Biomedical subjects

M D Maines

Publications and source records attributed to M D Maines.

At least 19 recordsLinked to original sources

PubMed1992

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.

Adrenal Glands↗

PubMed1992

Human heme oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal.

We show by Northern blot analysis that human HO-2 is encoded by two transcripts (1.3 and 1.7 kb) and is a single-copy gene as judged by Southern blot analysis. We further provide evidence based on Northern blot and sequence analysis of a cDNA representing the larger transcript that the transcripts differ in the 3' untranslated region. A 274-base-pair DNA fragment from the rat heme oxygenase-2 gene (I. Cruse and M.D. Maines, 1988, J. Biol. Chem. 263, 3348-3353) was used to isolate a human HO-2 cDNA from a fetal kidney library in lambda gt11. The clone, designated hK-1, was sequenced and the cDNA insert was determined to be 1625 base pairs in length, encoding a protein of 313 amino acids. Two consensus polyadenylation signals separated by 440 nucleotides were identified in the 3' untranslated region. The size of the cDNA insert closely approximated the larger of two mRNAs. The nucleotide sequence was 88% identical to the rat HO-2 gene within the predicted coding region and the putative translation product was also estimated to be 88% identical to the rat gene product (M. O. Rotenberg and D. Maines, 1990, J. Biol. Chem. 265, 7501). The predicted size, 36 kDa, corresponded well with HO-2 detected in human testis microsomes by Western blot analysis. Further, the fusion protein expressed in Escherichia coli displayed significant heme oxygenase activity, which was inhibited by Zn- and Sn-protoporphyrins, known inhibitors of eukaryotic heme oxygenase, but not by sulfhydryl reagents.

Amino Acid Sequence↗

PubMed1992

Expression and characterization of a cDNA for rat kidney biliverdin reductase. Evidence suggesting the liver and kidney enzymes are the same transcript product.

Biliverdin reductase is a unique dual cofactor- and pH-dependent enzyme that converts biliverdin to bilirubin and displays extensive inter-organ pI and molecular weight microheterogeneity. Presently we have explored the molecular basis for these properties. The amino acid composition and the sequences of NH2 termini plus five tryptic fragments of purified rat liver and kidney enzymes were obtained. A 62-nucleotide DNA probe was designed and in combination with antibody was used to screen a rat kidney cDNA library. A cDNA sequence of 1108 base pairs (bp) containing an 885-bp open reading frame was generated. The cloned cDNA probe detected a single mRNA of approximately 1500 bp in liver and kidney. The open reading frame encodes a 295 amino acid protein. Methionine and aspartic acid residues at positions 1 and 2 of the deduced protein are removed during processing. The deduced amino acid composition of the mature protein closely matched that of the purified rat liver and kidney enzymes. All liver peptides were found in the deduced amino acid sequence of kidney enzyme and the NH2 termini of both enzymes were identical. The expressed protein co-migrated with purified reductase and was recognized by antiserum to the enzyme. The expressed reductase displayed two distinct pH optima using a different cofactor at each pH: NADH at the lower pH 6.7-6.9 range and NADPH at pH 8.5-8.7. The findings suggest that the liver and kidney enzymes are the products of the same transcript(s) and that their microheterogeneity may reflect tissue-specific post-translational modifications.

Amino Acid Sequence↗

PubMed1992

Normal and heat-induced patterns of expression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein.

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

Animals↗

PubMed1992

Comparative effects of tin- and zinc-protoporphyrin on steroidogenesis: tin-protoporphyrin is a potent inhibitor of cytochrome P-450-dependent activities in the rat adrenals.

Synthetic metalloporphyins inhibit formation of bilirubin by the heme oxygenase system, an ability that is of considerable experimental and clinical interest for suppression of jaundice in the newborn. The present investigation compares the consequences of treatment with Sn- and Zn-protoporphyrin on hemoprotein-dependent enzymes of the rat adrenals and corticosterone production and defines Sn-protoporphyrin as a potent toxin to adrenal functions. Treatment of rats with Sn-protoporphyrin (two doses of 50 mumols/kg, in 7 d) resulted in a marked reduction of 30-40% in cytochrome P-450-dependent adrenal microsomal 21 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities. In the serum, the levels of corticosterone were reduced to about 70% of the control value. In addition, the mitochondrial cytochrome P-450SCC activity was decreased by about 50%. This decrease, however, could not be attributed to a reduced total heme level or an accelerated heme degradatory activity. Disruption by Sn-protoporphyrin of adrenal hemoprotein-dependent functions was not restricted to steroidogenic activities and encompassed drug metabolism activity of the organ; benzo(a)pyrene hydroxylase activity of both the microsomal and the mitochondrial fractions, as well as the microsomal NADPH-cytochrome P-450 reductase activity, were significantly reduced. Zn-protoporphyrin did not cause significant alterations in the above measured parameters although it too was effective in inhibiting the hepatic microsomal heme oxygenase activity. In light of the presently defined adverse effects of Sn-protoporphyrin on adrenal steroidogenesis, we suggest Zn-protoporphyrin is the agent of choice for potential use in treatment of hyperbilirubinemia in humans.

Adrenal Glands↗

PubMed1992

Tin-protoporphyrin-mediated disruption in vivo of heme oxygenase-2 protein integrity and activity in rat brain.

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

Animals↗

PubMed1992

Tin-protoporphyrin: a potent inhibitor of hemoprotein-dependent steroidogenesis in rat adrenals and testes.

The present investigation provides evidence of the ability of Sn-protoporphyrin to cause striking alterations in adrenal and testicular cytochrome P-450-dependent steroidogenesis and defines the potential of this metalloporphyrin to serve as a cellular toxin. Sn-protoporphyrin is currently used on an experimental basis for treatment of hyperbilirubinemias in humans, including newborn infants. Specifically, in the adrenals of rats treated with a moderate regimen of Sn-protoporphyrin (two doses of 50 mumol/kg, s.c.), marked decreases of 60 to 70% in the microsomal 21 alpha-hydroxylase and the mitochondrial 11 beta-hydroxylase activities were observed after 7 days. Concomitant with these decreases was a significant depression in the adrenal mitochondrial cytochrome P-450 content and a notable reduction (approximately 30%) in serum corticosterone levels. Similarly, in the testes, significant decreases in the microsomal and mitochondrial cytochrome P-450 contents and the microsomal 17 alpha-hydroxylase activity were observed. Serum testosterone level, however, was not decreased, reflecting an absence of decrease in side chain cleavage activity. Metalloporphyrin caused a striking decrease of 65 to 80% in the microsomal heme oxygenase activity in the testes and the adrenals, as well as significant reductions in NADPH-cytochrome P-450 reductase activity of the organs. The decrease in heme oxygenase activity, however, as suggested by Western immunoblotting, apparently resulted, to a large extent, from the loss of enzyme protein integrity rather than a competitive inhibition of activity. At the transcript level, Northern blot analysis using a full length rat testis cDNA probe for heme oxygenase-2 mRNA indicated that Sn-protoporphyrin treatment did not decrease the amount of message for either of the heme oxygenase-2 transcripts (1.3 and 1.9 Kb).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1991

Characterization of a cDNA-encoding rabbit brain heme oxygenase-2 and identification of a conserved domain among mammalian heme oxygenase isozymes: possible heme-binding site?

A 1.3-kb rat testis cDNA clone for heme oxygenase-2 (HO-2) was used as a Northern blot hybridization probe, and a single homologous mRNA species, of approximately 1.3 kb in rabbit brain and testis was detected. This contrasted with the observation made with rat brain in which two HO-2 transcripts of approximately 1.3 and 1.9 kb were detected. Use of the same rat HO-2 probe to screen a rabbit brain cDNA library in lambda gt11 resulted in the recovery of a single 1.2-kb cDNA clone. This cDNA exhibits 84% overall nucleotide sequence homology with rat HO-2 and encodes a protein of 35,352 Da, displaying 88% amino acid sequence homology with rat testis HO-2. Furthermore, when expressed in Escherichia coli, the rabbit cDNA-encoded protein displays heme oxygenase activity and cross-reactivity with antibody to rat HO-2. Based on findings obtained through Western immunoblot analysis of partially purified HO-2 protein prepared from rabbit testis and brain, the 35- to 36-kDa molecular form appears to be the major HO-2 form detected in the brain, whereas a 42-kDa species is the predominant form observed in rabbit testis. Having deduced the amino acid sequence of rabbit brain HO-2, we provide a comparison of this sequence with those of rat, mouse, and human HO-1 and rat HO-2, and thereby identify a 24-amino-acid-long peptide region which, except for one residue, is identical in all five species of HO-1 and HO-2 compared (96% similarity), and exhibits 100% similarity in predicted secondary structure (for this region) in all five proteins. We propose that this peptide may be important to the heme binding and isomer-specific tetrapyrrole cleavage activities of the heme oxygenase isozymes.

Amino Acid Sequence↗

PubMed1991

Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells.

Chinese hamster ovary cells cultured in vitro were used to assess the role of glutathione metabolism in the induction of the 32-kDa stress protein. Enhanced synthesis of the 32-kDa protein was observed after cells were incubated with CdCl2 or diethylmaleate and protein was subjected to SDS-PAGE followed by fluorography. Concomitantly, in both cell preparations an increase in heme oxygenase activity was observed. Proteins from CdCl2- and diethylmaleate-treated cells were subjected to Western blotting and protein crossreacting with either rabbit antibody to rat liver heme oxygenase-1 (32,000 Mr) or rat testis heme oxygenase-2 (36,000 Mr) quantitated. The analysis indicated that the CdCl2 treatment increased the intensity of the HO-1 band 5.5-fold while the diethylmaleate treatment increased it three-fold relative to control. Neither treatment affected the intensity of HO-2 antibody binding. Incubation of cells with buthionine sulfoximine, under conditions which resulted in greater than or equal to 90% of the intracellular glutathione being depleted, enhanced synthesis of a 32-kDa protein when assayed by SDS-PAGE. This protein exhibited a Mr similar to the 32-kDa protein induced by either CdCl2 or diethylmaleate treatment. Proteins from buthionine sulfoximine and diethylmaleate-treated cells were mixed together and subjected to 2D PAGE. The resulting fluorograph demonstrated that both treatments produced identical patterns. In contrast, incubation of cells in diamide, a thiol oxidizing compound, resulted in enhanced synthesis of the 110-, 90-, and 73-kDa heat shock proteins but not the 32-kDa protein. The data presented have shown that depletion of glutathione by two independent methods, conjugation and inhibition of synthesis, enhances the synthesis of a 32-kDa protein identified as heme oxygenase-1; oxidation of glutathione, on the other hand did not. We interpret this to indicate that glutathione depletion rather than conjugation or oxidation represents one pathway for induction of heme oxygenase-1.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

Heterogeneity of haem oxygenase 1 and 2 isoenzymes. Rat and primate transcripts for isoenzyme 2 differ in number and size.

In Cebus apella monkey, as with other mammalian species tested to date, two different forms of haem oxygenase, HO-1 and HO-2, are detected. With the use of cDNA fragment corresponding to HO-1 nucleotides +71 to +833, blot hybridization of RNA revealed the presence of only one HO-1 mRNA of approx. 1.8 kb in both rat and monkey liver, kidney and brain. With the use of a full-length HO-2 DNA probe, blot hybridization of RNA isolated from the same rat organs revealed the presence of two HO-2 homologous transcripts of approx. 1.3 kb and approx. 1.9 kb. The same probe detected only one message of approx. 1.7 kb in monkey organs. The rat 1.3 kb mRNA has been previously shown [Rotenberg & Maines (1990) J. Biol. Chem. 265, 7501-7506] to encode HO-2 (36 kDa). The monkey 1.7 kb mRNA and the rat 1.3 kb mRNA encode proteins with similar molecular masses and immunochemical properties as indicated by Western-immunoblotting analysis. In rat organs the relative abundance of the two mRNAs differed as follows: in the liver the 1.3 kb mRNA was by far the most abundant form; in the brain equal amounts of the two mRNAs were detected, whereas in the kidney the 1.3 kb mRNA was somewhat more abundant. The protein encoded by the 1.8 kb HO-1 mRNA in the monkey did not exhibit immunochemical reactivity with antibody to rat HO-1 in Western blotting and direct e.l.i.s.a. analysis. The data suggest that, at the primary structural level, both HO-1 and HO-2 share extensive base sequence similarity in the rat and the Cebus apella monkey. The HO-1 protein, however, appears to undergo differential post-translational and/or conformational modifications in the two species, whereas the secondary structure of HO-2 protein and antigenic epitopes are conserved among the two mammalian species.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

Heme oxygenase-2 mRNA: developmental expression in the rat liver and response to cobalt chloride.

We have previously identified two isozymes of heme oxygenase, HO-1 and HO-2, in the rat liver exhibiting vastly different molecular biochemical properties, including their responses to various chemical inducers. In the present study of the livers of fetal and newborn rats (-1 day to 21 days) we observed marked differences in the developmental pattern of expression of the transcripts for the two forms of heme oxygenase. In addition, the transcripts for HO-1 and HO-2 vary in their number and response to treatment with cobalt chloride. Specifically, using a full length cDNA probe for HO-2, we observed the 1.3-kb mRNA previously shown to encode HO-2 in the testis, as well as a second less abundant transcript of 1.9 kb. An HO-1 cDNA probe detected only the anticipated single transcript of 1.8 kb. The abundance of each HO-2 homologous transcript was unaffected by cobalt chloride treatment (4 or 24 h) in newborn (7 days old) and adult rats. The 1.8-kb HO-1 mRNA, however, was markedly increased in abundance in both age groups by this treatment. The 1.3-kb HO-2 mRNA level nearly doubled after parturition, but remained essentially unchanged during the first 2 weeks of life, and only modestly increased by age 21 days. At all stages of development, the relative abundance of the 1.3- and 1.9-kb transcripts remained unchanged. In the case of 1.8-kb HO-1 mRNA, the level of mRNA was relatively constant during the first week of life, but was substantially reduced by age 14 days, and was further decreased by age 21 days and in the adult animals. In adult rats, the abundance of the 1.3-kb HO-2 mRNA did not markedly increase over that detected in the course of the early postparturition developmental period. The data suggest that the abundance of the two HO-2 homologous transcripts is not readily subject to regulation by chemicals at any stage of development. The expression of HO-1, on the other hand, is subject to such regulation throughout life, and its high transcript abundance in newborn rats may well reflect availability of an endogenous inducer.

Animals↗

PubMed1990

Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: the olfactory bulb and the mitochondrial fraction have high levels of activity.

In brain of female monkey (M. fascicularis) the content of cytochrome P-450 and benzo(a)pyrene hydroxylase activity in the mitochondrial fraction exceeded that of the microsomes by more than 4-fold. The mitochondrial drug metabolism activity exhibited substrate specificity and, unlike the microsomes, did not catalyze 7-ethoxyresorufin O-deethylase reaction. Moreover, the rate of benzo(a)pyrene hydroxylase activity by both the mitochondrial and the microsomal fractions displayed regional variation with the olfactory bulb displaying the highest hydroxylase activity. In contrast, the microsomal 7-ethoxyresorufin O-deethylase activity was uniformally distributed in all brain regions.

Animals↗

PubMed1990

Promotion of trans-platinum in vivo effects on renal heme and hemoprotein metabolism by D,L-buthionine-S,R-sulfoximine. Possible role of glutathione.

We have examined the toxicity of trans-platinum (trans-diamminedichloroplatinum II) to heme and hemoprotein metabolism in the kidney of glutathione (GSH)-depleted rats and compared it with that produced by cis-platinum. Unlike cis-platinum treatment (7.0 mg/kg, i.v.) which caused after 7 days significant increases in cytochromes P450 and b5, and a marked decrease in porphyrin content of the kidney, trans-platinum alone (7 mg/kg, i.v.) did not elicit notable changes in these variables when measured 1 or 7 days after treatment. Also, cis-platinum treatment significantly altered the heme degradation pathway by increasing the activity of heme oxygenase and decreasing that of biliverdin reductase; trans-platinum treatment did not elicit a response in these activities. However, when rats were given the inhibitor of GSH synthesis, D,L-buthionine-S,R-sulfoximine (BSO), the subsequent administration (2 hr later) of trans-platinum produced, in 1 day, the spectrum of responses that were mediated by cis-platinum after 7 days. In the kidneys of rats treated with BSO plus trans-platinum the concentration of platinum measured only about 50% of that detected in the kidneys of rats treated with trans-platinum alone. In the liver, trans-platinum by itself or in combination with BSO was ineffective in altering the measured variables of heme metabolism. The possibility that similarity between cis-platinum and trans-platinum plus BSO may extend to systems other than heme metabolism, e.g. GSH synthesis and degradation, was examined. cis-Platinum caused significant inhibition of both renal gamma-glutamyl synthetase and gamma-glutamyl transpeptidase after 7 days, but not after 1 day. Twenty-four hours after treatment, BSO + trans-platinum caused inhibition of gamma-glutamylcysteine synthetase activity, whereas this activity in animals treated with BSO alone had returned to control values. At this time point, neither oxidized glutathione (GSSG)-reductase nor gamma-glutamyl transpeptidase activity was affected by trans-platinum + BSO treatment. The findings suggest that GSH constitutes an important defense mechanism against trans-platinum alteration of heme metabolism and may play a role in cellular accumulation of the drug in an inactive complex. It is proposed that BSO treatment, despite resulting in a diminished intracellular concentration of trans-platinum, allows reaction of the metal complex with target molecules by virtue of its ability to deplete GSH.

Animals↗

PubMed1990

Developmental expression of heme oxygenase isozymes in rat brain. Two HO-2 mRNAs are detected.

Blot hybridization of RNA isolated from rat brain revealed the presence of two HO-2 homologous transcripts (1.3 and 1.9 kilobases (kb] at all stages of development ranging from 1 day before birth to adulthood. The level of both HO-2 messages appeared to be developmentally regulated and a gradual increase was observed from prenatal day 1 to adulthood. The two transcripts were highly homologous as assayed through hybridization studies using probes derived from the 5' end, middle, and 3' end of a cloned rat testis HO-2 cDNA. The 1.3-kb mRNA was essentially identical in size to the testis HO-2 cDNA. The message was efficiently translated in the brain, and is believed to encode the HO-2 protein. It seems unlikely that the 1.9-kb species represents a precursor of the 1.3-kb mRNA, as it was also translated in vivo, although less efficiently than the smaller mRNA species. Neither of the two HO-2 mRNA species were induced by bacterial endotoxin. Unlike HO-2, only one HO-1 transcript of approximately 1.8 kb could be detected. This transcript was of very low abundance and was not developmentally regulated, but could be increased by bacterial endotoxin. The product of this induced message, however, was not detectable by Western immunoblot analysis using antibody raised against liver HO-1. An immunoprecipitate could be detected in brain microsomes by radioimmunoassay using the same antibody. This protein, however, exhibited antigenic properties different from that of the purified liver HO-1 or that of spleen microsomal HO-1. Brain heme oxygenase activity correlated well with the amount of immunoreactive HO-2 protein and both reflect the abundance of the 1.3-kb mRNA message over the course of development.

Animals↗

PubMed1990

Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2.

In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.

Amino Acid Sequence↗

PubMed1990

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.

Animals↗