HO-1 mRNA Levels Research Articles

CAMP induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle.

Recent studies indicate that vascular smooth muscle cells generate carbon monoxide (CO) via the action of heme oxygenase (HO). Because adenosine 3',5'-cyclic monophosphate (cAMP) is an important intracellular signaling molecule in the regulation of vascular cell function, we examined whether this second messenger modulates the expression of HO and the production of CO by rat aortic smooth muscle cells. Treatment of smooth muscle cells with the membrane-permeable cAMP derivative dibutyryl cAMP or with compounds that increase intracellular cAMP levels (isoproterenol and forskolin) resulted in a concentration- and time-dependent increase in the levels of HO-1 mRNA and protein, whereas the expression of HO-2 remained unchanged. Both actinomycin D and cycloheximide blocked the basal expression of HO-1 mRNA and protein and prevented the cAMP-mediated induction of HO-1. Incubation of platelets with cAMP-treated smooth muscle cells resulted in a significant increase in platelet cGMP concentration that was partially reversed by treatment of smooth muscle cells with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the HO blocker zinc protoporphyrin-IX. However, the combined addition of these two inhibitors to cAMP-treated smooth muscle cells or the addition of the CO and NO scavenger hemoglobin to platelets completely blocked the stimulatory effect on platelet cGMP levels. These results demonstrate that cAMP induces the expression of the HO-1 gene and stimulates the formation of CO and NO in vascular smooth muscle cells. The capacity of cAMP to induce the synthesis of guanylate cyclase-stimulatory CO from smooth muscle cells may represent a novel mechanism by which this nucleotide regulates vascular tone.

Regulation of heme oxygenase-2 by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element

Heme oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of heme to produce biliverdin IX α, CO and Fe. Detection of a consensus sequence of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and transcript development in the newborn rat brain and has examined the promoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14-day-old rats postnatally treated with corticosterone (5 μg/g, 4×, starting 24–36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs (∼1.3 and ∼1.9 kb) increased in HeLa cells treated with DX (5 μM), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.

Hyperoxic regulation of lung heme oxygenase in neonatal rats.

In the neonatal lung, hyperoxic exposure is associated with induction of various genes and critical antioxidants. Heme oxygenase, specifically the HO-1 isoenzyme, is regulated in oxidant stress and may also serve to limit oxidative damage. However, it is not known whether neonatal lung HO-1 is regulated in hyperoxia specifically and, if so, what type of regulation occurs. Therefore, we attempted to answer these questions using newly born (< 12 h) Wistar rats exposed to hyperoxia for 3 d. Neonatal rat lungs were evaluated daily for total HO activity, immunoreactive HO-1 protein, and steady state levels of HO-1 mRNA and compared with air-exposed controls. In neonatal rats, we noted an increased lung HO activity after 3 d of hyperoxic exposure. Additionally, evaluation of HO activity after immunoprecipitation of HO-1 protein suggested that HO-1 contributed most of the increase in lung total HO activity observed in hyperoxia. Nonetheless, we did not see a significant difference in immunoreactive HO-1 protein in neonatal lungs after 3 d of hyperoxic exposure, although we did so on d 2. Also, in contrast with previous reports, we did not detect any significant differences in steady state levels of HO-1 mRNA on any day of hyperoxic exposure compared with air. We therefore conclude that neonatal rat lung HO-1 is regulated in hyperoxia and speculate that the regulation of neonatal lung HO-1 occurs by posttranscriptional mechanisms, at least within the first days of hyperoxic exposure.

Stress response of the rat testis: in situ hydridization and immunohistochemical analysis of heme oxygenase-1 (HSP32) induction by hyperthermia.

By using in situ hybridization and immunohistochemistry, the distribution patterns of heme oxygenase (HO)-1 (HSP32) transcript and protein were studied, and their response to thermal stress was examined. And, by using an HO-1 cDNA probe and polyclonal antibody, the levels of HO-1 mRNA and protein in normal and heat-shocked testis were quantified. The digoxigenin-labeled probe detected a strong signal for HO-1 transcript in Leydig cells, and in the Sertoli cells, spermatogonia, primary spermatocytes, and spermatids of the seminiferous tubules. In all cell types, the transcript was predominantly concentrated in the nucleus in a defined pattern. Thermal stress (42 degrees C, 20 min) did not change the cell population pattern of HO-1 transcript expression; however, it did cause distortion of the nuclear pattern and diffusion of the transcript signal in cells. Hyperthermic treatment of rats resulted in a modest (2- to 2.8-fold), time-dependent, and sustained (1-16 h) increased in testicular 1.8-kb HO-1 mRNA. Immunohistochemical analysis of normal and heat shock patterns of testicular HO-1 expression showed robust staining of Sertoli ad leydig cells after heat shock; in normal tissue, immunoreactivity was low in these cell populations. As with the transcript distribution, hyperthermia did not affect the pattern of HO-1 immunoreactivity, and the protein was not detected in spermatogenic cells under control or stress conditions. In the Leydig cells, hyperthermia led to a more than 3-fold increase in the intensity of cytoplasmic staining for HO-1 protein. Consistent with the selective expression of HO-1, the level of the single HO-1 immunoreactive protein (approximately 32 kDa) detected in total testis microsomes showed a modest (1.5-fold) increase 6 h after heat shock. Data are consistent with te suggestion that differential distribution of HO-1 protein in the germ cell line and Sertoli cells reflects differential HO-1 mRNA processing in these cell types. The increase may be essential for the catalysis of the heme moiety of denatured hemoproteins such as cytochrome P450 and hemoglobin heme and hence may protect against heme-catalyzed free radical formation. We propose that induction of HO-1 protein in Sertoli and Leydig cells may function to protect the spermatogenic cells under conditions of thermal stress.

Expression of heme oxygenase-1 (HSP32) in human prostate: normal, hyperplastic, and tumor tissue distribution

Objectives. Heme oxygenase isozymes, HO-1 and HO-2, are members of the stress/heat shock (HSP) family of proteins, with the known function of cleaving the heme molecule to biliverdin, iron, and carbon monoxide. The aim of this study was to examine the pattern of tissue expression of HO-1 in the human prostate under different states of proliferation and differentiation and to investigate whether the pattern differs between these states.Methods. Presently, we have determined the pattern of tissue expression of the stress-inducible isozyme, HO-1 (HSP32), in human prostate under normal and pathologic conditions, by immunohistochemistry, using polyclonal antibodies, and have measured HO-1 and HO-2 mRNA levels in normal prostate and benign prostatic hyperplasia (BPH) by Northern blotting. The activity of prostate to catalyze heme degradation was also assessed.Results. In normal and BPH tissue, columnar epithelial cells of acini and ducts and cells in stroma displayed HO-1 immunoreactivity; in all cells, perinuclear staining was prominent. In BPH tissue, however, a more intense staining of the epithelial cells occurred, with notable staining of the basal cells. In undifferentiated malignant tumors, intense HO-1 staining was manifest in nearly all tumor cells, and also in the epithelial lining of blood vessels. HO-1 in the prostate tissue was found catalytically active and oxidatively cleaved the heme molecule (Fe-protoporphyrin IX) to biliverdin. Northern blot analysis shows that two forms of HO are present in the human prostate. Compared with normal tissue, predominantly hyperplastic tissue demonstrates a pronounced increase in the approximately 1.8 kb mRNA that hybridizes to the rat HO-1 probe. The levels of two transcripts, approximately 1.3 and approximately 1.7 kb, that hybridize to the rat HO-2 probe are not increased in BPH tissue.Conclusions. The finding that HO-1 expression is increased in BPH and malignant prostate tissue is consistent with a role for this stress protein in the pathogenesis of BPH and prostate cancer; in the context of iron metabolism, an argument is made in support of this possibility.

Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxygenase-1.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to bilirubin. HO-1 is highly induced by heme, its major substrate, and nonheme products, including metal ions and hormones. Interest in HO-1 has been stimulated recently by observations that HO-1 is also highly induced in response to oxidative stress in vitro. The physiologic significance of HO-1 induction following oxidant injury in vivo, however, is poorly understood. In a rat model of lipopolysaccharide endotoxin (LPS)-induced lung injury and sepsis, we demonstrate that the lung responds to LPS by expressing high levels of HO-1 mRNA and enzyme activity. We hypothesize that this HO-1 induction could play a critical role in the lung's defense against LPS. Pretreatment of rats with hemoglobin, a potent inducer of HO-1, resulted in HO-1 induction and more importantly provided complete protection against subsequent lethal endotoxemia (100% survival). Tin protoporphyrin, a competitive inhibitor of HO, blocked this protective effect of hemoglobin and rendered the rats more susceptible to a lethal dose of LPS. Taken together, these data strongly implicate HO-1 in playing an important role in the defense against endotoxic shock, with potential therapeutic implications.

Expression of the mRNA of Heme-Binding Protein 23 Is Coordinated with That of Heme Oxygenase-1 by Heme and Heavy Metals in Primary Rat Hepatocytes and Hepatoma Cells

A 23-kDa protein with high affinity for heme (KD = 55 nM), therefore termed heme-binding protein 23 kDa (HBP23), was purified from rat liver cytosol [Iwahara, S., et al. (1995) Biochemistry 34, 13398-13406]. Homology search of the cloned HBP23 cDNA revealed that this protein belongs to a recently recognized class of thiol peroxidases, the antioxidant peroxiredoxin family. Since HBP23 gene expression was highest in the liver, HBP23 mRNA regulation by heme and heavy metals was investigated in cultures of primary rat hepatocytes and mouse hepatoma Hepa 1-6 cells. In both cell cultures HBP23 mRNA levels were upregulated in a time- and dose-dependent manner by heme. Heme-dependent induction of HBP23 mRNA occurred coordinately with that of the heme-metabolizing enzyme heme oxygenase-1, which was recently identified as inducible by oxidative stress. Treatment of primary rat hepatocyte or hepatoma cell cultures with the heavy metals CdCl2 (10 microM) and CoCl2 (300 microM) induced in parallel HBP23 and HO-1 mRNA levels, in the case of CdCl2 to even higher levels than heme. By contrast, mRNA expression of another heme binding protein, hemopexin, was not induced in hepatocyte cell cultures by heme or heavy metals. The data suggest that the expression of HBP23 and HO-1 mRNA is regulated by (a) similar mechanism(s) in liver and that both genes could play a common physiological role as antioxidants and/or in heme metabolism.

Distribution of constitutive (HO-2) and heat-inducible (HO-1) heme oxygenase isozymes in rat testes: HO-2 displays stage-specific expression in germ cells

The heme oxygenase isozymes, HO-1 and HO-2, oxidatively cleave the heme molecule to produce antioxidants, the bile pigments, the gaseous cellular messenger, CO, and iron, a regulator of transferrin, ferritin, and nitric oxide synthase gene expression. HO-1 (hsp32) is a stress-inducible enzyme, whereas HO-2 is constitutively expressed at high levels in the testes and brain. In the present study, using immunohistochemical and in situ hybridization techniques, we report for the first time the cellular distribution of HO-1 and HO-2 in the testes of normal and heat-shocked rats and define a cell-specific expression of the isozymes and a stage-specific expression of HO-2 in the organ. In normal tissue, HO-1 was present at low levels in the Sertoli cells and could not be detected in germ or Leydig cells. HO-2, on the other hand, was most prominently expressed in residual bodies and was not detected in spermatogonia. Modest levels of HO-2 were observed in spermatocytes, spermatids, and select Leydig cells. In contrast, prominent expression of HO-2 messenger RNAs (mRNAs) was detected by in situ hybridization in spermatogonia, as well as spermatocytes, spermatids, and residual bodies of the seminiferous epithelium. The expression pattern of HO-2 protein and transcript in testes of heat-stressed (42 C; 20 min) rats did not differ from that in the control animals, whereas the expression pattern of HO-1 differed from that in the controls, in which distinct populations of Leydig and Sertoli cells displayed intense immunoreactivity. Thermal stress also resulted in an increase (2.8-fold) in the testicular HO-1 mRNA level within 1 h after treatment, followed by a significant increase (32%) in total microsomal heme oxygenase activity 6 h after treatment. Notably, this increase followed a significant depression (36%) in enzyme activity, which was detected 1 h after hyperthermia. The disparity between HO-2 mRNA and protein distribution clearly indicates cell-specific differences in the translational efficiency of HO-2 transcripts. It appears that HO-2 mRNA translation is linked to the maturation and expression of a factor(s) that regulates this process. This, in turn, appears to coincide with sperm development. HO-1 activity, on the other hand, which has a transcriptional component to its regulation, may have a role in maintenance of the conditions required for spermatogenesis.

Distribution of constitutive (HO-2) and heat-inducible (HO-1) heme oxygenase isozymes in rat testes: HO-2 displays stage-specific expression in germ cells.

The heme oxygenase isozymes, HO-1 and HO-2, oxidatively cleave the heme molecule to produce antioxidants, the bile pigments, the gaseous cellular messenger, CO, and iron, a regulator of transferrin, ferritin, and nitric oxide synthase gene expression. HO-1 (hsp32) is a stress-inducible enzyme, whereas HO-2 is constitutively expressed at high levels in the testes and brain. In the present study, using immunohistochemical and in situ hybridization techniques, we report for the first time the cellular distribution of HO-1 and HO-2 in the testes of normal and heat-shocked rats and define a cell-specific expression of the isozymes and a stage-specific expression of HO-2 in the organ. In normal tissue, HO-1 was present at low levels in the Sertoli cells and could not be detected in germ or Leydig cells. HO-2, on the other hand, was most prominently expressed in residual bodies and was not detected in spermatogonia. Modest levels of HO-2 were observed in spermatocytes, spermatids, and select Leydig cells. In contrast, prominent expression of HO-2 messenger RNAs (mRNAs) was detected by in situ hybridization in spermatogonia, as well as spermatocytes, spermatids, and residual bodies of the seminiferous epithelium. The expression pattern of HO-2 protein and transcript in testes of heat-stressed (42 C; 20 min) rats did not differ from that in the control animals, whereas the expression pattern of HO-1 differed from that in the controls, in which distinct populations of Leydig and Sertoli cells displayed intense immunoreactivity. Thermal stress also resulted in an increase (2.8-fold) in the testicular HO-1 mRNA level within 1 h after treatment, followed by a significant increase (32%) in total microsomal heme oxygenase activity 6 h after treatment. Notably, this increase followed a significant depression (36%) in enzyme activity, which was detected 1 h after hyperthermia. The disparity between HO-2 mRNA and protein distribution clearly indicates cell-specific differences in the translational efficiency of HO-2 transcripts. It appears that HO-2 mRNA translation is linked to the maturation and expression of a factor(s) that regulates this process. This, in turn, appears to coincide with sperm development. HO-1 activity, on the other hand, which has a transcriptional component to its regulation, may have a role in maintenance of the conditions required for spermatogenesis.

Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress.

Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.

Increased expression of heme oxygenase mRNA in rat brain following transient forebrain ischemia

Heme oxygenase is a rate-limiting enzyme in heme catabolism, the end products of which include iron, carbon monoxide and bilirubin. Expression of the inducible form of heme oxygenase (HO-1) was investigated in rat brain following 20 min of forebrain ischemia by Northern blot and in situ hybridization analyses. The level of HO-1 mRNA was undetectable in the cerebral cortex of sham control, but increased following ischemic insult, reached the maximum after 12 h of reperfusion, and then decreased. In sham control brain, HO-1 mRNA was detectable only in the scattered neuron-like cells within the dentate gyrus hilus. At 12 h of reperfusion, the remarkable increase in HO-1 mRNA levels was observed in both neuronal and glia-like cells disturbed in the neocortex, hippocampus and thalamus.

Hemeoxygenase expression after reversible ischemia of rat brain

Heme oxygenase (HO-1) gene expression was studied in the brains of rats subjected to 30 min global cerebral ischemia followed by recirculation of up to 24 h. Total RNA was isolated from the cerebral cortex, striatum and hippocampus and reverse-transcribed into cDNA. cDNA was taken as template for PCR using HO-1-specific primers. We found that, when PCR reactions were run for 22 cycles, the amount of PCR products correlated closely with the amount of cDNA. HO-1 gene expression was sharply increased after cerebral ischemia in all three brain structures studied. In the cortex and striatum, the HO-1 mRNA content increased constantly after cerebral ischemia up to 24 h of recovery, being 8- and 9-fold over control after 24 h of recirculation in the cortex and striatum, respectively. In the hippocampus, HO-1 mRNA levels peaked at 4 h after ischemia (9-fold over control) and declined thereafter to 4.5-fold over control 24 h after ischemia. Assuming that the observed increase in mRNA levels is paralled by increased HO-1 protein synthesis, formation of the products of HO reaction, biliverdin and carbon monoxide, is activated after ischemia. These products may produce different and divergent effects on the recovery from the metabolic stress produced by cerebral ischemia.

Tin-Mediated Heme Oxygenase Gene Activation and Cytochrome P450 Arachidonate Hydroxylase Inhibition in Spontaneously Hypertensive Rats

The effect of SnCl2 on the transcription of the heme oxygenase gene in spontaneously hypertensive rats was examined using cDNA for the rat heme oxygenase (HO-1). An increase in renal HO-1 mRNA levels was observed in response to SnCl2 treatment. Quantitative evaluation by scanning densitometry demonstrated a maximal increase in HO-1 mRNA 24-fold over control at 8 hours after SnCl2 administration. Nuclear runoff assay using isolated renal nuclei from SnCl2-treated rats revealed an active HO-1 gene transcription. Transcription of HO-1 in rat kidney was greatly increased within 3 hours of administration of SnCl2, as evidenced by the level of [alpha 32P]UTP incorporation into nuclear RNA. As a consequence of activation of the HO-1 gene transcription, renal enzyme activity increased eightfold at 16 hours after SnCl2, and reached maximal activity of 16-fold over control at 32 hours after injection. No significant change in cytochrome P450 fatty acid omega-hydroxylase (P450 4A) mRNA was observed after SnCl2 administration. Cytochrome P450-arachidonic acid omega/omega-1 hydroxylase(s) activity (formation of 20- and 19-HETE) was significantly reduced 24 hours after SnCl2 administration and remained lower than the control level 48 and 72 hours after injection. In addition, blood pressure was reduced from 151 +/- 2.5 mm Hg to 133 +/- 2.3 mm Hg after 48 hours of SnCl2 treatment. The reduction in blood pressure preceded natriuresis. It is concluded that SnCl2 induces activation of the HO-1 gene, which is followed by elevation in enzyme activity and a decrease in cytochrome P450-arachidonic acid omega-hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain.

In mammalian systems, the heme oxygenase (HO) isozymes HO-1 (HSP32) and HO-2 oxidatively cleave the heme molecule to produce bile pigments and carbon monoxide. Although HO-1 is inducible by various chemicals in systemic organs and cell culture systems, this communication reports for the first time the induction of this stress protein and its transcript by a chemical in the brain. In addition, this study demonstrates expression of HO-1 in select populations of cells in the brain in response to GSH depletion. Specifically, treatment of adult rats with diethyl maleate (DEM; 4.7 mmol/kg) caused a pronounced decrease in brain GSH content within 1 h. GSH levels remained significantly depressed for at least 24 h postinjection. Northern blot analysis of brain poly(A)+ mRNA following DEM treatment revealed on the average a sixfold increase in the 1.8-kb HO-1 mRNA level compared with that of controls; concomitant with this change was a decrease in GSH levels. Total brain HO activity was not significantly altered along with the increase in HO-1 mRNA level. The increase in transcription of HO-1 was a direct response to GSH depletion, as judged by the observation that treatment of neonatal rats with L-buthionine-(S,R)-sulfoximine (BSO) (3 mmol/kg, twice daily, for 2 days), a selective inhibitor of GSH synthesis, caused a marked depression in total brain GSH level and a concomitant increase in brain 1.8-kb HO-1 mRNA content. The magnitude of the increase was up to approximately 11.5-fold that of the control level, as evidenced by northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation.

We cloned a cDNA coding for a putative human heme oxygenase isozyme, designated type 2 (HO-2), and analyzed its function by transient expression assays. HeLa cells transfected with either HO-2 cDNAs or a cDNA coding for authentic heme oxygenase (HO-1) expressed the activity of heme oxygenase, although no activity was detected in the mock transfected cells. Using specific anti-HO-1 antibody, we showed that expression of a HO-1 cDNA resulted in the increase in its protein levels, but HO-1 protein was not detectable in the cells expressing HO-2 cDNAs. We thus confirmed the functional identity of HO-1 and HO-2. Then, we analyzed their expression in an erythroid cell line, YN-1-0-A. Treatment with hemin or by heat shock (42 degrees C) led to a remarkable increase in the HO-1 mRNA levels, while HO-2 mRNA expression was not induced at all, suggesting that they are under separate regulation.

Tin-Protoporphyrin-Mediated Disruption In Vivo of Heme Oxygenase-2 Protein Integrity and Activity in Rat Brain

The ability of synthetic metalloporphyrins to suppress heme oxygenase activity and bilirubin formation has recently become of considerable clinical and experimental interest for suppression of jaundice in humans, including neonatal hyperbilirubinemia. The present investigation compares the biochemical effects of Sn- and Zn-protoporphyrins on the predominant heme oxygenase isozyme present in the brain (HO-2) at activity, protein, and transcript levels and describes the ability of Sn-protoporphyrin to adversely affect this isozyme. Specifically, 6 h after a modest dose (50 mumol/kg, i.v.) of Sn-protoporphyrin, heme oxygenase activity in rat brain was nearly undetectable. In addition, as revealed by Western blot analysis, HO-2 protein level was decreased by 20% and the electrophoretic behavior of the protein in the microsomal membranes was altered. Moreover, the activity of NADPH-cytochrome P-450 reductase, which is required for the oxidation of heme molecule, was markedly decreased (60% of control). Western immunoblot analysis revealed also a pronounced decrease in the reductase protein level. The inducible form of heme oxygenase, HO-1, was not detectable by immunoblotting in brain microsomes of either control or Sn-protoporphyrin-treated animals. Northern blot analyses did not reveal decreases in the levels of the single HO-1 mRNA (1.8 kb) or the two HO-2 transcripts (1.3 and 1.9 kb), suggesting that Sn-protoporphyrin mediates its effects on heme oxygenase isozymes at the protein level. Zn-protoporphyrin, on the other hand, had no deleterious effect on brain parameters presently investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of heme oxygenase isozymes by Sn- and Zn-protoporphyrins: Possible relevance to suppression of hyperbilirubinemia

Synthetic metalloporphyrins decrease heme oxygenase (HO)-dependent bilirubin formation. Presently, the effects in vivo and in vitro of Sn- and Zn-protoporphyrins on HO-1 (HSP-32) and HO-2 at the protein and transcript levels were examined. Western blot analysis of HO-2 in testes microsomes of Sn-protoporphyrin-treated rats revealed a dramatic disruption of the integrity of the HO-2 protein. Similar observations were made with the liver and adrenal HO-2 and the NADPH-cytochrome P-450 reductase of treated rats. Northern blot analysis, however, suggested unaltered tissue levels of HO-2 transcripts (∼ 1.9 and ∼ 1.3 kb). The HO-1 protein integrity in organs of treated rats was less dramatically affected by the metalloporphyrin and an increase in its 1.8 kb mRNA level in the testes was detected. Zn-protoporphyrin also increased HO-1 mRNA level in the testes, but did not affect HO-2 protein integrity. In in vitro studies with purified HO-1 and HO-2, both Sn- and Zn-protoporphyrins were equally inhibitory to HO-1 activity; Sn-protoporphyrin, however, was by far more inhibitory to HO-2-dependent activity than to that of HO-1. Together, these findings and the fact that HO-2 under normal conditions is the predominant form of the enzyme in most organs suggest that loss of HO-2 protein integrity may to a significant degree account for suppression of bilirubin formation by Sn-protoporphyrin. These in turn may reflect differences between HO-1 and HO-2, both at the transcriptional level with HO-2 being noninducible, and in structure/composition of the isozymes, with HO-2 being more labile.

Normal and Heat‐Induced Patterns of Expression of Heme Oxygenase‐1 (HSP32) in Rat Brain: Hyperthermia Causes Rapid Induction of mRNA and Protein

Most cells possess a variety of mechanisms, such as high levels of glutathione, that guard against cytotoxic free radicals, which are suspected in the etiology of various neurological deficits. Neurons, however, are deficient in this antioxidant source. The list of other potent antioxidants includes the bile pigments biliverdin and bilirubin. Heme oxygenase (HO) isozymes, HO-1 (HSP32) and HO-2, catalyze the rate-limiting step in the only biological pathway by which bile pigments are produced. In this study, heat shock is identified as the only stimulus reported to date that can alter expression in brain HO-1 of protein and mRNA in vivo. Using a HO-1 cDNA probe, we examined the level of HO-1 mRNA in normal rat brain and in brain 1 and 6 h following heat shock. Exposure of male rats to 42 degrees C for 20 min caused a 20-fold increase in brain HO-1 1.8-kb mRNA within 1 h after treatment. Quantification of brain HO-1 protein by HO-1 radioimmunoassay revealed a fourfold increase at 6 h posttreatment. In normal brain, HO-1 protein was sparsely expressed in few select neuronal and nonneuronal cell populations in forebrain, diencephalon, cerebellum, and brainstem regions. Six hours following heat shock, an intense increase in HO-1 protein in glia throughout the brain, ependyma lining the ventricles of the brain, paraventricular nucleus, Purkinje cell layer of the cerebellum, and cochlear nucleus of brainstem was observed. We suggest that increases in HO-1 transcript and protein reflect a means to elevate levels of antioxidants in cells with compromised defense mechanisms caused by stress.