protoporphyrin IX, and extremely poor substrate.

A reconstituted heme oxygenase system which was composed of a purified heme oxygenase from pig spleen microsomes and a partially purified NADPH-cytochrome c reductase from pig liver microsomes could not catalyze the conversion of cobaltic protoporphyrin IX (Co-heme) to biliverdin, although Co-heme could bind with the heme oxygenase protein to form a complex. The heme oxygenase system in the microsomes from pig spleen, rat spleen, and rat kidney also failed to oxidize Co-heme to biliverdin. Properties of the complex of Co-heme and heme oxygenase closely resembled those of cobalt myoglobin and cobalt hemoglobin; the Co-heme bound to the heme oxygenase protein did not react with cyanide and azide, the Co-heme moiety was reduced but only slowly with sodium dithionite, and the reduced form of the Co-heme did not appear to bind carbon monoxide. The co-heme bound to heme oxygenase was not reduced with the NADPH-cytochrome c reductase system in air. These findings further support the views that heme oxygenase may have a heme-binding crevice similar to those of myoglobin and hemoglobin and that reduction of heme is the prerequisite for the oxidative degradation of heme in the heme oxygenase reaction.

Animals↗

PubMed1992

Intestinal heme oxygenase inhibition and increased biliary iron excretion by metalloporphyrins.

The effects of synthetic metalloporphyrins on heme oxygenase activity in the epithelium of the proximal region of the small intestine were examined both in vitro and in vivo in male Sprague-Dawley rats. Metalloporphyrins, which inhibit hepatic heme oxygenase in vitro, also inhibit intestinal heme oxygenase. Chromium and tin porphyrins are the most potent inhibitors of the intestinal enzyme in vitro. Oral administration of Sn-protoporphyrin (25 mumol/kg body weight) resulted in inhibition of intestinal heme oxygenase; however, no effect was observed on the splenic, hepatic, or renal enzymes. Metal analyses in these tissues showed essentially no intestinal absorption of the metalloporphyrin. Oral administration of Cr-mesoporphyrin (25 mumol/kg body wt) also resulted in inhibition of intestinal heme oxygenase activity. Zinc and manganese mesoporphyrin did not inhibit intestinal heme oxygenase activity when administered orally. Microsomal intestinal heme oxygenase activity was inhibited in a dose-dependent manner by antiserum raised in rabbit against rat hepatic heme oxygenase. The parenteral administration of metalloporphyrin inhibitors of heme oxygenase to bile duct-cannulated rats resulted in a significant increase in iron levels in the bile.

Animals↗

PubMed1976

Pretranslational control of tryptophan oxygenase levels in Morris hepatoma and host liver.

Tryptophan oxygenase is present and hormonally inducible in host livers but is absent in transplanted Morris hepatomas examined under basal conditions as well as in hormonally induced animals. Studies were performed to determine whether the absence of tryptophan oxygenase in hepatomas is mediated by an alteration in the translational efficiency or the level of the messenger RNA (mRNA) for tryptophan oxygenase. The tissue level of the specific mRNA coding for tryptophan oxygenase was quantitated in an mRNA-dependent Krebs ascites cell-free protein-synthesizing system. The enzyme levels and mRNA activities in host livers and hepatomas from control rats and rats given injections of an inducing dose of hydrocortisone were compared; they indicate that the induction of tryptophan oxygenase in host livers by hormones is accompanied by a proportional increase in the level of its mRNA, whereas in the transplanted hepatomas the tryptophan oxygenase catalytic activity and the mRNA coding for this enzyme were undetectable in both control and glucocorticoid-induced animals. No functional mRNA for tryptophan oxygenase could be detected in the total polyadenylate-containing mRNA isolated from the Morris hepatoma cells. The hepatomas contained normal levels of cytoplasmic glucocorticoid receptor that could bind glucocorticoid, undergo "activation," and translocate to both normal and neoplastic nuclei. Thus, deletion of tryptophan oxygenase in hepatomas is a consequence of the absence of the gene product, i.e., the tryptophan oxygenase mRNA, which codes for its synthesis; this is not due to detectable alterations in the ability of the glucocorticoid receptor to bind the steroid hormone, or of the hormone-receptor complex to undergo activation, or of the activated steroid-receptor complex to bind to nuclei derived from the hepatoma or normal liver.

Animals↗

PubMed1979

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.

Animals↗

PubMed1987

Identification of heme oxygenase and cytochrome P-450 in the rabbit heart.

The regulation of cardiac heme oxygenase and cytochrome P-450 mixed function oxidase was studied in the rabbit heart. Heme oxygenase activity is found in ventricular and atrial microsomal fractions. This activity is NADPH dependent, and is inhibited by tin and zinc protoporphyrin, but not by either SKF 525A or 7,8-benzoflavone. Immunologic studies of cardiac heme oxygenase demonstrate that antibodies prepared against human purified hepatic heme oxygenase recognize rabbit atrial heme oxygenase and inhibit the enzyme activity by 92%. In contrast, control immunoglobulin does not inhibit heme oxygenase activity. Further, the western blotting technique demonstrates that a similar band of protein with a molecular weight of 32,000 exists in cardiac microsomes and that no protein cross-reacts with purified hepatocyte heme oxygenase. Marked induction of atrial heme oxygenase is observed in microsomal fractions prepared from rabbits treated with cobalt chloride. Atrial microsomes possess 0.24 nmol of cytochrome P-450 as compared to 0.68 nmol/mg protein in microsomes from the liver. The levels of aryl hydrocarbon hydroxylase (AHH) activity, a cytochrome P-450-dependent enzyme, in ventricle and atrium are stimulated by a NADPH-generating system and are sensitive to 7,8-benzoflavone, and SKF 525A, known inhibitors of cytochrome P-450 mixed function oxidase. AHH activity in ventricular and atrial microsomes is 2-3% of that seen in liver microsomes whereas the P-450 content/mg protein is about 20% of that observed in the liver. AHH activity is mediated by a form of cytochrome P-450 that is inducible by 3-methylcholanthrene/beta-naphthoflavone. A possible new role of the heart cytochrome P-450 system in cardiac function is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy.

Cyclo-oxygenase is expressed in cells in two distinct isoforms. Cyclo-oxygenase-1 is present constitutively whilst cyclo-oxygenase-2 is expressed primarily after inflammatory insult. The activity of cyclo-oxygenase-1 and -2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo-oxygenase include the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo-oxygenase-2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo-oxygenase-1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo-oxygenase-2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo-oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Alzheimer Disease↗

PubMed2002

Inhibition of Helicobacter pylori-induced cyclo-oxygenase-2 aggravates NSAID-caused gastric damage in Mongolian gerbils.

BACKGROUND: The effect of Helicobacter pylori infection on non-steroidal anti-inflammatory drug-induced gastric mucosal injury is controversial. AIM: To examine the effect of the interaction between H. pylori and non-steroidal anti-inflammatory drugs on gastric mucosal injury. METHODS: Mongolian gerbils infected with H. pylori were treated with indometacin at 8 mg/kg for 2 days or 7 days. Mucosal damage was assessed by macroscopic and histological examination, and myeloperoxidase activity was measured as an index of neutrophil infiltration. The expression levels of cyclo-oxygenase proteins were determined by Western blot analysis and cyclo-oxygenase activity. RESULTS: A 2-day course of indometacin did not cause an increase in gastric damage in H. pylori-infected Mongolian gerbils compared to uninfected gerbils, while a 7-day course of indometacin caused additive gastric damage in H. pylori-infected animals. H. pylori infection induced cyclo-oxygenase-2 expression in the stomach. Treatment with indometacin for 2 days did not significantly affect cyclo-oxygenase activity in H. pylori-infected animals, while treatment for 7 days inhibited both cyclo-oxygenase-1 and cyclo-oxygenase-2 activities. Pre-treatment with a selective cyclo-oxygenase-2 inhibitor aggravated mucosal injury in H. pylori-infected animals treated or not treated with indometacin for 2 days. CONCLUSIONS: Our results suggest that cyclo-oxygenase-2 protein induced by H. pylori infection may be involved in the defence of the gastric mucosa against damage caused by non-steroidal anti-inflammatory drugs. Therefore, inhibition of cyclo-oxygenase-2 activity may enhance non-steroidal anti-inflammatory drug-caused gastric damage in H. pylori-infected animals.

Animals↗

PubMed2004

Barrett's oesophagus with predominant intestinal metaplasia correlates with superficial cyclo-oxygenase-2 expression, increased proliferation and reduced apoptosis: changes that are partially reversed by non-steroidal anti-inflammatory drugs usage.

BACKGROUND: Cyclo-oxygenase-2 expression has been reported to play an important role in the metaplasia-dysplasia-carcinoma sequence in Barrett's oesophagus. However, the existence of cyclo-oxygenase-2 expressing cells in Barrett's epithelium is still uncertain. AIM: To identify the cells that express cyclo-oxygenase-2 protein and to investigate the relationship between cyclo-oxygenase-2 expression and mucin-phenotype of Barrett's epithelium. METHODS: Sections from 466 biopsy samples of Barrett's epithelium from 358 non-medicated patients were immunohistochemically examined for the cyclo-oxygenase-2 expression, mucin-phenotype, cell proliferation and apoptosis. RESULTS: Cyclo-oxygenase-2 expression was detected in 71.0% of Barrett's epithelium biopsy samples. In Barrett's epithelium with the gastric predominant mucin-phenotype, cyclo-oxygenase-2 expression was mainly found in stromal and deep epithelial cells, whereas in intestinal predominant mucin-phenotype, it was mostly in superficial epithelial cell. A significant elevation of proliferating cell nuclear antigen index and suppression of apoptotic index was observed in Barrett's epithelium with superficial epithelial cyclo-oxygenase-2 expression. Neither such elevation of proliferating cell nuclear antigen index nor the suppression of apoptotic index could be found in chronic non-steroidal anti-inflammatory drugs users. CONCLUSIONS: Barrett's epithelium with intestinal mucin and superficial epithelial cyclo-oxygenase-2 expression possess a higher proliferation potential, but this risk may be thwarted by non-steroidal anti-inflammatory drugs administration.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1983

Differential response of testicular and ovarian heme oxygenase activity to metal ions.

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.

Animals↗

PubMed1989

Expression of rat liver heme oxygenase gene during development.

We studied the genetic expression during fetal development of heme oxygenase, the rate-limiting enzyme in the oxidation of heme to bilirubin. The transcription of the heme oxygenase gene in livers of fetal and neonatal rats (9 days before birth to 28 days after birth) was examined. Hybridization analyses of total RNA from livers of these animals using cDNA for rat heme oxygenase as the probe revealed a single mRNA species of approximately 18 S in every sample examined. The mRNA level was above the adult level throughout the course of study and reached a maximum 24 h after birth. The high level of heme oxygenase mRNA in fetuses was unaffected when Sn-protoporphyrin, a potent inhibitor of heme oxygenase, was administered to their mothers. On the other hand, the mRNA levels in the mothers treated with this heme analog were substantially increased, possibly by the same mechanism as for the induction by heme. Sn-protoporphyrin potentiates induction of heme oxygenase mRNA in cobalt chloride-pretreated rats, and also acts as a potent inhibitor of heme oxygenase enzyme activities. Our results also indicate that high heme oxygenase levels during fetal maturation are due to an increase in transcription of the gene. Thus, Sn-protoporphyrin which crosses the placenta controls fetal hyperbilirubinemia by direct enzyme inhibition.

Age Factors↗

PubMed1988

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.

Blotting, Western↗

PubMed2003

Exhaled carbon monoxide and inducible heme oxygenase expression in a rat model of postperfusion acute lung injury.

OBJECTIVE: Expression of inducible heme oxygenase has been shown to be increased in various visceral inflammatory disorders, which may confer a protective role. The purpose of our study was to determine whether the expression of inducible heme oxygenase was up-regulated within lungs in a rat model of extracorporeal circulation. METHODS: Wistar rats underwent either a partial femoro-femoral extracorporeal circulation in normothermia for 3 hours (n = 5) or a sham procedure (n = 5). Exhaled carbon monoxide concentration was monitored with an infrared analyzer. After the rats were killed, lungs were harvested for determination of heme oxygenase activity and inducible heme oxygenase expression (by Western blot and immunohistochemistry). Lung injury was also assessed by arterial blood gas analysis and microscopic study. RESULTS: Extracorporeal circulation was responsible for a lung injury characterized by decreased arterial blood oxygen saturation and typical morphologic findings (marked alveolar neutrophil infiltration; interstitial edema). Exhaled carbon monoxide concentration remained stable throughout the experiment in all sham rats, whereas it increased after extracorporeal circulation (from 0.16 +/- 0.05 ppm at baseline to 0.7 +/- 0.2 ppm at end of experiment; P =.0001). Pulmonary heme oxygenase activity and inducible heme oxygenase content (assessed by Western blot) were increased within lungs of rats that underwent extracorporeal circulation. Immunohistochemistry revealed that the expression of inducible heme oxygenase was mainly localized to inflammatory cells. CONCLUSIONS: Post-extracorporeal circulation acute lung injury in rats was associated with an increased expression of inducible heme oxygenase, the functional significance of which remains to be determined.

Acute Disease↗

PubMed1988

Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals.

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.

Animals↗

PubMed1998

Acute phencyclidine neurotoxicity in rat forebrain: induction of haem oxygenase-1 and attenuation by the antioxidant dimethylthiourea.

Phencyclidine and other N-methyl-D-aspartate receptor antagonists are toxic to pyramidal neurons in the posterior cingulate/retrosplenial cortex of rat brain. Previous studies have shown induction of heat shock protein 70 in affected neurons. In this study, expression of haem oxygenase-1, a heat shock protein induced by oxidative stress, was examined in rat forebrain after administration of a single intraperitoneal dose of phencyclidine (50 mg/kg). Northern and Western blot analyses of brain tissue extracts from phencyclidine-treated rats revealed a marked induction of haem oxygenase-1 mRNA and protein, respectively. Immunohistochemistry studies revealed that phencyclidine increased haem oxygenase-1 immunoreactivity primarily in posterior cingulate/retrosplenial, piriform and entorhinal cortices, striatum and hippocampus. Haem oxygenase-1 protein was induced in non-neuronal cells, mainly astrocytes. Some microglia expressing haem oxygenase-1 protein were also found in the posterior cingulate/retrosplenial cortex. Haem oxygenase-1 immunoreactive astrocytes and microglia were present in close proximity to the heat shock protein 70-positive neurons in the posterior cingulate/retrosplenial cortex following phencyclidine. Pretreatment of rats with 1,3-dimethylthiourea, an antioxidant, significantly reduced haem oxygenase-1 protein induction by phencyclidine. Thus, induction of haem oxygenase-1 in glia by phencyclidine appears to be mediated mostly by oxidative stress. Experiments with the amino cupric silver stain for neuronal degeneration revealed phencyclidine-induced neurotoxicity in the posterior cingulate/retrosplenial cortex. The number of affected neurons was significantly reduced after 1,3-dimethylthiourea pretreatment. This suggests that the neurotoxicity of N-methyl-D-aspartate antagonists is due in part to the oxidative stress and may be amenable to therapeutic interventions.

Animals↗

PubMed1999

Suppression of heme oxygenase-1 mRNA expression by interferon-gamma in human glioblastoma cells.

Heme oxygenase is a rate-limiting enzyme in heme catabolism that cleaves heme to form biliverdin, carbon monoxide, and iron. Heme oxygenase-1 is an inducible isozyme and is expressed in many types of cells and tissues. Large amounts of these heme degradation products may be noxious to the host, especially in the brain. We therefore searched for the factors that suppress the expression of heme oxygenase-1. Northern blot analysis showed that treatment with interferon-gamma and with interleukin-1beta for 24 h decreased the expression levels of heme oxygenase-1 mRNA to approximately 20 and approximately 50% of the control levels, respectively, in a human glioblastoma cell line, T98G. Treatment with a combination of these two cytokines additively decreased the expression levels of heme oxygenase-1 mRNA. Western blot analysis showed that the expression level of heme oxygenase-1 protein was also decreased by treatment with interferon-gamma, but not with interleukin-1beta. Moreover, pretreatment with interferon-gamma partially suppressed the induction of heme oxygenase-1 mRNA expression caused by either sodium nitroprusside, cadmium, or hemin. These findings raise the possibility that the expression of heme oxygenase-1 is down-regulated by interferon-gamma in the nervous system.

Cell Survival↗

PubMed1985

Cloning and expression of cDNA for rat heme oxygenase.

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in lambda gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

Amino Acid Sequence↗