Regulation and expression of heme oxygenase enzymes in aged-rat brain: age related depression in HO-1 and HO-2 expression and altered stress-response.

The heme oxygenase isozymes, HO-1 and HO-2, oxidatively cleave the heme molecule to produce biliverdin and the gaseous messenger, CO. The cleavage results in the release of iron, a regulator of transferrin, ferritin, and nitric oxide (NO) synthase gene expression. Biliverdin reductase (BVR) then catalyzes the reduction of biliverdin, generating the potent intracellular antioxidant, bilirubin. We report an age-related decrease in HO-1 and HO-2 expression present in select brain regions including the hippocampus and the substantia nigra, that are involved in the high order cognitive processes of learning and memory. The age-related loss of monoxide-producing potential in select regions of the brain was not specific to the HO system but was also observed in neuronal NO-generating system. Furthermore, compared to 2-month old rats, the ability of aged brain tissue to respond to hypoxic/hyperthermia was compromised at both the protein and the transcription levels as judged by attenuated induction of HO-1 immunoreactive protein and its 1.8 Kb transcript. Neotrofin (AIT), a cognitive-enhancing and neuroprotective drug, caused a robust increase in HO-1 immunoreactive protein in select neuronal regions and increased the expression of HO-2 transcripts. The potential interplay between regulation of HO-2 gene expression and the serum levels of the adrenal steroids is discussed. We suggest the search for therapeutic agents that reverse the decline and aberrant stress response of HO enzymes may lead to effective treatment regimens for age-associated neuronal deficits.

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.

Multiple transcripts encoding heme oxygenase-2 in rat testis: developmental and cell-specific regulation of transcripts and protein.

We report for the first time that heme oxygenase-2 (HO-2) expression is regulated by developmental and cell type-specific factors in the testis, and we describe the presence of three unique sizes of HO-2 transcripts in the testis. HO-2, together with HO-1 (HSP32), catalyzes oxidative cleavage of the heme molecule to biliverdin, carbon monoxide, and iron; HO-2 is the major isozyme of the testis. Northern blot analysis was used to demonstrate the presence of five transcripts for HO-2 in rat testis mRNA; they range from approximately 1.3 to approximately 2.1 kg in length with a predominant 1.45-kb message; three of the transcripts, approximately 1.45 kb, approximately 1.7 kb, and approximately 2.1 kg, are unique to testis. The two other transcripts of approximately 1.3 and approximately 1.9 kb are common to every tissue examined, including the testis. Analysis of three distinct cDNAs isolated from rat libraries in phage lambda indicates that all are identical from -37, relative to translation initiation through the coding region to the first of two poly(A) signals previously identified in the HO-2 gene (McCoubrey and Maines, 1994). Upstream of -37, the 5' untranslated sequences of the isolates differ in both length and sequence. Comparison with the genomic sequence suggests that the multiple transcripts arise by splicing of alternative first exons as well as use of alternate poly(A) signals. Northern hybridization with probes specific for the unique portion of each cDNA are consistent with this interpretation. Further, unlike HO-1, HO-2 messages are developmentally regulated; only approximately 1.3- and approximately 1.9-kb transcripts were detected, at minute levels, in the testis RNA of 7-day-old rats. A pronounced increase in total message level was observed by Day 28 postpartum, although the level had not reached the marked amplification seen in the adult testis. Further, the transcript patterns differed when Day 28 and adult testis were compared to Day 7 testis. The very predominant approximately 1.45-kb band and the approximately 1.7- and 2.1-kb bands were absent from Day 7 testis. Heme oxygenase activity and HO-2 protein levels, as assessed by Western blot, reflect the increases at the RNA level. Interestingly, although abundant HO-2 mRNA can be detected by in situ hybridization in spermatogonia, spermatocytes, and spermatids, HO-2 protein was detected, by immunocytochemistry, only in spermatids. These observations demonstrate tissue and cell specificity of HO-2 gene expression and suggest that in the testis, HO-2 expression is regulated at the transcriptional and translational levels.

Coordinated expression and mechanism of induction of HSP32 (heme oxygenase-1) mRNA by hyperthermia in rat organs.

Heme oxygenase isozymes, HO-1 and HO-2, catalyze the cleavage of heme b (Fe-protoporphyrin-IX) at the alpha-meso carbon bridge to form the antioxidant, biliverdin IX alpha, and the putative cellular messenger, carbon monoxide. HO-1 is a heat shock (HSP32) or stress protein, while HO-2 is a noninducible enzyme. Presently, we have examined the time course of expression of HSP32 in liver, kidney, and heart of rats exposed to hyperthermia and investigated the mechanism of induction of HO-1 by hyperthermia. We report a coordinated induction response of all organs to elevated ambient temperature (42 degrees C, 20 min). Specifically, the maximum induction of the 1.8 kb HO-1 mRNA was observed 1 h after hyperthermia and reached a value 20-40-fold that of the control; the transcript level approximated the control value by 6 h after heat stress. In contrast, the levels and the ratio of the 1.3 and 1.9 kb HO-2 transcripts were not affected by hyperthermia. As judged by in vitro nuclear transcription run-on assays, thermal stress caused the stimulation of HO-1 gene transcription. The increase in HO-1 mRNA transcription was accompanied by an increase in binding of nuclear factor(s) to the heat shock element in the promoter region of the gene. The increase of the HO-1 mRNA was reflected in increases in both heme oxygenase activity and in immunoreactive HO-1 protein. We suggest that the induction of heme oxygenase by heat stress is a physiologically relevant defense mechanism whereby both the degradation of heme of denatured hemoproteins and the generation of biologically active products of heme catabolism are enhanced.

Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein.

Catalytic activity of heme oxygenase (heme, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.3) isozymes, HO-1 and HO-2, permits production of physiologic isomers of bile pigments. In turn, bile pigments biliverdin and bilirubin are effective antioxidants in biological systems. In the rat brain we have identified only the HO-1 isozyme of heme oxygenase as a heat shock protein and defined hyperthermia as a stimulus that causes an increase in brain HO-1 protein. Exposure of male rats to 42 degrees C for 20 min caused a rapid and marked increase in brain 1.8-kilobase HO-1 mRNA. Specifically, a 33-fold increase in brain HO-1 mRNA was observed within 1 h and sustained for at least 6 h posttreatment. In contrast, the two HO-2 homologous transcripts (1.3 and 1.9 kilobases) did not respond to heat shock; neither the ratio nor the level of the two messages differed from that of the control when measured either at 1, 6, or 24 h after hyperthermia. The induction of a 1.8-kilobase HO-1 mRNA resulted in a pronounced increase in HO-1 protein 6 h after hyperthermia, as detected by both Western immunoblot and RIA. Immunocytochemistry of rat brain showed discrete localization of HO-1-like protein only in neurons of select brain regions. Six hours after heat shock, an intense increase in HO-1-like protein was observed in both Purkinje cells of the cerebellum and epithelial cells lining the cerebral aqueduct of the brain. We suggest that the increase in HO-1 protein, hence increased capacity to form bile pigments, represents a neuronal defense mechanism against heat shock stress.

Differential effect of cadmium on GSH-peroxidase activity in the Leydig and the Sertoli cells of rat testis. Suppression by selenium and the possible relationship to heme concentration.

In the testes of rats treated with cadmium acetate (7 or 20 mumoles/kg, 24 hr, s.c.), the activity of glutathione (GSH)-peroxidase was increased. At the same time, the activity of glutathione disulfide (GSSG)-reductase and the cellular GSH concentration were decreased significantly. The basal activity of peroxidase in the Leydig and the Sertoli cell populations was comparable. However, the magnitude of increases in the activities markedly differed in the two cell populations, with that of the Sertoli cells increasing to nearly 450% of the control value in response to treatment with 20 mumoles/kg Cd2+. In the Leydig cells, the enzyme activity in response to the same treatment increased to only about 170% of the control value. Cd2+ treatment increased the concentration of heme in the microsomal and the smooth and rough endoplasmic reticulum fractions of the whole testis, as well as in the microsomal fractions of the Leydig and the Sertoli cells. As with the peroxidase activity, the two cell populations vastly differed in their susceptibilities to Cd2+ treatment, with the Sertoli cells being more severely affected by the metal. In the Sertoli cells the microsomal heme concentration was increased by approximately 11-fold, whereas only a 2-fold increase in the Leydig cells was noted. The increase in GSH-peroxidase activity was not due to the peroxidase activity of GSH-S-transferases, insofar as an increase in transferase activity was not observed in the Leydig and the Sertoli cells. Treatment of rats with sodium selenite (10 mumoles/kg, s.c.) 30 min before Cd2+ treatment (20 mumoles/kg) fully suppressed the above-described spectrum of effects of Cd2+ in the testis. Also, sodium selenite at a lower dose of 5 mumoles/kg prevented an increase in GSH-peroxidase activity. It is hypothesized that increased GSH-peroxidase activity in the Leydig and the Sertoli cells constitutes an adaptive response to increased cellular levels of heme and to the free radicals generated by the heme molecule. Selenium prevents the increase in GSH-peroxidase activity by circumventing the increase in cellular heme concentration. The protection is believed to be related, at least in part, to increased production of cellular GSH.

Biliverdin reductase: characterization in the rat kidney and the inhibition of activity by mercuric chloride.

The effects of metal ions on the activities of biliverdin reductase in the rat kidney and liver were examined; the pH optimum and the cofactor requirement for the enzyme activity in the kidney were also studied. The reduction of biliverdin IX alpha by biliverdin reductase in the rat kidney cytosol fraction could be supported by NADPH and NADH. The activity was optimal around pH 8.7 when NADPH was the cofactor. The activity with NADH was undetectable at this pH. NADH-dependent biliverdin reductase was optimal at pH 7.0, where the NADPH-dependent activity was negligible. Biliverdin reductase activity was not inducible in the kidney or liver in response to treatment of rats with metal ions--Co2+, Ni2+, Pb2+, Sn2+, Zn2+, Cd2+, and Cu2+ or sodium selenite. Rather, both NADPH- and NADH-dependent activities in the kidney were decreased markedly in a time- and dose-related manner following the administration of HgCl2 (10-30 mumoles/kg, 24 hr). The pretreatment of rats (30 min) with sodium selenite (5 mumoles/kg) effectively blocked the Hg2+ (20 mumoles/kg, 24 hr) inhibition of the kidney cytosol biliverdin reductase activity. Similarly, in vitro Hg2+ was an effective inhibitor of the kidney biliverdin reductase. In addition, highly purified biliverdin reductase also was extremely sensitive to Hg2+ and the thiol reagent, 5,5'-dithiobis-(2-nitrobenzoic acid). The inhibition of purified reductase by 5,5'-dithiobis-(2-nitrobenzoic acid), but not by Hg2+, could be reversed by dithiothreitol.

Inhibition of the enzymes of glutathione metabolism by mercuric chloride in the rat kidney: reversal by selenium.

The treatment of rats with 10 mumoles/kg (s.c.) of mercuric chloride (Hg2+) caused time-dependent decreases in the activities of the enzymes of the glutathione (GSH) metabolism pathway in the kidney. Twenty-four hours after administration of Hg2+, the activities of gamma-glutamylcysteine synthetase and glutathione disulfide (GSSG)-reductase in the kidney were decreased by 50-60%, and the activities of the GSH catabolic enzymes, gamma-glutamyl transpeptidase and GSH-peroxidase, were decreased by 25-35%. In the liver, only the activity of GSSG-reductase was decreased at this time. The observed decreases in the enzyme activities were not accompanied by a depression in the cellular protein concentration. The same pattern of enzyme response was noted when rats were given 30 mumoles/kg Hg2+; however, the decreases in the specific activity of the enzymes were accompanied by great losses in the cellular protein concentrations in both the liver and the kidney (35-40%). This dose of Hg2+ also caused significant decreases in the concentration of GSH in both organs. In vitro, Hg2+ only inhibited the activity of GSSG-reductase. When rats were given sodium selenite (Na2SeO3; 5, 10 or 20 mumoles/kg, s.c.) 30 min after Hg2+ treatment (10 mumoles/kg), the Hg2+-related depressions in the activities of the enzymes of GSH metabolism in the liver and the kidney were blocked. Also, in rats treated with 30 mumoles/kg Hg2+, the administration of 10 mumoles/kg selenium significantly decreased the magnitude of depression in the concentration of GSH in the kidney.

Enzymatic basis of metal ion alterations of cellular heme and glutathione metabolism.

It is becoming increasingly apparent that the enzymes of heme and GSH metabolism pathways are extremely sensitive to metal ions. It follows that alterations in the activities of the enzymes of heme metabolism are often reflected in the heme dependent cellular functions, particularly those which depend on cytochrome P-450. Moreover, perturbations in cellular GSH levels may alter the biological inactivation of the intermediates of cytochrome P-450 activity normally inactivated by GSH-conjugation. The effects of transition and heavy metal ions on the heme metabolism pathway are perhaps of particular significance when exerted on the two key enzymes of the pathway: the delta-aminolevulinate synthetase, the initial and the rate-limiting enzyme of the heme biosynthetic pathway, and heme oxygenase, the rate-limiting enzyme of heme degradation pathway. The activities of other enzymes of the heme metabolism pathway are also effected by metal ions; the nature of the effect is generally that of inhibition. Similarly, the inhibition by heavy metal ions of the activities of GSSG-reductase, gamma-glutamylcysteine synthetase, and gamma-glutamyl transpeptidase, which are the key enzymes of GSH metabolism, have toxicological significance. However, it is important to recognize that the presently discussed effects of metal ions do not necessarily have negative biological implications. The rather intricate and interrelated effects of metal ions on heme and GSH metabolism pathway under certain circumstances may have positive ramifications.

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase.

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.