Abstract
In the neonatal lung, hyperoxic exposure is associated with induction of various genes and critical antioxidants. Heme oxygenase, specifically the HO-1 isoenzyme, is regulated in oxidant stress and may also serve to limit oxidative damage. However, it is not known whether neonatal lung HO-1 is regulated in hyperoxia specifically and, if so, what type of regulation occurs. Therefore, we attempted to answer these questions using newly born(< 12 h) Wistar rats exposed to hyperoxia for 3 d. Neonatal rat lungs were evaluated daily for total HO activity, immunore-active HO-1 protein, and steady state levels of HO-1 mRNA and compared with air-exposed controls. In neonatal rats, we noted an increased lung HO activity after 3 d of hyperoxic exposure. Additionally, evaluation of HO activity after immunoprecipitation of HO-1 protein suggested that HO-1 contributed most of the increase in lung total HO activity observed in hyperoxia. Nonetheless, we did not see a significant difference in immunoreactive HO-1 protein in neonatal lungs after 3 d of hyperoxic exposure, although we did so on d 2. Also, in contrast with previous reports, we did not detect any significant differences in steady state levels of HO-1 mRNA on any day of hyperoxic exposure compared with air. We therefore conclude that neonatal rat lung HO-1 is regulated in hyperoxia and speculate that the regulation of neonatal lung HO-1 occurs by posttranscriptional mechanisms, at least within the first days of hyperoxic exposure.
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Abbreviations
- HO:
-
heme oxygenase
- TBS:
-
Tris-buffered saline
- GAPD:
-
glyceraldehyde-3-phosphate dehydrogenase
- Do:
-
preexposure control
- CCR:
-
NADPH-cytochrome c2 P450 reductase
- PCR:
-
polymerase chain reaction
- UVA:
-
UV long wavelength
References
Frank L 1991 Developmental aspects of experimental pulmonary oxygen toxicity. Free Radic Biol Med 11: 463–494
Jornot L, Junod AF 1993 Variable glutathione levels and expression of antioxidant enzymes in human endothelial cells. Am J Physiol 264:L482–L489
Keyse SM, Tyrrell RM 1989 Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 86: 99–103
Balla J, Jacob HS, Balla G, Nath K, Eaton J.W, Vercellotti GM 1993 Endothelial-cell heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage. Proc Natl Acad Sci USA 90: 9285–9289
Stocker R, Yamamoto Y, McDonagh AF, Glazzer AN, Ames BN 1987 Bilirubin is an antioxidant of possible physiologic importance. Science 235: 1043–1046
Dennery PA, McDonagh, AF, Spitz DR, Rodgers PA, Stevenson, DK 1995 Hyper-bilirubinemia results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia. Free Radic Biol Med 19: 395–404
Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, and Vercellotti GM 1992 Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 267: 18148–18153
Ewing J.F, Maines MD 1993 Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain. J Neurochem 60: 1512–1519
Maines MD 1988 Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2: 2557–2568
Tyrrell RM, Applegate LA, Tromvoukis Y 1993 The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants. Carcinogenesis 14: 761–765
Keyse SM, Applegate LA, Tromvoukis Y, Tyrrell RM 1990 Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts. Mol Cell Biol 10: 4967–4969
Srivastava KK., Cable EE., Donohue SE, Bonkovsky HL 1993 Molecular basis for heme-dependent induction of heme oxygenase in primary cultures of chick embryo hepatocytes. Demonstration of acquired refractoriness to heme. Eur J Biochem 213: 909–917
Clerch, LB, Massaro D 1992 Rat lung antioxidant enzymes: differences in perinatal gene expression and regulation. Am J Physiol 263: L466–L470
Clerch LB, Massaro D 1992 Oxidation-reduction-sensitive binding of lung protein to rat catalase mRNA. J Biol Chem 267: 2853–2855
Clerch LB, Iqbal J, Massaro D 1991 Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia. Am J Physiol 260:L428–L433
Cresteil T, Flinois JP, Pfister A, Leroux JP 1979 Effects of microsomal preparations and induction on cytochrome P-450 dependent monooxygenases in fetal and neonatal rat liver. Biochem Pharmacol 28: 2057–2063
Vreman HJ, Stevenson DK 1988 Heme oxygenase activity as measured by carbon monoxide production. Anal Biochem 168: 31–38
Asakawa T, Matsushita S 1980 Thiobarbituric acid test for detecting lipid peroxides. Lipids 14: 401–406
Tateishi T, Yoshimine N, Kuzuya F 1987 Serum lipid peroxide assayed by a new colorimetric method. Exp Gerontol 22: 103–111
Lowry O, Rosebrough H, Farr A, Randall R 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–272
Wilks A, Ortiz de Montellano PR 1993 Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species. J Biol Chem 268: 22357–22362
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellular sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354
Shibahara S, Muller R, Taguchi H, Yoshida T 1985 Cloning and expression of cDNA for rat heme oxygenase. Proc Natl Acad Sci USA 82: 7865–7869
Adams MD, Dubnick M, Kerlavage AR, Moreno R, Kelley JM, Utterback TR, Nagle JW, Fields C, Venter JC 1992 Sequence identification of 2,375 human brain genes. Nature 355: 632–634
Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde BR, Moreno F, Venter JC 1991 Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252: 1651–1656
Sambrook J, Fritsch E, Maniatis T. 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 1.21–1.105
Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13
Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159
Harlow E, Lane D 1988 Immunoprecipitation. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 462–463
Shibahara S, Yoshizawa M, Suzuki H, Takeda K, Meguro K, Endo K 1993 Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J Biochem (Tokyo) 113: 214–218
Kikkawa Y, Yano S, Skoza L 1984 Protective effect of interferon inducers against hyperoxic pulmonary damage. Lab Invest 50: 62–71
Coutant-Perrone VH, Mansour C, Marquetty B, Rouveix B, Pocidalo JJ 1991 Activities of lung NADPH-cytochrome C-reductase and of cytochrome P-450 peroxidase during the protection of rat from hyperoxic injury in polyriboinosinic acid-polyribocytidilic acid. J Pharmacol Exp Ther 259: 1059–1063
Blanco P, Machado A, Satrustegui J 1987 Variations due to hyperoxia and ageing in the activities of glutathioneS- transferase and NADPH-cytochrome c reductase. Mech Ageing Dev 39: 11–19
Choi, AM, Sylvester S, Otterbein L, Holbrook NJ 1995 Molecular responses to hyperoxia in vivo: relationship to increased tolerance in aged rats. Am J Respir Cell Mol Biol 13: 74–82
Tom DJ, Rodgers PA, Shokooki V, Stevenson DK, Dennery PA 1996 Heme oxygenase is inducible in neonatal rats during the early postnatal period. Pediatr Res 40: 288–293
Applegate, LA., Luscher P, Tyrrell RM 1991 Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 51: 974–978
Lutton JD, da Silva J, Moqattash S, Brown AC, Levere RD, Abraham NG 1992 Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3B) by environmental agents. J Cell Biochem 49: 259–265
Smith A, Alam J, Escriba PV, Morgan WT 1993 Regulation of heme oxygenase and metallothionein gene expression by the heme analogs, cobalt-, and tin-protoporphyrin. J Biol Chem 268: 7365–7371
Drummond GS, Kappas A 1986 sn-Protoporphyrin inhibition of fetal and neonatal brain heme oxygenase. Transplacental passage of the metalloporphyrin and prenatal suppression of hyperbilirubinemia in the newborn animal. J Clin Invest 77: 971–977
Rodgers PA, Lee CS, Stevenson DK, Dennery PA 1995 Ontogeny of lung heme oxygenase in the neonatal Wistar rat. Pediatr Res 37: 234A
Dennery PA, Wong HE, Sridhar KJ, Rodgers PA, Sim JE, Spitz DR 1996 Differences in basal and hyperoxia associated heme oxygenase expression in oxidant resistant fibroblasts. Am J Physiol 271:L672–L679
Applegate LA, Noel A, Vile G, Frenk E, Tyrrell RM 1995 Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress. Photochem Photobiol 61: 285–291
Basu-Modak S, Luscher P, Tyrrell RM 1996 Lipid metabolite involvement in the activation of the human heme oxygenase-1 gene. Free Radic Biol Med 20: 887–897
Acknowledgements
The authors thank Harvey Cohen, M.D., for his thoughtful comments and Tonya Gonzales for her excellent secretarial assistance.
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Supported in part by a grant from the American Lung Association (176A857), Grant HD 14426-13SI from the National Institute of Child Health and Human Development, and Grant HL-52701 from the National Heart, Lung, and Blood Institute.
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Dennery, P., Rodgers, P., Lum, M. et al. Hyperoxic Regulation of Lung Heme Oxygenase in Neonatal Rats. Pediatr Res 40, 815–821 (1996). https://doi.org/10.1203/00006450-199612000-00007
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DOI: https://doi.org/10.1203/00006450-199612000-00007
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