Regulation of Heme Oxygenase Expression by Cyclopentenone Prostaglandins

Abstract

Prostaglandins (PGs) originate from the degradation of membranar arachidonic acid by cyclooxygenases (COX-1 and COX-2). The prostaglandin actions in the nervous system are multiple and have been suggested to play a significant role in neurodegenerative disorders. Some PGs have been reported to be toxic and, interestingly, the cyclopentenone PGs have been reported to be cytoprotective at low concentration and could play a significant role in neuronal plasticity. They have been shown to be protective against oxidative stress injury; however, the cellular mechanisms of protection afforded by these PGs are still unclear. It is postulated that the cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer’s disease, would be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of cyclopentanone could be caused partially by an induction of heme oxygenase 1 (HO-1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer’s disease. HO acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin that is rapidly converted into bilirubin. Using mouse primary neuronal cultures, we demonstrated that PGs of the J series induce HO-1 in a dose-dependent manner (0, 0.5, 5, 10, 20, and 50 μg/ml) and that PGJ₂ and dPGJ₂ were more potent than PGA₂, dPGA₂, PGD₂, and PGE₂. No significant effects were observed for HO-2 and actin expression. In regard to HO-3 expression found in rat, with its protein deducted sequence highly homologous to HO-2, no detection was observed in HO-2^−/− mice, suggesting that HO-3 protein would not be present in mouse brain. We are proposing that several of the protective effects of PGJ₂ could be mediated through beneficial actions of heme degradation and its metabolites. The design of new mimetics based on the cyclopentenone structure could be very useful as neuroprotective agents and be tested in animal models of stroke and Alzheimer’s disease.

Prostaglandins (PGs) were first discovered in the 1930s and it is now known that they have various intrinsic biological actions. PGs are generated from the enzymatic degradation of 20-carbon arachidonic acid by cyclooxygenases. There are two isoforms of cyclooxygenase: COX-1 is expressed constitutively in most tissues and is present under normal conditions at very low levels in the brain. COX-2, the inducible isoform, is found acutely expressed in several cell types after head injury, in cerebral ischemia, and in Alzheimer’s disease. Ischemic infarct, especially the reperfusion phase, is associated with significant formation and release of arachidonic acid and its metabolites. Within the PG family, the PGs of A and J series contain a cyclopentenone ring structure that is characterized by α,β-unsaturated carbonyl group (1). Evidence indicates that this reactive unsaturated group is required for many of the biological actions (1, 2). The cyclopentenone PGs PGA₂ and PGJ₂ are made from dehydration within the cyclopentane ring of PGE₂ and PGD₂, respectively. Note that PGD₂ is the major prostanoid in the murine central nervous system (3, 4). Some of the effects of these eicosanoids have been described as an increase in blood flow, inhibition of platelet function, and inhibition of the activation/extravasation of granulocytes (5). Many of these effects are receptor-mediated through either stimulation of phospholipase C to produce inositol 1, 4, 5-trisphosphate (IP₃) and diacylglycerol or through modulation of adenylyl cyclase via the guanine nucleotide-binding regulator proteins (G-proteins) (6–14). In contrast with other PGs, most of the cyclopentenones interact with other specific cellular targets. These cyclopentenone PG members have been shown to be “cytoprotective” and have antineoplastic, anti-inflammatory and antiviral properties (see review in ref. 1), although the mechanisms of action are still unclear.

In this study we investigated whether some of the biological actions of these cyclopentenone PGs could be partially mediated through modulation of HO. HO catalyzes the cleavage of the heme to form iron, carbon monoxide, and biliverdin/bilirubin. We and others have previously shown that modulation of HO activity could afford neuroprotection (see Refs. 15–18 for reviews). There are abundant heme-containing enzymes in mitochondria and endoplasmic reticulum, which presumably undergo turnover during oxidative stress; as such, HO would play an important role in assuring that pro-oxidant heme does not increase to toxic levels. Furthermore, in cultured neurons, HO could make biliverdin/bilirubin at sufficient concentrations to act as antioxidants (19). A specific significant increase of HO levels is observed after treatment of primary neuronal cultures with PGA₂ and PGJ₂, or their derivatives, while no induction is detectable with PGE₂ and PGD₂.

This family of compounds offers unique mechanisms of action and their roles and potential therapeutic applications in acute and/or chronic neurological conditions are still under investigation. We are proposing that several of the protective effects of PGA₂ and PGJ₂ could be mediated through beneficial actions of heme degradation and its metabolites.

Materials and Methods

Primary Cultures of Neuronal Cells.

Cultures of cortical neuronal cells were obtained from 17-day-old embryos of timed pregnant mice. Animal care was administered according to protocols and guidelines of the Johns Hopkins University Animal Care Committee. Cultures were prepared in serum-free conditions. Neurons were plated onto poly-D-lysine coated 24-well dishes at a density of 1 × 10⁶ cells/well in the B27 supplement HEPES-buffered high glucose Neurobasal medium, as previously described (19). Half of the initial medium was removed at day 4 and replaced with the prewarmed medium. Cells were maintained in growth medium at 37°C in a 95% air/5% CO₂ humidified atmosphere until the day of experiment. Materials used for cell cultures were obtained from InvitroGen (Carlsbad, CA). Unless stated otherwise, all other chemicals were purchased from Sigma Co. (St. Louis, MO).

Experimental Treatments.

After 8 days in culture, cells were incubated in fresh medium containing PGs obtained from Cayman Chemical (Ann Arbor, MI). Experimental treatments with PGs, or with vehicle-control, were conducted in the B27 minus antioxidant supplement (InvitroGen) HEPES-buffered high-glucose Neurobasal medium. All experiments were conducted under a dim light to avoid heme pigment photodegradation.

Assessment of Cell Survival.

After treatments, neurons were maintained for an additional period of 23 hr and their survival was assessed by phase-contrast microscopy with Trypan Blue exclusion assay and quantified using MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] (Sigma) colorimetric assay. MTT is an indicator of the mitochondrial activity of living cells and is widely used as an index of cell survival (19). After a 2-hr incubation at 37°C, living cells containing MTT formazan crystals were solubilized in a solution of anhydrous isopropanol, HCl 0.1 N, and 0.1% Triton X100. The optical density was determined at 570 nm. Experiments were performed in triplicates and repeated with at least three separate batches of cultures.

Preparation of Mouse Brain Homogenates.

Cortical brain region from mouse brains was dissected on ice, rinsed with cold PBS (phosphate buffered saline), add 600 μl of lysis buffer [phosphate-buffered saline, containing protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma)]. The mixtures were sonicated for 7 sec twice, and then centrifuged at 7000g at 4°C for 30 min. The supernatants collected and centrifuged at 100,000g at 4°C for 40 min. The supernatant (cytosol-enriched fraction) and pellet (microsome-enriched fraction) were collected. Microsomal fractions were resuspended into 600 μl of lysis buffer, and protein concentration was determined and 25 μg was loaded onto the gel.

Western Blot Analysis.

Neuronal cultures were solubilized with 250 μl of lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl, 0.5% Triton X-100) including protease inhibitor cocktail (Roche) on ice for 30 min and then centrifuged for 10 min at 12,000g then collected the supernatant. Protein quantification was accomplished using BCA assay (Pierce, Rockford, IL). Western blots (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were performed using 12% gels (Novex, San Diego, CA) and proteins were transferred to nitrocellulose membranes (Novex) (19). Membranes were blocked for 1h at RT with 5% milk in PBS with 0.1% Tween 20 before incubation at 4°C overnight with primary antibodies. Blots were washed and incubated with second antibodies for 1 hr at room temperature and then developed by ECL (Amersham Biosciences, Piscataway, NJ). Gels were stained with Ponceau S Solution to verify that equal amounts of proteins were loaded in each lane. Primary polyclonal antibodies to HO-1 and HO-2 were obtained from StressGen Inc. (Victoria, BC) and anti-actin was obtained from Sigma, and used at a dilution of 1:3,500, 1:2,500, and 1:5,000 respectively.

Results and Discussion

Our data demonstrate that the cyclopentenones (Fig. 1), and especially the ones of the J series can induce HO-1 protein expression in mouse primary cortical cells (Figs. 2 and 3 ). Our results indicate that the HO-1 induction effect of dPGJ₂ was dose dependent. It was observed as low as 0.5 μM with a maximal effect at 20 μM after a 6-hr incubation (Fig. 2 ). Previous studies have shown that induction of HO-1 could act as an antioxidant system. Moreover, no significant effect was observed on the HO-2 expression levels, which was also confirmed by similar expression levels of actin (Fig. 1 ). We also observed that at these concentrations no toxicity effect would be measured, and this up to 24 hr of treatment (data not shown). It has been previously reported that a cDNA coding for a transcript, with a predicted amino acid sequence close to 90% similar to HO-2 and a similar pattern of distribution, could be present in rat tissues (20). This transcript was referred as HO-3, although its mRNA expression is present in several tissues, its protein expression has not been detected. Using different polyclonal antibodies against HO-2 (raised against peptides which shares approximately 75% homology and against a rat purified native HO-2), we could not detect the presence of other cross-reacting proteins in mouse brain tissues obtained from HO-2^−/− mice (Fig. 4). This would suggest that, at least in mouse, HO3 protein would not be detectable.

PGJ₂ have been shown to have intrinsic cytoprotective properties in addition to anti-inflammatory indications. It has been suggested that they could also reduce ischemic brain damage (2). It is possible that these protective effects could be extended to the treatment of impairments and symptoms in traumatic brain injury, stroke, multi-infarct dementia, cerebral atherosclerosis, cerebral insufficiency, cerebral edema, inflammation, as well as Alzheimer’s and age-associated dementia. It is postulated that the determinants of neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer’s disease, may be mediated by free radical damage. PGJ₂ does not appear to have significant direct antioxidant properties, but its protective cellular mechanism is still unclear. HO cleaves the heme ring to form biliverdin, which is rapidly reduced to bilirubin, carbon monoxide, and iron (Fig. 5). We questioned whether HO activity could play a role in dPGJ₂’s neuroprotective function.

As represented in Figure 5 , HO degrades the pro-oxidant heme. Free heme in the cell can be rapidly generated from heme-containing proteins/enzymes (such as myoglobin, catalase, glutathione peroxidase, cytochrome, soluble guanylate cyclase, superoxide dismutase, nitric oxide synthase, etc.). For example, hypoxia during ischemic injury may trigger micromolar concentrations of heme to be released in the intracellular pool. HO, through rapid degradation of pro-oxidant heme, can limit its capacity to enter into a generation of free radical cycle. HO, by directly metabolizing heme, acts by itself as a potent antioxidant enzyme. The observation reported here that PGJ₂ can increase HO-1 levels in neuronal cultures is likely to be an important factor in degradation of the pro-oxidant heme. In addition, this HO enzymatic reaction generates several catalytic metabolites with intrinsic actions (i.e., iron, carbon monoxide and biliverdin).

The degradation of hemin/heme by HO generates iron. Then, modulation of HO activity could be a limiting factor in controlling the rate by which iron is being eliminated from the cells (21). Controlling the iron homeostasis within the cell is essential. For example, free iron is a key element in the traditional Fenton reaction, which, by reaction with H₂O₂, generates free radicals. The iron cellular homeostasis is a complex system and is regulated by abundant proteins, some of which are being identified and characterized. The strict control of the iron level within a cell is critical, and we believe that our previous work suggests that HO is an important enzyme in this regard. The direct increase of HO-1 by dPGJ₂ is likely to directly affect the intracellular iron levels. In the opposite scenario, one would expect that a decrease in HO activity would be sufficient to change the iron levels within cells. In fact, this is what was revealed by the use of the HO-1 knockout mice generated by Poss and Tonegawa, in which they observed accumulation of iron in several organs (22). PGJ₂, and its analogues, could then be protective by acting indirectly through the modulation of iron free levels via other iron binding proteins. Numerous iron-binding proteins have been found and their expression can modulate the iron intracellular free pool (23). To cite only one example, ferritin in the cell can sequester free iron, and its intracellular levels can be very rapidly induced by free iron (24), which could represent by itself another pathway by which dPGJ₂ can be protective. The therapeutic implications of controlling iron levels are numerous. For instance, one study revealed that the administration of desferroxiamine, a trivalent ion chelator, over a two-year period slowed the clinical progression of symptoms in AD (25). By demonstrating here that dPGJ₂ significantly alters HO-1 levels, it could subsequently protect several cells and organs against iron-mediated toxicity.

The opening of the heme porphyrin ring by HO generates biliverdin. We and others have demonstrated that biliverdin by itself could be a potent antioxidant (26–28). It is generally believed that biliverdin is almost immediately converted into bilirubin by biliverdin reductase (29). Moreover, it has been recently proposed that the production of bilirubin (BR) is dependent on the autophosphorylation of biliverdin reductase (30). BR is well known as a toxic agent in infants because an accumulation of micromolar concentrations in the brain tends to aggregate. Yellowing of several brain regions is a landmark of kinecterus. Similarly, we have observed in neuronal cultures at high micromolar concentrations that BR can aggregate and stick to cellular membranes (28), lending a yellow appearance to the cells. Aggregates of BR incorporated to the cell membrane are likely to affect the normal cellular functions. However, when BR is used at physiological levels, it is protective against oxidative stress injury (19, 31–33). Interestingly, BR, by reducing free radicals, would partially be converted into biliverdin (Fig. 5). A reducing loop can then be an effective way to act as a radical scavenger; in addition, its has been suggested that BR metabolites can have intrinsic vasodilating properties (34).

Carbon monoxide (CO) is a gas that in cells is mostly generated by the degradation of heme from HO (35, 36). CO can travel freely throughout intracellular and extracellular compartments. The literature regarding the role of carbon monoxide is complex, with many controversies that have yet to be resolved (18, 37–41). CO is known to be toxic and the inhalation of high concentrations can cause death. Some of this toxicity has been attributed to the fact that it saturates hemoglobin and decreases the ability to transport oxygen. The affinity of CO to several proteins is generally lower than nitric oxide, though its longer half-life could be a key factor in modifying several key enzymes containing a heme moiety (42). At a cellular level, physiological levels of CO generated from degradation of intracellular heme are then likely to have biological actions on several heme-containing proteins. For example, CO may act as a vasodilator by binding to soluble guanylate cyclase and modulate its activity (18, 38). CO can also act by opening calcium-activated potassium channels (K_Ca channels; ref. 43). CO has also been reported to have specific anti-inflammatory and antiapoptotic effects (44, 45). Further investigations are necessary to clarify the cellular effect of physiologic concentrations of CO. dPGJ₂, by increasing HO activity, would generate more CO, within physiological levels, allowing cells and tissues to benefit from many of its biological actions especially in a scenario where blood flow is reduced and cell survival is triggered.

Puzzling effects of the PGs have been reported (46), sometimes good and sometimes bad. We are proposing here that significant induction of HO-1 in neuronal cultures would participate in the protective actions of dPGJ₂. At this point, we have not yet identified the specific cascade that triggers HO-1 upregulation: influence of HO-1 mRNA levels, longer mRNA half-life or longer HO-1 half-life (47). An increase of HO activity in cells provides a way to degrade free heme generated from heme-containing proteins, and generation of the different metabolites (i.e., iron, biliverdin/bilirubin, carbon monoxide). These are likely to effect cell survival and may contribute to the beneficial effects of dPGJ₂ in therapeutic situations. Design of new mimetics based on the cyclopentenone structure could be very useful as neuroprotective agents (48). Further studies will be necessary to determine whether the cyclopentenone PGs or analogues can be of use as preventive agents against acute neurodegenerative conditions, especially in limiting damage following a stroke, or reducing the progression of diseases with chronic neurodegeneration like Alzheimer’s disease and possibly even retarding signs of “normal” aging.

Figure 1.

Diagram of the cyclopentanone PG synthesis pathway.

Figure 2.

Effect of different concentrations of the cyclopentenone prostaglandin of the J series, dPGJ₂ on the expression of HO-1, HO-2, and actin in mouse primary cortical neuronal cultures. Cells were treated with different concentrations (0, 0.5, 5, 10, 20, and 50 μM) of dPGJ₂ for 6 hr before being harvested and analyzed. (A) Dose-dependent HO-1 protein induction by dPGJ₂. (B) No significant differences in the HO-2 protein levels were found after treatment with dPGJ₂. Using antibodies against actin probed on the same blot. (C) Similar loading in different lanes.

Figure 3.

Effect of different PGs on HO expression in mouse primary cortical neuronal cultures. Cells were treated with 5 μM of PGJ₂, dPGJ₂, PGA₂, dPGA₂, PGD₂, and PGE₂ for 6 hr before being harvested and analyzed. (A) HO-1 protein induction by different PGs and predominantly by PGJ₂ and dPGJ₂. (B) No significant effect of these PGs on HO-2 protein was revealed. (C) Using antibodies against actin probed on the same blot, similar loading in different lanes is indicated.

Figure 4.

Detection of immunoreactive HO-2-related protein in wild-type (WT) and HO-2 gene knockout (HO-2^−/−) mouse brain homogenates. On this representative Western blot, mouse brain tissues were processed for microsomal and cytosolic fractions as described in the Materials and Methods section.

Figure 5.

Schematic representation of heme degradation by HO. An interesting visual example of this enzymatic reaction is a “black eye.” Hemolysis occurs as a result of physical shock at which point heme is released (black). Then, through the action of the HO and cytochrome P₄₅₀ reductase (CP₄₅₀R), heme degrades into biliverdin (green). And finally, through the action of biliverdin reductase (BVR), BR is generated (yellow).

Footnotes

This work was supported by a Grant-in-Aid from the American Heart Association, the Alzheimer’s Association and the American Heath Assistance Foundation.

1

To whom requests for reprints should be addressed at Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, 600 N. Wolfe Street, (Blalock 1404A), Baltimore, MD 21217. E-mail:

Acknowledgements

The authors wish to thank James M. Carlisle for his assistance.

References

Straus DS, Glass CK. Cyclopentenone prostaglandins: New insights on biological activities and cellular targets. Med Res Rev 21 : 185–210, 2001.

Satoh T, Furuta K, Tomokiyo K, Namura S, Nakatsuka D, Sugie Y, Ishikawa Y, Hatanaka H, Suzuki M, Watanabe Y. Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins. J Neurochem 77 : 50–62, 2001.

Ito S, Negishi M, Sugama K, Okuda-Ashitaka E, Hayaishi O. Signal transduction coupled to prostaglandin D2. Adv Prostaglandin Thromboxane Leukot Res 21A : 371–374, 1991.

Urade Y, Kitahama K, Ohishi H, Kaneko T, Mizuno N, Hayaishi O. Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. Proc Natl Acad Sci USA 90 : 9070–9074, 1993.

Simpson PJ, Mickelson J, Fantone JC, Gallagher KP, Lucchesi BR. Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart. Circ Res 60 : 666–673, 1987.

Di Battista JA, Doré S, Morin N, He Y, Pelletier JP, Martel-Pelletier J. Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca(++)-calmodulin regulated processes. J Cell Biochem 65 : 408–419, 1997.

Pierce KL, Fujino H, Srinivasan D, Regan JW. Activation of FP prostanoid receptor isoforms leads to Rho-mediated changes in cell morphology and in the cell cytoskeleton. J Biol Chem 274 : 35944–35949, 1999.

Pierce KL, Bailey TJ, Hoyer PB, Gil DW, Woodward DF, Regan JW. Cloning of a carboxyl-terminal isoform of the prostanoid FP receptor. J Biol Chem 272 : 883–887, 1997.

Irie A, Segi E, Sugimoto Y, Ichikawa A, Negishi M. Mouse prostaglandin E receptor EP3 subtype mediates calcium signals via Gi in cDNA-transfected Chinese hamster ovary cells. Biochem Biophys Res Commun 204 : 303–309, 1994.

10.

Katoh H, Watabe A, Sugimoto Y, Ichikawa A, Negishi M. Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells. Biochim Biophys Acta 1244 : 41–48, 1995.

11.

Crider JY, Griffin BW, Sharif NA. Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: Pharmacological characterization using agonists and antagonists. Br J Pharmacol 127 : 204–210, 1999.

12.

Hatae N, Yamaoka K, Sugimoto Y, Negishi M, Ichikawa A. Augmentation of receptor-mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a Gbetagamma subunit-independent manner. Biochem Biophys Res Commun 290 : 162–168, 2002.

13.

Negishi M, Harazono A, Sugimoto Y, Hazato A, Kurozumi S, Ichikawa A. Selective coupling of prostaglandin E receptor EP3D to multiple G proteins depending on interaction of the carboxylic acid of agonist and arginine residue of seventh transmembrane domain. Biochem Biophys Res Commun 212 : 279–285, 1995.

14.

Negishi M, Irie A, Sugimoto Y, Namba T, Ichikawa A. Selective coupling of prostaglandin E receptor EP3D to Gi and Gs through interaction of alpha-carboxylic acid of agonist and arginine residue of seventh transmembrane domain. J Biol Chem 270 : 16122–16127, 1995.

15.

Doré S. Decreased activity of the antioxidant heme oxygenase enzyme: Implications in ischemia and in Alzheimer’s disease. Free Radic Biol Med 32 : 1276–1282, 2002.

16.

Maines MD. The heme oxygenase system and its functions in the brain. Cell Mol Biol (Noisy-le-grand) 46 : 573–585, 2000.

17.

Abraham NG, Lin JH, Schwartzman ML, Levere RD, Shibahara S. The physiological significance of heme oxygenase. Int J Biochem 20 : 543–558, 1988.

18.

Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH. Carbon monoxide: A putative neural messenger. Science 259 : 381–384, 1993.

19.

Doré S, Takahashi M, Ferris CD, Zakhary R, Hester LD, Guastella D, Snyder SH. Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad Sci USA 96 : 2445–2450, 1999.

20.

McCoubrey WK Jr, Huang TJ, Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247 : 725–732, 1997.

21.

Ferris CD, Jaffrey SR, Sawa A, Takahashi M, Brady SD, Barrow RK, Tysoe SA, Wolosker H, Baranano DE, Doré S, Poss KD, Snyder SH. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol 1 : 152–157, 1999.

22.

Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci USA 94 : 10919–10924, 1997.

23.

Andrews NC. Metal transporters and disease. Curr Opin Chem Biol 6 : 181–186, 2002.

24.

Quinlan GJ, Chen Y, Evans TW, Gutteridge JM. Iron signalling regulated directly and through oxygen: Implications for sepsis and the acute respiratory distress syndrome. Clin Sci (Lond) 100 : 169–182, 2001.

25.

Crapper McLachlan DR, Dalton AJ, Kruck TP, Bell MY, Smith WL, Kalow W, Andrews DF. Intramuscular desferrioxamine in patients with Alzheimer’s disease. Lancet 337 : 1304–1308, 1991.

26.

Wu TW, Carey D, Wu J, Sugiyama H. The cytoprotective effects of bilirubin and biliverdin on rat hepatocytes and human erythrocytes and the impact of albumin. Biochem Cell Biol 69 : 828–834, 1991.

27.

Van Bergen P, Rauhala P, Spooner CM, Chiueh CC. Hemoglobin and iron-evoked oxidative stress in the brain: protection by bile pigments, manganese and S-nitrosoglutathione. Free Radic Res 31 : 631–640, 1999.

28.

Doré S, Snyder SH. Neuroprotective action of bilirubin against oxidative stress in primary hippocampal cultures. Ann NY Acad Sci 890 : 167–172, 1999.

29.

Noguchi M, Yoshida T, Kikuchi G. Identification of the product of heme degradation catalyzed by the heme oxygenase system as biliverdin IX alpha by reversed-phase high- performance liquid chromatography. J Biochem (Tokyo) 91 : 1479–1483, 1982.

30.

Salim M, Brown-Kipphut BA, Maines MD. Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation. J Biol Chem 276 : 10929–10934, 2001.

31.

Wu TW, Wu J, Li RK, Mickle D, Carey D. Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage. Biochem Cell Biol 69 : 683–688, 1991.

32.

Stocker R, McDonagh AF, Glazer AN, Ames BN. Antioxidant activities of bile pigments: biliverdin and bilirubin. Methods Enzymol 186 : 301–309, 1990.

33.

Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ, Motterlini R. Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol Heart Circ Physiol 278 : H643– H651, 2000.

34.

Kranc KR, Pyne GJ, Tao L, Claridge TD, Harris DA, Cadoux-Hudson TA, Turnbull JJ, Schofield CJ, Clark JF. Oxidative degradation of bilirubin produces vasoactive compounds. Eur J Biochem 267 : 7094–7101, 2000.

35.

Vreman HJ, Wong RJ, Sanesi CA, Dennery PA, Stevenson DK. Simultaneous production of carbon monoxide and thiobarbituric acid reactive substances in rat tissue preparations by an iron-ascorbate system. Can J Physiol Pharmacol 76 : 1057–1065, 1998.

36.

Yoshida T, Noguchi M, Kikuchi G. The step of carbon monoxide liberation in the sequence of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system. J Biol Chem 257 : 9345–9348, 1982.

37.

Alkadhi KA, Al-Hijailan RS, Malik K, Hogan YH. Retrograde carbon monoxide is required for induction of long-term potentiation in rat superior cervical ganglion. J Neurosci 21 : 3515–3520, 2001.

38.

Maines M. Carbon monoxide and nitric oxide homology: Differential modulation of heme oxygenases in brain and detection of protein and activity. Methods Enzymol 268 : 473–488, 1996.

39.

Meffert MK, Haley JE, Schuman EM, Schulman H, Madison DV. Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins. Neuron 13 : 1225–1233, 1994.

40.

Poss KD, Thomas MJ, Ebralidze AK, O’Dell TJ, Tonegawa S. Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice. Neuron 15 : 867–873, 1995.

41.

Linden DJ, Narasimhan K, Gurfel D. Protoporphyrins modulate voltage-gated Ca current in AtT-20 pituitary cells. J Neurophysiol 70 : 2673–2677, 1993.

42.

Hartsfield CL, Alam J, Cook JL, Choi AM. Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide. Am J Physiol 273 : L980–L988, 1997.

43.

Wang R, Wu L. The chemical modification of KCa channels by carbon monoxide in vascular smooth muscle cells. J Biol Chem 272 : 8222–8226, 1997.

44.

Otterbein LE. Carbon monoxide: Innovative anti-inflammatory properties of an age-old gas molecule. Antioxid Redox Signal 4 : 309–319, 2002.

45.

Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH, Soares MP. Heme oxygenase-1-derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cells from tumor necrosis factor-alpha-mediated apoptosis. J Biol Chem 277 : 17950–17961, 2002.

46.

Schror K. Eicosanoids and myocardial ischaemia. Basic Res Cardiol 82 : 235–243, 1987.

47.

Dwyer BE, Nishimura RN, De Vellis J, Yoshida T. Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells. Glia 5 : 300–305, 1992.

48.

Saragovi HU, Gehring K. Development of pharmacological agents for targeting neurotrophins and their receptors. Trends Pharmacol Sci 21 : 93–98, 2000.