Biomedical subjects

M D Maines

Publications and source records attributed to M D Maines.

At least 19 recordsLinked to original sources

PubMed1999

Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice.

Heme oxygenase-1 (HO-1, HSP32) is an early gene that is responsive to an array of pathological conditions including, but not limited to, hypoxia and cerebral ischemia. HO-1 cleaves the heme molecule and produces carbon monoxide (CO) and biliverdin (an antioxidant) and is essential for iron homeostasis. The purpose of this study was to investigate, using transgenic (Tg) mice, whether overexpression of HO-1 in the brain augments or attenuates cellular injury caused by ischemic stroke. Homozygous HO-1 Tg mice that overexpress HO-1 under the control of the neuron-specific enolase promoter (characterized previously) were used. Under halothane anesthesia and normothermic conditions, wild-type nontransgenic (nTg; n = 22) and HO-1 Tg (n = 24) mice were subjected to middle cerebral artery occlusion (MCAo). Six hours after induction of ischemia, Tg and nTg mice developed infarcts that were 39 +/- 6 and 63 +/- 9 mm3, respectively (p < 0.01). No significant difference between the two strains was observed in the values of brain edema (11.3 +/- 4% in Tg vs. 14.6 +/- 5% in nTg; p < 0.1). At 24 h after MCAo, Tg mice exhibited significant neuroprotection as determined by the stroke volumes (41 +/- 2 mm3 in Tg vs. 74 +/- 5 mm3 in nTg; p < 0.01) and values of ischemic cerebral edema (21 +/- 6% in Tg vs. 35 +/- 11% in nTg; p < 0.01). Data suggest that neuroprotection in Tg mice was, at least in part, related to the following findings: (a) constitutively up-regulated cyclic GMP and bcl-2 levels in neurons; (b) inhibition of nuclear localization of p53 protein; and (c) antioxidant action of HO-1, as detected by postischemic neuronal expression of ferritin, and decreases in iron staining and tissue lipid peroxidation. We suggest that pharmacological stimulation of HO-1 activity may constitute a novel therapeutic approach in the amelioration of ischemic injury during the acute period of stroke.

Animals↗

PubMed1998

Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine.

BACKGROUND & AIMS: The interstitial cell (IC) network may be of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells are depolarized in the absence of ICs, and there are no spontaneous slow waves. The messenger molecules between IC network and smooth muscle are unknown. Exogenous administration of CO relaxes the opossum internal anal sphincter and the guinea pig ileum, and it modulates potassium current and membrane potential of circular smooth muscle cells of the human jejunum. The aim of this study was to determine whether heme oxygenase (HO)-1 and HO-2, enzymes that catalyze the production of CO, are present in the IC network of the mouse small intestine. METHODS: Antibodies specific for c-Kit, HO-1, and HO-2 were used for immunohistochemistry. Confocal images were obtained and were volume rendered, and the images were converted into three-dimensional images. RESULTS: HO-2-like but not HO-1-like immunoreactivity was found in IC networks associated with the myenteric plexus and the deep muscular plexus. CONCLUSIONS: HO-2 but not HO-1 is present in the IC cell network of the mouse small intestine. The enzymatic activity of HO-2 will result in the endogenous production of CO in IC networks of the mouse small intestine.

Animals↗

PubMed1998

Neuronal overexpression of heme oxygenase-1 correlates with an attenuated exploratory behavior and causes an increase in neuronal NADPH diaphorase staining.

Heme oxygenase isozymes, HO-1 (also known as hsp32) and HO-2, are the source for the formation of the putative messenger molecule carbon monoxide (CO), reactive iron, and the in vitro antioxidant bilirubin. We have developed and characterized transgenic (Tg) mice that overexpress the stress protein in neurons in various brain regions. The Tg mice were generated by the use of rat HO-1 cDNA under the control of the neuron-specific enolase promoter. Except for a tendency to have an enlarged spleen, Tg mice did not show gross anatomical changes. Increase in HO-1 mRNA, which was demonstrated by northern blot analysis and in situ hybridization, was accompanied by an increase in neuronal HO-1 protein expression, shown by immunohistochemistry and western blotting, and an increase in HO activity. Expression of the transgene correlated with an attenuation of exploratory behavior and increased circling activity and coincided with enhanced neuronal NADPH diaphorase staining. Those changes were not accompanied by an increase in DNA damage or significant change in whole-brain NO synthase activity. The HO-1 Tg mice potentially represent a good model to examine the function of CO as a neuromodulator, iron as a gene regulator, and bile pigments as in vivo antioxidants.

Animals↗

PubMed1998

Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum.

Carbon monoxide (CO) has been postulated to be a messenger in the gastrointestinal tract. The aims of this study were to determine the distribution of heme oxygenase (HO), the source for endogenous CO in the canine jejunum, and to determine the effects of CO on jejunal circular smooth muscle cells. HO-2 isoform was present in a population of myenteric and submucosal neuronal cell bodies, in nerve fibers innervating the muscle layers, and in smooth muscle cells. HO-1 isozyme was not detected in the canine jejunum. Exogenous CO increased whole cell current by 285 +/- 86%, hyperpolarized the membrane potential by 8.5 +/- 2.9 mV, and increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in smooth muscle cells. 8-Bromo-cGMP also increased the whole cell current. The data suggest that endogenous activity of HO-2 may be a source of CO in the canine jejunum and that exogenously applied CO can modulate intestinal smooth muscle electrical activity. It is therefore reasonable to suggest a role for endogenously produced CO as a messenger in the canine jejunum.

Alkaloids↗

PubMed1997

Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3.

Two isozymes of heme oxygenase (HO), HO-1 or HSP32 and the constitutive form HO-2, have been characterized to date. We report the discovery of a third protein species and refer to it as HO-3. HO-3 is the product of a single transcript of approximately 2.4 kb and can encode a protein of approximately 33 kDa. The HO-3 transcript is found in the spleen, liver, thymus, prostate, heart, kidney, brain and testis and is the product of a single-copy gene. The predicted amino acid structure of HO-3 differs from both HO-1 (HSP32) and HO-2 but is closely related to HO-2 (approximately 90%). Escherichia coli expressed and purified HO-3 protein does not cross react with polyclonal antibodies to either rat HO-1 or HO-2, is a poor heme catalyst, and displays hemoprotein spectral characteristics. The predicted protein has two heme regulatory motifs that may be involved in heme binding. These motifs and the hemoprotein nature of HO-3 suggest a potential regulatory role for the protein in cellular processes which are heme-dependent.

Amino Acid Sequence↗

PubMed1997

Heme oxygenase-2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis.

The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 --> Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 --> Ala mutation inactivates HO-2, Cys264 --> Ala and Cys281 --> Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 --> Ala or Pro282 --> Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell.

Animals↗

PubMed1997

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 alpha, 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 microg/g, 4 x, 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 (approximately 1.3 and approximately 1.9 kb) increased in HeLa cells treated with DX (5 microM), 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.

Animals↗

PubMed1997

Histochemical localization of heme oxygenase-2 protein and mRNA expression in rat brain.

Heme oxygenase (HO) proteins are members of the HSP30 family and consist of 2 isozymes identified to date, termed HO-1 and HO-2. Separate genes encode the isozymes and protein products which are immunochemically distinct, share less than 50% similarity at the amino acid sequence level. Each form, however, shows greater than 90% similarity among species, including human and the rat (reviewed in ref.). Furthermore, these isozymes function in a well-defined role to carry out oxidation of the heme molecule (Fe-protoporphyrin IX) in concert with NADPH-cytochrome P450 reductase. The oxidation of heme is isomer specific and results in the formation of bile pigments, carbon monoxide, and iron. The heme molecule constitutes the prosthetic moiety of hemoproteins, such as hemoglobin, myoglobin, catalase, soluble guanylate cyclase, cytochrome b5, cytochromes P450 and NO synthase. HO-1 also known as heat shock protein (HSP) 32 is encoded by a gene which is exquisitely stress-responsive and a host of stimuli that mediate oxidative stress cause induction of the protein both in vivo and in vitro. The HO-2 form shows a unique pattern of regulation from that of HO-1. HO-2 is a constitutive protein and its expression is not affected by the inducers of HO-1 tested to date; rather, the only known regulator of HO-2 yet identified is adrenal glucocorticoids. The two isozymes display vast differences in tissue distribution and under normal conditions HO-1 is present in the whole brain at the limit of immunodetection and is discreetly localized in select neuronal populations. HO-1 protein (approximately 32 kDa) and its approximately 1.8 kb transcript are increased, however, in response to stressful stimuli primarily in non-neuronal cell populations. The heme oxygenase system serves in both a catabolic and anabolic capacity in the cell. In the former capacity, it down-regulates cellular heme and hemoprotein levels. And, as such it inactivates the most effective catalyst for formation of free radicals, the heme molecule. In its anabolic role, as noted above, heme oxygenase produces bile pigments, carbon monoxide, and iron, all of which are biologically active: bile pigments function as antioxidants; the carbon monoxide generated by HO activity has been correlated with the generation of cGMP; and iron regulates expression of various genes, including that of HO-1 itself, as well as transferrin receptors, ferritin, and NO synthase. We used rabbit anti-rat HO-2 polyclonal antibody and HO-2 cDNA to localize HO-2 immunoreactive protein and the 1.3- and 1.9 kb homologous transcripts, respectively, in rodent brain as visualized by histochemical staining procedures. These protocols provide the first detailed description of methodologies successfully used to define the pattern of HO-2 expression at the transcriptional and translational levels in the adult rat brain and glucocorticoid-treated newborn rats. The procedures described herein have the virtue of being non-radioactive, as well as applicability to the systemic organs, such as the cardiovascular system and the male reproductive organs. Visualization of cellular HO-2 expression aids in assessment of potential sites of carbon monoxide, iron, and bilirubin production within the nervous system.

Animals↗

PubMed1997

Regulation of photomorphogenesis by expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.

The photoregulatory activity of the phytochrome photoreceptor requires the synthesis and covalent attachment of the linear tetrapyrrole prosthetic group phytochromobilin. Because the mammalian enzyme biliverdin IX alpha reductase (BVR) is able to functionally inactivate phytochromobilin in vitro, this investigation was undertaken to determine whether BVR expression in transgenic plants would prevent the synthesis of functionally active phytochrome in vivo. Here, we show that plastid-targeted, constitutive expression of BVR in Arabidopsis yields plants that display aberrant photomorphogenesis throughout their life cycle. Photobiological and biochemical analyses of three transgenic BVR lines exhibiting a 25-fold range of BVR expression established that the BVR-dependent phenotypes are light dependent, pleiotropic, and consonant with the loss of multiple phytochrome activities. Chlorophyll accumulation in BVR-expressing transgenic plants was particularly sensitive to increased light fluence rates, which is consistent with an important role for phytochrome in light tolerance. Under blue light, transgenic BVR plants displayed elongated hypocotyls but retained phototropic behavior and the ability to fully deetiolate. Directed BVR expression may prove to be useful for probing the cellular and developmental basis of phytochrome-mediated responses and for selective control of individual aspects of light-mediated plant growth and development.

Animals↗

PubMed1997

The heme oxygenase system: a regulator of second messenger gases.

The heme oxygenase (HO) system consists of two forms identified to date: the oxidative stress-inducible protein HO-1 (HSP32) and the constitutive isozyme HO-2. These proteins, which are different gene products, have little in common in primary structure, regulation, or tissue distribution. Both, however, catalyze oxidation of heme to biologically active molecules: iron, a gene regulator; biliverdin, an antioxidant; and carbon monoxide, a heme ligand. Finding the impressive heme-degrading activity of brain led to the suggestion that "HO in brain has functions aside from heme degradation" and to subsequent exploration of carbon monoxide as a promising and potentially significant messenger molecule. There is much parallelism between the biological actions and functions of the CO- and NO-generating systems; and their regulation is intimately linked. This review highlights the current information on molecular and biochemical properties of HO-1 and HO-2 and addresses the possible mechanisms for mutual regulatory interactions between the CO- and NO-generating systems.

Animals↗

PubMed1996

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain.

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.

Animals↗

PubMed1996

Corticosterone promotes increased heme oxygenase-2 protein and transcript expression in the newborn rat brain.

Heme oxygenase-2 (HO-2) is the predominant heme oxygenase isozyme in neurons in the brain, the enzyme cleaves the heme molecule at the alpha-meso carbon bridge to form CO, Fe and biliverdin. Recently, in the promotor region of the HO-2 gene a consensus sequence of the glucocorticoid response element (GRE) has been identified. Presently, we have investigated the potential relevance of the GRE to the expression of the isozyme, at the transcript and protein levels, in the 14 day old rat brain, by examining the effect of postparturition corticosterone treatment (4 days, starting 24-36 h after birth) on the developmental pattern of HO-2 expression. Northern blot analysis showed that HO-2 transcripts (approximately 1.3 and approximately 1.9 kb) in brain increase with age. In many brain nuclei, HO-2 protein, as visualized by immunohistochemistry, was detected at low levels in neurons in the 14 day old rat brain. Postparturition exposure to corticosterone resulted in a marked enhancement of HO-2 immunoreactivity in several neuronal populations, including, among others, the cerebellum, the hippocampal formation, and the oculomotor and red nuclei. The response to elevated levels of corticosterone was particularly striking in the Purkinje neurons of the cerebellum and the CA3 region of the hippocampus. This was linked to an increase in gene transcription, as indicated by in situ hybridization analysis, which revealed an increase in the signal for HO-2 transcripts in these regions. Elevated levels of heme oxygenase activity and HO-2 protein were consistent with an increase in catalytically active protein expression. These data point to the intimate involvement of the adrenal steroids in developmentally-linked HO-2 expression in the neurons involved in motor function and cognition, and hence, identify a potentially important aspect of the adrenal steroids' effect on brain growth and differentiation.

Aging↗

PubMed1996

Human biliverdin IXalpha reductase is a zinc-metalloprotein. Characterization of purified and Escherichia coli expressed enzymes.

Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity. A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene. Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity.

Amino Acid Sequence↗

PubMed1996

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.

Aged↗

PubMed1996

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.

Animals↗

PubMed1996

Renal ischemia/reperfusion up-regulates heme oxygenase-1 (HSP32) expression and increases cGMP in rat heart.

For the first time, the authors report an intimate link between kidney and heart functions as it pertains to the regulation of stress protein gene expression in the heart. They show that the stress on the target organ, the kidney, is translated into a response in the cardiovascular system, as reflected by the induction of heme oxygenase (HO)-1 gene expression, which, in turn, may be a cellular defense response as suggested by an increase in cGMP level in the heart, and an increase in the rate of bilirubin formation by the kidney and the heart. HO-1 is a stress protein (HSP32) and, together with HO-2, catalyzes oxidation of the heme molecule to generate CO, a likely signal molecule for the generation of cGMP, and bilirubin, an antioxidant. Specifically, bilateral renal ischemia for 30 min caused a 3-fold increase in the approximately 1.8-kb HO-1 mRNA in the heart within 4 h after reperfusion and remained essentially at this level for 24 h, at which point, a 2.6-fold increase in HO-1 mRNA in the descending aorta was also detected. Heart HO-1 mRNA remained elevated for more than 48 h; in contrast, at the 48-h time point, the transcript level in the kidney, which had increased by 10-fold 24 h after reperfusion, had returned to the control level. Neither in the heart nor in the kidney did HO-2 transcripts (approximately 1.3 and 1.9 kb) respond to renal ischemia/ reperfusion. The increase in heart HO-1 transcript level was accompanied by an increase in HO-1 protein, as judged by Western blot and immunohistochemical analysis, and in enzyme activity, as judged by bilirubin formation. In addition, cGMP concentration in the heart was elevated when measured at 24 h and 48 h after reperfusion of the kidney, in the absence of an increase in the activity of NO. Data suggest that hemodynamic stress caused by the occlusion of the renal artery is responsible for activation of HO-1 gene expression in the heart. An argument is made for the role of HO-1 in the defense mechanisms of the heart pertaining to the enzyme's function in a hemoprotein regulatory capacity, along with the biological activity of its products.

Animals↗

PubMed1995

The structure, organization and differential expression of the rat gene encoding biliverdin reductase.

Screening of phage lambda libraries and genomic polymerase chain reactions were employed to generate clones of the rat gene encoding biliverdin reductase (BVR), the penultimate enzyme in the heme metabolic pathway. This enzyme, which converts biliverdin to bilirubin, is unique among enzymes characterized to date in that it exhibits two pH optima, 6.75 and 8.7, and utilizes a different cofactor, NADH and NADPH, respectively, at each optimum. The gene, which is 12270 bp in length, consists of five exons and four introns; two introns are > or = 4 kb. Only two of the four splice sites conform to consensus donor/acceptor sequences. Primer extension indicates the presence of two distinct transcription start points (tsp) in kidney and brain, as well as an additional tsp present in kidney, but not in brain RNA. The gene lacks a conventional TATA-box; however, an overlapping pair of TATA-like sequences is found 80 nt upstream from the kidney-specific tsp. The promoter region contains binding sites for several known regulatory factors, including AP-1, HNF-5 and INF-1, as well as two partial (7/8) matches to the heat-shock (HS) transcription factor-binding site. However, the time-course of the increase in message level, as determined by Northern blot analysis, indicates that BVR is not an early HS protein in that the relative abundance of mRNA is increased 6 h after hyperthermia and not at 1 h after HS. The approx. 1.6-kb BVR message is abundantly expressed in kidney, spleen, liver and brain, and at lower levels in the thymus, with minimal levels being detected in testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Immunohistochemical localization of biliverdin reductase in rat brain: age related expression of protein and transcript.

Biliverdin reductase regulates heme oxygenase activity by removing the inhibitory product of the oxygenase activity, biliverdin; and reducing it to bilirubin. The other products of the oxygenase are carbon monoxide and Fe. To date, biliverdin reductase remains unique among all enzymes described by using 2 different cofactors (NADPH and NADH) at different pH ranges. The present study reports on the developmentally regulated changes in the pattern of protein expression and the level of biliverdin reductase transcript in rat brain. Biliverdin reductase activity of the brain cytosol with both NADPH (pH 8.7) and NADH (pH 6.7) exhibited developmental changes with the activity increasing after birth, reaching an adult level by day 28 postpartum. When analyzed by Western blotting the immunoreactive protein detected increased as the animal matured (day 1 to 28 postparturition). Northern blot hybridization of RNA isolated from rat brain revealed the presence of approximately 1.5 kb biliverdin reductase transcript at all stages of development ranging from 1 day post partum to 20 months. The level of the transcript was developmentally regulated and a gradual increase ( approximately 4-fold) was observed from day 1 after birth to adulthood and was maintained in 20 month old animals. Cellular localization, using immunohistochemical technique, revealed age-related pattern of expression of the reductase in select regions such as the cortex, substantia nigra, hippocampus and in the cerebellum; the changes, however, did not follow the same pattern. To elaborate, in the cortex, the reductase expression increased when 7-day-old animals were compared with young adults (2 months old) and then declined in the 20-month-old animals. In the substantia nigra the level of reductase expression progressively declined with age when 7-day-old neonate, 2- and 20-month-old animals were compared. In the hippocampus, a distinct reductase-expressing cell population residing between CA1 and the dentate gyrus was observed in the 7-day-old animals; these cells were not detected in the adults (2 or 20 months old). In the cerebellum, the expression of the reductase reflected the developmental organization of this region. We postulate that age-dependent increase of the brain reductase at the transcript and protein levels in the course of maturation serves to control heme oxygenase activity which also displays a developmental pattern in the organ. As such, the reductase modulates generation of biologically active heme degradation products; bilirubin, carbon monoxide and Fe.

Aging↗