Evidence for the catabolism of polychlorinated biphenyl-induced cytochrome P-448 by microsomal heme oxygenase, and the inhibition of delta-aminolevulinate dehydratase by polychlorinated biphenyls.

Polychlorinated biphenyls (PCB) are potent inducers of hepatic microsomal CO-binding hemoprotein P-448 (P1-450) and of delta-aminolevulinate synthetase (ALAS) activity. Inorganic cobalt was able to block PCB induction of cytochrome P-448 and to modify the PCB effect on ALAS activity in a time-dependent manner. PCB were also found to decrease the activity of delta-aminolevulinic acid dehydratase (ALAD) in liver. Pretreatment of rats with cobalt (30 min) produced the following changes in PCB actions on heme metabolism in liver: (a) augmentation of the porphyrinogenic effect of PCB, as determined by the total porphyrin content and ALAS activity; (b) augmentation of PCB inhibition of ALAD activity; and (c) blockade of induction of microsomal hemoprotein (cytochrome P-448). PCB did not interfere with cobalt induction of hepatic heme oxygenase activity. The sequence of administration of the metal and the PCB was important in relation to the changes produced in hepatic ALAS activity and microsomal hemoprotein and heme contents. When cobalt was administered 24 h after PCB treatment, the magnitude of induction of ALAS by PCB was lowered, and there was a great reduction in microsomal hemoprotein and heme contents. The renal response to PCB was different than that of the liver. In the kidney, PCB blocked the induction of heme oxygenase and depletion of cellular heme produced by cobalt. Furthermore, renal microsomal heme content was increased by PCB treatment alone or in combination with cobalt. It is concluded that (a) the heme moiety of microsomal cytochrome P-448 is metabolized by the heme oxygenase system, and it is suggested that for this catabolism to take place, the hemoprotein must be first converted to the denatured form of the hemoprotein, cytochrome P-420; (b) that the synthesis of heme in the kidney and the liver are regulated through different mechanisms; and (c) that ionic cobalt controls activity of ALAS by first inhibiting synthesis of the enzyme followed by the indirect induction of the enzyme as a result of the catabolism of heme, the physiological repressor of ALAS, by the metal-induced heme oxygenase. Thus microsomal heme oxygenase may be viewed as having an overall regulatory role in relation to mictochondrial ALAS by virtue of its ability to catabolize endogenous heme.

Study of the developmental pattern of heme catabolism in liver and the effects of cobalt on cytochrome P-450 and the rate of heme oxidation during the neonatal period.

The comparative development patterns of heme oxidation andof cytochrome P-450 dependent drug oxidation in rat liver were examined. High levels of heme oxygenase activity were present in whole embryo preparations at day 13 of gestation. At birth this enzyme activity in liver was approximately equal to that of normal adult liver. In the immediate postnatal period the rate of hepatic heme oxidation increased sharply, reaching levels 3-5 times normal during the first week postpartum. Thereafter, this enzyme activity progressively decreased and returned to normal adult levels by the 28th postpartum day. The development of microsomal heme oxidation and of P-450 dependent drug oxidation exhibited reciprocal patterns, with the latter being at low levels of activity during the immediate postnatal period and reaching adult activity only 4 or more wk after birth. Cobalt injected into pregnant animals or in to nursing mothers did not induce heme oxygenase in the fetus or suckling neonate. However, when treated directly with the metal, 4-day old neonates exhibited a small induction response of this enzyme; and the inducibility of heme oxygenase increased gradually to fully adult levels by the end of the 4th postpartum week. Cobalt at all postnatal developmental stages was capable of diminishing hepatic contents of total microsomal heme and P-450; however this effect of the metal was small in the immediate period after birth and increased progressively with maturation. These findings demonstrate that the patterns of development of hepatic capacity for carrying out the oxidation of heme and the P-450 dependent oxidation of drugs are different and thus provide further evidence that these microsomal enzyme systems are distinct from each other and under separate regulatory mechanisms. The degree of induction response for hepatic heme oxygenase evoked by the trace metal, cobalt, was also shown to have developmental determinants as did the susceptibility of hepatic cytochrome P-450 to degradation by this metal. The very high levels of hepatic heme oxygenase activity which characterize neonates during the first week of life indicate that over-production of bilirubin contributes significantly to the mechanism of neonatal jaundice.

Enzymes of heme metabolism in the kidney: regulation by trace metals which do not form heme complexes.

The in vivo regulation by metal ions of the enzymes of heme metabolism in kidney-particularly of ALAS, the rate-limiting enzyme in heine formation- was investigated. Ni(2+) and Pt(4+), metals which do not enzymatically form metalloporphyrins, were found to regulate ALAS in kidney as they do in liver. The pattern of this regulation was generally similar to that observed with heme and metal ions in liver, i.e., a late increase in enzyme activity after an early period in which ALAS activity was unaltered or inhibited. The metals did not interact with the enzyme in vitro to alter its activity. In this study no direct reciprocal relationship between ALAS activity and total cellular heine content was demonstrated. The metal ions, particularly Pt(4+), also altered the activity of other enzymes of heme biosynthesis in kidney. Pt(4+) severely inhibited the activity of ALAD and UROS. Ni(2+) and Pt(4+) were potent inducers of heme oxygenase, the initial and rate-limiting enzyme in heine degradation. It is proposed that the physiological regulation of ALAS is mediated through the action of metal ions, rather than by the cellular content of heine, and that the regulation of ALAS by heine reflects the action of the central metal ion of heme rather than that of the entire metalloporphyrin complex. In this proposed mechanism for metal ion regulation of ALAS, the tetrapyrrole moiety of heine is considered to function principally as an efficient carrier of metal to the regulatory site for ALAS production, inasmuch as the tetrapyrrole ring itself has been shown in earlier studies not to have any effect on ALAS activity. The production of heine oxygenase is believed to be similarly regulated.

Bile pigment formation by skin heme oxygenase: studies on the response of the enzyme to heme compounds and tissue injury.

Skin heme oxygenase is locally elevated by stimuli such as tissue injury and injections of whole blood, myoglobin, and hematin. The enzyme activity is also increased at the proximity of the injection site of chemicals such as cobalt and cobalt-protoporphyrin-IX (cobalt-heme). Protoporphyrin-IX, the tetrapyrrole nucleus of type-b heme compounds, was ineffective in altering the enzyme activity in vivo. The developmental pattern of heme oxygenase in skin was compared to that of the enzyme in liver. The enzyme activity in both organs was greatest during the 1st postpartum wk and declined to adult levels after 2 wk. The physiological implications of the increased activity of skin heme oxygenase are discussed, and it is concluded that the activity of the hepatic heme oxygenase system and that of the skin are regulated by the same mechanism.

Metals as regulators of heme metabolism.

Heme is essential for cell respiration, energy generation, and oxidative biotransformations. The latter function is exemplified by the oxidative metabolism of various endogenous and exogenous chemicals catalyzed by the heme protein cytochrome P-450. Recent studies have established that metal ions directly regulate cellular content of heme, and thus of heme proteins by controlling production of delta-aminolevulinate synthetase and heme oxygenase, the rate-limiting enzymes for heme synthesis and degradation, respectively. Metal ions also alter cellular content of glutathione. In excess amounts, metal ions greatly accelerate the turnover and degradation of heme and substantially impair the oxidative functions of cells--particularly those dependent on cytochrone P-450. As a result, the biological impact of chemicals which are detoxified or metabolically transformed by the P-450 system is greatly altered.

Tin: a potent inducer of heme oxygenase in kidney.

Tin greatly enhances heme breakdown in kidney, thus impairing heme-dependent cellular functions, such as cytochrome P-450 mediated drug biotransformation. This novel action of the metal results from a potent induction effect on heme oxygenase, the enzyme that catalyzes heme oxidation in microsomes. The possible toxicological implications of this tin effect in the kidney merit further investigation.

Zinc . protoporphyrin is a selective inhibitor of heme oxygenase activity in the neonatal rat.

The present study was undertaken to examine the liver, spleen and kidney heme oxygenase activity in the rat, and also to investigate the response of the enzyme to a variety of metalloporphyrin complexes. The enzyme activity in the liver and the kidney of 3--4 day-old rats was several-fold greater than the corresponding values in the adult animals; however, the splenic enzyme activity was markedly depressed in comparison to that of adult rats. During the first 2--3 weeks post-parturation period, the activity of heme oxygenase in the spleen progressively increased, and in 4 weeks approached the adult values. The treatment of the newborn animals with the metalloporphyrin complex. Zn . protoporphyrin-IX, inhibited heme oxygenase activity in the spleen, liver and the kidney. Sn . protoporphyrin treatment also inhibited the activity of the enzyme in the liver and the spleen. The mechanism of the inhibition appeared to be competitive in nature. In contrast, the treatment of the newborn animals with Co . protoporphyrin increased the activity of the enzyme in the tested organs. The treatment of newborn animals with Fe . protoporphyrin (heme) also increased heme oxygenase activity in the spleen and the kidney. In addition, Co . and Fe . protoporphyrin complexes inhibited the activity of delta-aminolevulinate synthetase in the spleen; Sn . protoporphyrin and Zn . protoporphyrin, however, did not alter the activity of this enzyme. The effects of Co . protoporphyrin and Zn. protoporphyrin on the microsomal contents of cytochromes P-450, b5, the total heme, and the microsomal drug metabolism activity in the liver were compared. Zn . protoporphyrin was ineffective in altering the indicated cellular variables. According to these findings Zn . protoporphyrin may be useful as an experimental tool for the selective suppression of heme degradation activity.

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.

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 (approximately 1.9 and approximately 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.

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.

Regulation of the activity of heme degradative enzymes in K562 erythroleukemic cells: induction by thymidine.

Heme oxygenase is rate-limiting in the heme degradative pathway, and its activity is induced by a host of chemicals. In K562 human erythroleukemic cells, heme oxygenase activity was not increased by exposure to potent inducers, such as cobalt chloride, bromobenzene, and heme. Indeed heme treatment severely suppressed the enzyme activity, and at 18 h the activity measured less than 5% of the control. Heme and cobalt chloride did not inhibit activities of NADPH-cytochrome c (P-450) reductase and biliverdin reductase to a marked degree. In contrast, treatment of cells with thymidine/hypoxanthine alone, or in combination with cobalt chloride, caused an increase in the activity of three enzymes of heme degradation. It is suggested that with thymidine, which is a committing inducer of hemoglobin synthesis, the induction of activity of the three enzymes of the heme degradation pathway is coupled with cell differentiation. On the other hand, in the case of heme, a noncommitting inducer of hemoglobin synthesis, induction of hemoglobin synthesis and increase in heme degradation activity may be independent.

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.

cis-platinum-mediated decrease in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis.

Previously we had shown that cis-platinum decreases testosterone levels in rat serum and that hCG reverses this effect. The purpose of these studies was to determine the biochemical basis of cis-platinum-mediated effects on testicular testosterone production. In the testis of rats treated with cis-platinum (7 mg/kg, iv), the mitochondrial P-450scc concentration and side-chain cleavage activity were depressed by 40%. Also, the microsomal 17 alpha-hydroxylase activity and cytochrome P-450 concentration were decreased. Testicular binding capacity (in vitro) for [125I]hCG was decreased by 75-80%. On the other hand, FSH binding to Sertoli cell membrane receptors was not appreciably changed. hCG (25 IU/100 g daily) in treated rats caused complete occupancy of the remaining 20-25% LH receptors and caused a 20- to 30-fold increase in serum and testicular testosterone, a 2-fold increase in mitochondrial P-450scc, and a 5-fold acceleration of side-chain cleavage activity. 17 alpha-Hydroxylase activity and microsomal cytochrome P-450 were not increased over the control values. In addition to testicular functions, pituitary glycoprotein hormone production was assessed. Treatment of rats with cis-platinum (7 mg/kg, iv) did not change serum LH or FSH, but caused a 50% decrease in serum and testicular testosterone levels. A GnRH challenge test (1.5 micrograms/100 g, in 30 min) of treated rats caused prompt increases of 10- to 15-fold in serum LH and resulted in increases in serum and testicular testosterone. Thus, there was little evidence for cis-platinum effects at the level of hypothalamus or pituitary that could account for the decreased testosterone production. Reversal of the cis-platinum effect on steroidogenesis by hCG or GnRH appears to be due to the induction of suprasaturating levels of LH with full occupancy of remaining Leydig cell LH receptors. This, in turn, would reverse the diminished levels of mitochondrial side-chain cleavage activity and cytochrome P-450scc. These data suggest that cis-platinum causes a depression in serum testosterone, mainly by decreasing the number of LH receptors and inhibiting side-chain cleavage activity.

Cyclosporin-mediated depression of luteinizing hormone receptors and heme biosynthesis in rat testes: a possible mechanism for decrease in serum testosterone.

The toxic side-effects of the immunosuppressive drug cyclosporin (CsA) include testicular dysfunction and a decline in circulating testosterone. However, mechanisms for the consistently observed CsA-mediated depression of serum testosterone levels are unclear because of conflicting reports concerning circulating gonadotropin levels and incomplete studies of intratesticular steroidogenesis. To elucidate these mechanisms, endocrine-regulated testicular steroidogenesis and heme metabolic parameters were studied in male rats given sc injections of either 25 or 40 mg/kg.day CsA for 6 days and then killed on the seventh day. Consistent with earlier reports, CsA treatment dramatically suppressed serum testosterone levels (less than 20% of control at both CsA doses). Additionally, the intratesticular testosterone content declined with the higher CsA dose. Serum LH and FSH levels were elevated up to 2- to 4-fold after the higher CsA treatment regimen. Measurement of decreases in testicular receptors for LH revealed for the first time that CsA treatment significantly reduced the ability of the testes to respond to normal or elevated circulating levels of LH. In animals receiving higher dose of the drug, cytochrome P-450-dependent mitochondrial cholesterol side-chain cleavage activity, which is the rate-limiting step in steroidogenesis, was markedly reduced to a mere 30% of the control value. Additionally, the activity of the microsomal cytochrome P-450-dependent 17 alpha-hydroxylase was decreased to less than half of the control value. Biotransformation of the prototype drug, benzo(a)pyrene, as well as microsomal cytochrome P450 levels declined significantly after the higher CsA dose, suggesting that CsA has an adverse affect on testicular cytochromes P-450 in general. In addition, CsA treatment altered heme metabolic parameters; significant increases in the activity of uroporphyrinogen-I synthetase and total porphyrin content were noted. Conversely, the activity of ferrochelatase, the enzyme that incorporates iron into porphyrin to form heme molecule, decreased significantly, as did the total heme levels. The latter was reduced to only 61% of control values. The findings suggest the likelihood that the observed inhibition of heme formation may contribute substantially to the reduced levels of microsomal cytochromes P-450 and steroidogenic activities that depend on them. Taken collectively, these data suggest a plausible mechanism by which CsA may induce testicular dysfunction; as the result of a combination of reduction in the number of LH receptors and a suppression of heme formation, the hemoprotein-dependent steroidogenic enzymes activities are compromised, leading to an impairment of normal testicular function.

Regulation of cytochrome P-450-dependent microsomal drug-metabolizing enzymes by nickel, cobalt, and iron.

The effects of metals as modifiers of the activity of microsomal drug-metabolizing enzymes were studied with the use of nickel, cobalt, and iron. These metals were found to impair cellular heme-dependent metabolism by affecting both the heme biosynthetic and heme degradative pathways, inhibiting the former and inducing the latter. As powerful cellular toxins, metals depress respiratory activity and indirectly reduce drug-detoxifying ability of cells. Metals also perturb cellular glutathione content and thus may alter the activity of glutathione-dependent enzymes. The toxicity of metals is cumulative depending on concentration and degree of cellular exposure to one or to several closely related metals. On the other hand, these metal effects on cellular heme metabolism could also have selective therapeutic application in circumstances in which it may be desirable to suppress heme synthesis in order to decrease drug biotransformation, i.e., when a certain drug metabolite is more toxic than the parent compound.

The structure, organization and differential expression of the gene encoding rat heme oxygenase-2.

Overlapping phage lambda clones were utilized to determine the complete nucleotide (nt) sequence of the rat gene encoding HO-2, the major heme oxygenase isozyme in the brain. This isozyme is the constitutive cognate of HSP32 (HO-1). The 12,563-bp gene consists of five exons and four introns, the first two exons are separated by a large intron of 8429 nt. The minus strand of intron 1 contains a nested sequence of 1046 nt with 87% identity to the cDNAs encoding the mouse and human non-histone chromosomal protein, HMG-17. In addition to the coding region, the similarity includes 40 bp upstream from the putative start codon and 800 bp of 3' untranslated sequence. The HO-2 gene lacks a conventional TATA box, but a TATA-like sequence (TAACTA) is found 26 nt upstream from the major transcription start point (tsp), as determined by primer extension. Upstream of the tsp, only a glucocorticoid-response element is found. The structure of the regulatory region is consistent with the previously demonstrated refractory nature of this isozyme to common inducers of gene expression and its apparent response to developmental changes in the adrenal steroid hormone profile. HO-2 is encoded by two transcripts (approx. 1.3 and approx. 1.9 kb), the larger of which is translated less efficiently than the smaller. Presently, we show that the transcripts are the products of a single gene and differ in the use of the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Developmentally regulated expression of two transcripts for heme oxygenase-2 with a first exon unique to rat testis: control by corticosterone of the oxygenase protein expression.

Heme oxygenase (HO)-2, the constitutive cognate of oxidative stress inducible HO-1 (HSP32), degrades heme to biliverdin, carbon monoxide, and iron. The highest levels of HO-2 are found in the testis. Previously we identified multiple HO-2 homologous transcripts that differ in size and use three different 5' UTRs that form the untranslated first exon of the gene (referred to as rHO-2, rHO-2-1 and rHO-2-2) and two poly(A) signals. Also, we have characterized a functional glucocorticoid response element (GRE) in the promoter region of rHO-2. In this study, we have examined the structural basis for size heterogeneity of HO-2 transcripts and whether expression of HO-2 at mRNA and protein levels is subject to regulation by corticosterone. Age and tissue-dependence of transcript expression were examined as well. Our data indicate that the remarkable increase in HO-2 mRNA in adult rat testis is due primarily to generation of two HO-2 homologous transcripts of approx. 2.1kb and approx. 1.45kb size that use rHO-2 and are unique to this tissue, and that rHO-2 is not used within other organs. These transcripts are not present in the brain, kidney, thymus, heart, spleen, liver, or in prepubertal 14day old rat testis. The testis-specific transcripts contain all of the coding region exons present in the approx. 1.3kb and approx. 1.9kb transcripts that are common to all organs, including the adult and prepubertal rat testis. Differential use of the poly(A) signals accounts for the difference in size of these two transcripts. Treatment of newborn rats with corticosterone for 5days, starting on day 2 after birth, induced HO-2 protein expression in the testis as detected by Western blotting. In adult rat testis, corticosterone treatment, however, was not an effective regulator of HO-2 transcript populations or levels. The findings suggest that HO-2 levels in the testis are controlled by glucocorticoids; and that developmental and tissue-specific factor(s) determine generation of transcripts unique to the organ. The apparent exclusive use of rHO-2 by the mature testis is consistent with the possibility that HO-2 may play a role in male reproduction.

Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs.

Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys264-Pro265 and Cys281-Pro282. We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP nor PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each HRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys264-Pro265 result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.

Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons.

Two isoforms of the enzyme heme oxygenase are expressed in distinct populations of neurons in the brain. These enzymes catalyse the oxidative cleavage of heme to the cellular antioxidant biliverdin resulting in the release of carbon monoxide in the process. Both heme and carbon monoxide may play important roles in regulating the nitric oxide-cyclic guanosine monophosphate signal transduction system. Thus we have examined the distributions of both isoforms of heme oxygenase in the rat brain, and compared their localizations with that of nitric oxide synthase determined with the NADPH-diaphorase histochemical technique. Heme oxygenase-1 is highly expressed in a few select populations of neurons including cells in the hilus of the dentate gyrus, in the hypothalamus, cerebellum and brainstem. This enzyme appears to be coexpressed with nitric oxide synthase only in a few cells in the dentate gyrus. Heme oxygenase-2 is much more widely expressed. It is present in mitral cells in the olfactory bulb, pyramidal cells in the cortex and hippocampus, granule cells in the dentate gyrus, many neurons in the thalamus, hypothalamus, cerebellum and caudal brainstem. However, only some of these labelled neurons also displayed nitric oxide synthase. Instead, many neurons expressing heme oxygenase-2 correspond to those known to express high levels of the hemoprotein soluble guanylyl cyclase. These results suggest that heme oxygenase may play a role in modulating guanylyl cyclase independent of nitric oxide synthase. This may result from regulation of intracellular heme and carbon monoxide levels by the heme oxygenase system.