![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Departments of Pharmacology and Toxicology (R.T.K., Y.J., J.F.B., K.N.) and Chemistry (J.Z.V., W.A.S.), Queen's University, Kingston, Ontario, Canada; and Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, United Kingdom (R.M.)
Received January 12, 2007; accepted August 29, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
(TNF-) produced cytotoxicity in wild-type LLC-PK1 cells (P < 0.05) but not in HO-1 and HO-2 overexpressing or wild type cells pretreated with hemin (10 µM). The presence of imidazole-dioxolane HO inhibitors (2–25 µM) decreased cell viability (P < 0.05). A CO-releasing molecule reversed, in a dose-dependent manner, the cytotoxic effects and caspase-3 activation induced by the combination of gliotoxin/TNF- and the HO inhibitors, suggesting an important role for CO in protection against renal toxicity. These data demonstrate a protective role of both HO-1 and HO-2 against gliotoxin/TNF--induced cytotoxicity in LLC-PK1 cells. The novel imidazole-dioxolane compounds can be used as effective inhibitors of HO activity in cell culture.
). The heme-degrading enzymes are the source of biliverdin and bilirubin, and most of the endogenous CO (85%) produced in mammals (Tenhunen et al., 1969); HO activity, which is attributed to two distinct HO genes, arises from HO-1 (32-kDa stress-inducible protein) and HO-2 (36-kDa constitutive isoform) (Maines, 1988). Whereas HO-2 may be involved in the regulation of normal physiological cell functions, HO-1 is induced in response to an array of physical and chemical stimuli, including heat shock, heavy metals, heme, ionizing radiation, reactive oxygen species, and proinflammatory cytokines. This induction of HO activity, with its resulting increase in CO, bilirubin, and biliverdin, is thought to contribute to the cytoprotective response of organs to noxious stimuli. Part of the protective effects of the HO system is related to the removal of free heme because this tetrapyrrole is a pro-oxidant that catalyzes the decomposition of organic peroxides leading to the generation of alkyl peroxyl radicals (Akaike et al., 1992). Thus, by breaking down heme, HO prevents the overproduction of reactive oxygen species in the circulation. Furthermore, bilirubin and biliverdin are products of HO activity with strong antioxidant properties and can scavenge free radicals including superoxide, peroxyl radicals, and peroxynitrite (Stocker et al., 1990; Foresti et al., 1997, 1999; Clark et al., 2000a). Accumulating evidence points to the protective role of bilirubin against oxidative stress-induced neurotoxicity (Doré et al., 1999), myocardial ischemia (Clark et al., 2000b), and nephrotoxicity (Polte et al., 2002). CO, on the other hand, exerts several effects, including a decrease in the stimulation of immune responses by cytokines (Lee and Chau, 2002), antiapoptotic or anti-inflammatory responses mainly through the stimulation of soluble guanylyl cyclase (sGC) (Liu et al., 2002), and the activation of mitogen-activated protein kinase signaling pathways (Brouard et al., 2002, 2000).
Much of our current knowledge of the CO/HO system has been derived from experiments that have exploited the metalloporphyrin HO inhibitors, such as tin protoporphyrin. Nonetheless, these compounds have some limitations, mainly related to their close structural analogy to the HO substrate (heme), which gives rise to a lack of selectivity. Thus, the metalloporphyrins are known to affect the activities of heme-dependent enzymes such as nitric oxide synthase and soluble guanylyl cyclase. This lack of selectivity has led to some uncertainty in the interpretation of experimental data generated using metalloporphyrins. The resulting ambiguities have led us to design nonporphyrin inhibitors of HO that are significantly more selective, i.e., imidazole-dioxolanes (Vlahakis et al., 2005). In general, these compounds inhibit HO activity in vitro with little or no effect on nitric oxide synthase and soluble guanylyl cyclase (Kinobe et al., 2006).
To establish the utility of these drugs more broadly, it is necessary to explore their pharmacology in experimental conditions other than broken-cell preparations. Ideally, the range should span from intact cells to whole organs and animals in vivo. Herein, we addressed the hypothesis that these novel HO inhibitors are effective in intact cells. The present communication describes studies of some imidazole-dioxolane-based HO inhibitors, extending their scope into cultured renal cells to explore the contribution of the HO pathway and its products (i.e., biliverdin and CO) in protection against gliotoxin/TNF--induced oxidative stress-mediated toxicity. The rationale for choosing renal tubule epithelial cells (LLC-PK1) is based on previous reports demonstrating that these cells are sensitive to some of the stressors described above (Nath et al., 2001) and promptly respond by activating the HO system (Balogun et al., 2003). These imidazole-dioxolane compounds will be useful tools in the studies on the physiology and pharmacology of the HO system.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Cells were studied as 80 to 90% confluent monolayer in all experiments. To enhance the expression and activity of HO-2, cells were transfected with a plasmid containing rat HO-2 (HO-2/cpRc/CMV) (2 µg/ml), and the transfectants were selected in G418 (700 µg/ml). Controls were transfected with an empty mammalian expression vector (cpRc/CMV). To transiently overexpress HO-1, cells were grown to 80% confluence and then exposed to 15 multiplicities of infection of the recombinant adenovirus encoding green fluorescence protein (GFP) or the bicistronic recombinant adenovirus encoding human HO-1 and GFP (GFP-HO-1) for 6 h. Transduction efficiency was assessed 24 h after exposure to the virus using fluorescence microscopy to detect GFP expression. HO-1 expression was also induced by treating cells with hemin chloride (10 µM) for 12 to 24 h as described previously (Liang et al., 2000).
SDS-PAGE and Western Blotting. To examine the expression of HO-1, HO-2, iNOS and sGC, LLC-PK1 cells were washed in 10 mM phosphate-buffered saline, scraped into centrifuge tubes using a rubber policeman, and then centrifuged at 1000g for 10 min at 4°C. The cell pellet was resuspended in 100 mM phosphate buffer (pH 7.4), containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 5 mM dithiothreitol. Cells were broken by sonication on ice; then the sonicate was centrifuged at 12,000g for 15 min at 4°C. Protein concentration of the cell lysate (supernatant) was determined by a modification of the biuret method as described by Marks et al. (1997). Sixty micrograms of the cell lysate was subjected to SDS-PAGE under reducing conditions, and the protein was transferred onto nitrocellulose Immobilon-P membranes (Millipore Corporation, Bedford, MA) according to the method of Laemmli (1970). Blots were then incubated with polyclonal antibodies against HO-1, HO-2, iNOS, or sGC, and membranes were subsequently incubated with a peroxidase-labeled goat anti-rabbit IgG secondary antibody (Vector Laboratories, Burlingame, CA). Peroxidase activity was detected by an enhanced chemiluminescence detection kit according to the manufacturer's instructions (GE Healthcare, Toronto, ON, Canada). Relative protein expression was quantified by optical densitometry using an NIH imager. Densitometric units for HO-1, HO-2, iNOS, and sGC expression were normalized to 
-actin protein expression.
Measurement of HO Enzymatic Activity. Total HO activity in the cell lysate was determined by quantitation of CO formed from the degradation of methemalbumin, i.e., heme complexed with albumin, according to published methods (Vreman and Stevenson, 1999). In brief, reaction mixtures (150 µl) consisted of 100 mM phosphate buffer (pH 7.4), 50 µM methemalbumin, 0.3 to 0.5 mg/ml protein, and 1.5 mM -NADPH, and the incubations were carried out for 45 min at 37°C. Reactions were stopped by instantly freezing the reaction mixture on pulverized dry ice, and CO formation was determined by gas chromatography using a TA 3000R Process Gas Analyzer (Trace Analytical, Newark, DE).
Cell Treatment and Viability Assay. The cytotoxicity of imidazole-based HO inhibitors on the LLC-PK1 cell line was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay as previously described (Hall and Heckel, 1990). In brief, cells were seeded at a density of 
1.0 x 105 cells/well in a 12-well plate and allowed to attach overnight. The medium was replaced, and cells were incubated with fresh medium containing the various agents (cytotoxic compounds or HO inhibitors) for 12 to 24 h. After 24 h, cells were washed in phosphate-buffered saline and then incubated with serum-free medium containing 0.5 mg/ml MTT for 2 h. Formazan formed from the reduction of MTT by mitochondrial dehydrogenases was dissolved in anhydrous isopropanol containing 0.1 M hydrochloric acid and 0.1% v/v Triton X-100, and the absorbance was measured spectrophotometrically at 570 nm. A water-soluble, CO-releasing molecule (CORM-3) (Clark et al., 2003; Motterlini et al., 2005) was used as a source of exogenous CO to assess the contribution of this HO product on oxidant-mediated toxicity. In a control experiment, an inactive form of CORM-3 (iCORM-3) that is not capable of releasing CO was used as described before (Clark et al., 2003; Motterlini et al., 2005; Tayem et al., 2006). Cell viability was expressed as a percentage of the optical density in untreated cells from three experiments carried out in triplicate.
|
. Peroxidase-conjugated polyclonal anti-active caspase-3 antibody was obtained from Abcam Inc. (Cambridge, MA) and used at a dilution of 1:250 (v/v). Relative activated caspase-3 expression was quantified by optical densitometry and then normalized to -actin protein expression using an NIH imager. LogP Determination. Theoretical values of the octanol/water partition coefficient were calculated using ChemDraw Ultra version 6.0 and Molinspiration version 2.2. The mean values for QC-1 and QC-13 were 3.97 and 3.27, respectively.
Data Analysis. Data are presented as the mean ± S.D. from three experiments performed in triplicate. Statistical analyses were performed by a completely randomized, one-way analysis of variance followed by a post hoc Newman-Keuls or Dunnett's test. P values <0.05 were considered to be statistically significant.
Materials. Hemin, -NADPH, MTT, TNF-, gliotoxin, Dulbecco's modified Eagle's medium, and nutrient mixture F-12 were obtained from Sigma-Aldrich (St. Louis, MO). CORM-3 was synthesized and prepared freshly in distilled water as reported previously (Clark et al., 2003). G418 was purchased from Invitrogen (Burlington, ON, Canada) and FuGENE 6 cell transfection reagent was obtained from Roche Applied Sciences (Laval, QC, Canada). Chromium mesoporphyrin IX chloride (CrMP) was purchased from Frontier Scientific, Inc. (Logan, UT). Anti-HO-1 and HO-2 antibodies were obtained from Stressgen Biotechnologies Corp. (Vancouver, BC, Canada), polyclonal anti-iNOS antibody was obtained from Santa Cruz Biochemicals, Inc. (Santa Cruz, CA), and polyclonal anti-sGC antibody was obtained from Dr. E. Martin, University of Texas Houston, TX. The plasmid (HO-2/pRc/CMV) containing rat HO-2 was received through the generosity of Dr. Phyllis Dennery, Hospital of the University of Pennsylvania (Philadelphia, PA). The recombinant adenovirus encoding GFP as well as the bicistronic recombinant adenovirus encoding human HO-1 and GFP were obtained from Dr. Luis Melo (Department of Physiology, Queen's University, Kingston, ON, Canada). (2S,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[{(4-aminophenyl)thio}methyl]-1,3-dioxolane dihydrochloride) (QC-1) and (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-methyl-1,3-dioxolane hydrochloride (QC-13) were synthesized and characterized as described previously (Vlahakis et al., 2005; 2006). All other chemicals were at least reagent grade and were obtained from Fisher Scientific (Ottawa, ON, Canada).
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-induced cytotoxicity experiments ranged from 1 to 50 µM.
Effects of HO Inhibition on Gliotoxin/TNF--Induced Cytotoxicity. Gliotoxin/TNF--induced cytotoxicity in LLC-PK1 cells proceeds via apoptosis and is mediated by increased generation of reactive oxygen species and activation of caspases (Zhou et al., 2000). The effects of various treatments on cell viability are summarized in Tables 1 and 2. Treatment with the combination of gliotoxin and TNF- resulted in cytotoxicity in wild-type LLC-PK1 cells and those transfected with the empty cpRc/CMV vector or those transduced with GFP only. In comparison, stable transfection resulting in the overexpression of HO-2 enzyme (Table 1) and the transduction with GFP-HO-1 adenovirus (Table 2) was found to be protective against this combination of gliotoxin and TNF-. Likewise, a cytoprotective effect was observed after the induction of HO-1 by pretreating cells with hemin chloride, whereas the inhibition of total HO activity was found to enhance gliotoxin/TNF--induced cytotoxicity (Table 1). Treatment with the metalloporphyrin, CrMP (2–50 µM) resulted in enhanced cytotoxicity of gliotoxin/TNF- with cell viability in all cell types being less than half of that in its absence. Likewise, QC-1, which inhibits both HO-1 and HO-2, increased the cytotoxicity in all cell types to approximately the same extent. In comparison, QC-13, which is an HO-1 selective inhibitor, induced a smaller increase in cytotoxicity and was least effective in cells in which HO-2 was overexpressed. Hemin chloride, CrMP, QC-1, and QC-13, at concentrations that induced changes in the cytotoxicity of the gliotoxin/TNF- treatment, were not cytotoxic.
|
|
|
-induced cytotoxicity in wild-type LLC-PK1 cells. Incubation of cells with nontoxic concentrations (2–50 µM) of CORM-3, a water-soluble CO prodrug, promoted concentration-dependent protection against the cytotoxic effects of QC-1, gliotoxin, and TNF- (Fig. 3). Control experiments, in which an inactive form of CORM-3, iCORM-3, that does not release CO was used, revealed no cytoprotective effect against a combination of QC-1, gliotoxin, and TNF-. Furthermore, the cytoprotection mediated by CORM-3 was blocked by a soluble guanylyl cyclase inhibitor (ODQ). The other organic product of HO activity, biliverdin, did not provide any cytoprotective effect at concentrations between 2 and 10 µM, (Fig. 4). To investigate mechanisms of cell death as well as provide an alternative assay for assessing the effects of QC-1- and QC-13-induced inhibition of HO activity on gliotoxin/TNF--induced cytotoxicity, the expression of activated caspase-3 (a marker of apoptosis in mammalian cells) was determined. Consistent with the findings of Zhou et al. (2000), we observed that gliotoxin/TNF- induced the expression of activated caspase-3 (Fig. 5). Moreover, this increased expression of activated caspase-3 was enhanced in the presence of QC-1 (Fig. 5) but was completely antagonized by the presence of CORM-3 (Fig. 5), indicating that CO contributes to the antiapoptotic effects of the HO system.
|
|
Effects of Imidazole-Dioxolane Compounds on HO, iNOS, and sGC Protein Expression. The HO protein expression profile was also determined in renal cells exposed to the treatments described above. As observed previously in numerous studies, treatment of the cells with hemin chloride (10 µM) was found to cause an increase in the expression of HO-1. Similarly the expression of HO-1 was significantly enhanced by transduction of LLC-PK1 cells with GFP-HO-1 adenovirus (Fig. 6). In contrast, neither QC-1 nor QC-13 at 25 µM showed any effect on the expression of HO-1 (Fig. 7). Likewise, QC-1 showed no effect on the expression of iNOS and sGC (Fig. 7). The expression of the constitutive HO isoform (HO-2) was not altered by hemin or any of the imidazole-based HO inhibitors tested.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Vlahakis et al., 2006). The calculated logP values for these inhibitors (QC-1 = 3.97; QC-13 = 3.27) suggested that they should cross membrane barriers and be effective in intact cell preparations as well as in vivo. Thus, the goal of the present study was to determine whether these drugs were effective in cultured proximal tubule epithelial cells (LLC-PK1) and whether they manifested the inhibitory activity against HO as indicated by their in vitro properties. The main findings of our present study are that cytotoxicity induced by treatment of LLC-PK1 cells with gliotoxin/TNF- was prevented by pretreatment with hemin to induce HO-1 or overexpression of HO-1 and HO-2; the cytotoxic effect was enhanced by sublethal concentrations (2–50 µM) of imidazole-dioxolane; and gliotoxin/TNF--mediated cytotoxicity was blocked by treatment with a CO releasing agent (CORM-3) in a concentration-dependent manner.
To address the above goal, LLC-PK1 cells were selected because extensive prior pharmacological studies indicated the presence of both HO-1 and HO-2 activities (Liang et al., 2000; Nath et al., 2001) and their sensitivity to the cytotoxic effects of gliotoxin/TNF- (Zhou et al., 2000). In addition, their complement of HO-1 and HO-2 would be susceptible to manipulation by transfection with HO-2 cDNA to elevate HO-2 activity and treatment with hemin to elevate HO-1 activity (Braggins et al., 1986; Maines 1988). The results from the present study confirm the appropriateness of this model in that total HO activity of broken cell preparations of these cells was elevated with hemin treatment, transduction with GFP-HO-1 adenovirus, or HO-2 transfection. Furthermore, both QC-1 and CrMP inhibited HO activity from cells exposed to the various treatments; in comparison, QC-13 inhibited HO activity only in preparations made from hemin-treated, and GFP-HO-1 transduced cells. These data demonstrate that, in broken LLC-PK1 cell preparations, QC-13 was selective for the inhibition of the inducible HO-1 isozyme, whereas QC-1 and CrMP were nonselective, inhibiting both HO-1 and HO-2 activities. These observations are in agreement with our previous in vitro studies using rat spleen and brain microsomes that showed the selectivity of QC-13 as an HO-1 inhibitor compared with QC-1 and CrMP (Kinobe et al., 2006; Vlahakis et al., 2006).
The experiments to evaluate the susceptibility of cells to oxidant-induced cytotoxicity in the presence of the HO inhibitors indicate that both QC-1 and QC-13 are effective because they enhanced the toxicity of gliotoxin/TNF- in cultured LLC-PK1 cells. The toxic effects elicited by gliotoxin/TNF- are known to be mediated by increased oxidative stress and apoptotic activities (Zhou et al., 2000). We also know that by degrading the pro-oxidant heme and producing the antioxidant biliverdin, the HO pathway becomes a dynamic protective system against oxidative stress (Clark et al., 2000a). The higher cell viability observed in this study after stable transfection with HO-2 cDNA or the pharmacological induction of HO-1 by pretreating cells with hemin supports the defensive role of HO activity in these cells. These data are consistent with those from several studies demonstrating that, although many of the cytoprotective effects of HO are attributable to the inducible isoform (HO-1), under basal conditions the constitutive isoform (HO-2) may also serve in a protective manner in a variety of cell types including LLC-PK1 cells (Shiraishi et al., 2000), human embryo kidney cells (Kim et al., 2005), and cerebral vascular endothelial cells (Parfenova et al., 2006). The increased cytotoxicity resulting from the presence of CrMP, QC-1, and QC-13 was anticipated because treatment with these drugs would interfere with the protective effects of HO activity. Moreover, the lesser enhancement in cytotoxicity induced by QC-13 is predicted because in vitro it is selective for HO-1 inhibition with little or no effect on HO-2 (Kinobe et al., 2006). These observations also indicate a cytoprotective role of HO-2 because the drugs that inhibit this isoform as well as HO-1 had the ability to augment the cytotoxic effects of gliotoxin/TNF- to a greater extent.
The cytotoxicity-enhancing effects of QC-1 and QC-13 found in this study can be interpreted to be due to their inhibition of CO production. In fact, gliotoxin/TNF--induced cytotoxicity was abrogated by treatment of cells with a CO-releasing molecule (CORM-3). CO liberated from this compound was directly responsible for the observed cytoprotective effects because control experiments using an inactive compound that does not release CO (iCORM-3 as a negative control) failed to prevent the loss in viability caused by gliotoxin/TNF- treatment. Likewise, the increase in caspase-3 expression, a recognized marker of apoptosis in mammalian cells, was completely abolished in cells challenged with gliotoxin/TNF- and treated with the CO-releasing agent; even in this case, the inactive compound (iCORM-3) was without effect. In contrast to the effects observed with CO, biliverdin, another product of HO activity that is known to protect against oxidative stress in many organ systems including the heart, kidney, and liver (Abraham and Kappas, 2005; Nath, 2006) was not cytoprotective in the LLC-PK1 cell culture model used herein. Collectively, these data indicate that CO and not the ruthenium metal complex used as the carrier of this gas is the cytoprotective principle in CORM-3. It appears therefore that, in this model, cytoprotection by HO was mediated by mechanisms that are dependent on CO signaling but independent of the antioxidant properties of biliverdin. This notion is supported by the fact that similar renoprotective actions of CO liberated from CORM-3 (but not biliverdin) were observed after treatment of LLC-PK1 cells with cisplatin, a commonly used antineoplastic drug that is well known for its oxidative stress-inducing properties and nephrotoxic effects (Tayem et al., 2006). As part of the mechanism(s) involved in this cytoprotection is elicited by CO, it is relevant that several of the signaling effects of CO depend on stimulation of sGC and/or activation of mitogen-activated protein kinase (Ramos et al., 1989; Utz and Ullirich, 1991). The data of the present study show that nontoxic concentrations of ODQ, a selective sGC inhibitor abolished the cytoprotective effects of CORM-3. This finding is consistent with the reported renoprotective effects of CO liberated from CORM-3 in a model of cisplatin-induced nephrotoxicity (Tayem et al., 2006).
In summary, the newer HO inhibitors, QC-1 and QC-13, were found to affect intact cultured cells in the manner expected for nonselective HO inhibitors and HO-1-selective inhibitors, respectively. Thus, these drugs can penetrate biological membranes adequately and should be useful for pharmacological studies in intact organs and whole animals as well. With their enhanced selectivity compared with the classic metalloporphyrin HO inhibitors, the imidazole-dioxolane HO inhibitors will be useful in enhancing our knowledge of the biological function of the CO/HO system.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HO, heme oxygenase; CO, carbon monoxide; sGC, soluble guanylyl cyclase; TNF-, tumor necrosis factor-; GFP, green fluorescent protein; G418, geneticin; PAGE, polyacrylamide gel electrophoresis; iNOS, inducible nitric oxide; MTT, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CORM-3, CO-releasing molecule-3; iCORM-3, inactive form of CORM-3; CrMP, chromium mesoporphyrin IX chloride; QC-1, (2S,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[{(4-aminophenyl)thio}methyl]-1,3-dioxolane dihydrochloride); QC-13, (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-methyl-1,3-dioxolane hydrochloride; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one.
Address correspondence to: Dr. Kanji Nakatsu, Department of Pharmacology and Toxicology, Queen's University, Kingston, ON K7L 3N5, Canada. E-mail: nakatsuk{at}post.queensu.ca
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abraham NG and Kappas A (2005) Heme oxygenase and the cardiovascular-renal system. Free Radic Biol Med 39: 1–25.[Medline]
Akaike T, Sato K, Ijiri S, Miyamoto Y, Kohno M, Ando M, and Maeda H (1992) Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides. Arch Biochem Biophys 294: 55–63.[CrossRef][Medline]
Balogun E, Foresti R, Green CJ, and Motterlini R (2003) Changes in temperature modulate heme oxygenase-1 induction by curcumin in renal epithelial cells. Biochem Biophys Res Commun 308: 950–955.[CrossRef][Medline]
Braggins PE, Trakshel GM, Kutty RK, and Maines MD (1986) Characterisation of two heme oxygenase isoforms in rat spleen: comparison with the hematin-induced and constitutive isoforms of the liver. Biochem Biophys Res Commun 141: 528–533.[CrossRef][Medline]
Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH, and Soares MP (2002) Heme oxygenase-1-derived carbon monoxide requires activation of transcription factor NF-
B to protect endothelial cells from tumor necrosis factor--mediated apoptosis. J Biol Chem 277: 17950–17961.
Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, and Soares MP (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 192: 1015–1026.
Clark JE, Foresti R, Green CJ, and Motterlini R (2000a) Dynamics of haem oxygenase-1 expression and bilirubin production in cellular protection against oxidative stress. Biochem J 348: 615–619.[CrossRef][Medline]
Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ, and Motterlini R (2000b) Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol Heart Circ Physiol 278: H643–H651.
Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R, and Motterlini R (2003) Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 93: 2–8.[CrossRef]
Doré S, Takahashi M, Ferris CD, Hester LD, Guastella D, and Snyder SH (1999) Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad SciUSA 96: 2445–2450.
Foresti R, Clark JE, Green CJ, and Motterlini R (1997) Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells. Involvement of superoxide and peroxynitrite anions. J Biol Chem 272: 18411–18417.
Foresti R, Sarathchandra P, Clark JE, Green CJ, and Motterlini R (1999) Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J 337: 729–736.
Hall TJ and Heckel C (1990) Lack of effect of verapamil and MDL 646, a cytoprotective PGE1 analogue on cyclosporin A nephrotoxicity in vitro. Res Commun Chem Pathol Pharmacol 67: 293–296.[Medline]
Ishido M, Suzuki T, Adachi T, and Kunimoto M (1999) Zinc stimulates DNA synthesis during its antiapoptotic action independently with increments of an antiapoptotic protein, Bcl-2, in porcine kidney LLC-PK1 cells. J Pharmacol Exp Ther 290: 923–928.
Kim Y, Zhuang H, Koehler RC, and Doré S (2005) Distinct protective mechanisms of HO-1 and HO-2 against hydroperoxide-induced cytotoxicity. Free Radic Biol Med 38: 85–92.[CrossRef][Medline]
Kinobe RT, Vlahakis JZ, Vreman HJ, Stevenson DK, Brien JF, Szarek WA, and Nakatsu K (2006) Selectivity of imidazole-dioxolane compounds for in vitro inhibition of microsomal haem oxygenase isoforms. Br J Pharmacol 147: 307–315.[CrossRef]
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 277: 680–685.
Lee T and Chau L (2002) Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med 8: 240–246.[CrossRef][Medline]
Liang M, Croatt AJ, and Nath KA (2000) Mechanisms underlying induction of heme oxygenase-1 by nitric oxide in renal tubular epithelial cells. Am J Physiol 279: F728–F735.
Liu XM, Chapman GB, Peyton KJ, Schafer AI, and Durante W (2002) Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc Res 55: 396–405.
Maines M (1997) The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517–554.[CrossRef][Medline]
Maines M (1988) Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2: 2557–2568.[Abstract]
Marks GS, McLaughlin BE, Vreman HJ, Stevenson DK, Nakatsu K, Brien JF, and Pang SC (1997) Heme oxygenase activity and immunohistochemical localization in bovine pulmonary artery and vein. J Cardiovasc Pharmacol 30: 1–6.[CrossRef][Medline]
Motterlini R, Mann BE, and Foresti R (2005) Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 14: 1305–1318.[CrossRef][Medline]
Nagothu KK, Bhatt R, Kaushal GP, and Portilla D (2005) Fibrate prevents cisplatin-induced proximal tubule cell death. Kidney Int 68: 2680–2693.[CrossRef][Medline]
Nath KA (2006) Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues. Kidney Int 70: 432–443.[Medline]
Nath KA, Vercellotti GM, Grande JP, Miyoshi H, Paya CV, Manivel JC, Haggard JJ, Croatt AJ, Payne WD, and Alam J (2001) Heme protein-induced chronic renal inflammation: suppressive effect of induced heme oxygenase-1. Kidney Int 59: 106–117.[CrossRef][Medline]
Parfenova P, Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, Regan RF, and Leffler CW (2006) Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection. Am J Physiol Cell Physiol 290: C1399–C1410.
Polte T, Hemmerle A, Bernd G, Grosser G, Abate A, and Schröder H (2002) Atrial natriuretic peptide reduces cyclosporin toxicity in renal cells: role of cGMP and heme oxygenase-1. Free Radic Biol Med 32: 56–63.[CrossRef][Medline]
Ramos KS, Lin H, and McGrath JJ (1989) Modulation of cyclic guanosine monophosphate levels in cultured aortic smooth muscle cells by carbon monoxide. Biochem Pharmacol 38: 1368–1370.[CrossRef][Medline]
Shiraishi F, Curtis LM, Truong L, Poss K, Visner GA, Madsen K, Nick HS, and Agarwal A (2000) Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol Renal Physiol 278: F726–F736.
Stocker R, McDonaugh AF, Glazer AN, and Ames BN (1990) Antioxidant activities of bile pigments: biliverdin and bilirubin. Methods Enzymol 186: 301–309.[Medline]
Tayem Y, Johnson TR, Mann BE, Green CJ, and Motterlini R (2006) Protection against cisplatin-induced nephrotoxicity by a carbon monoxide-releasing molecule. Am J Physiol Renal Physiol 290: F789–F794.
Tenhunen R, Marver HS, and Schmid R (1969) Heme oxygenase: characterisation of the enzyme. J Biol Chem 244: 6388–6394.
Utz J and Ullirich V (1991) Carbon monoxide relaxes ileal smooth muscle through activation of guanylate cyclase. Biochem Pharmacol 41: 1195–1201.[CrossRef][Medline]
Vlahakis JZ, Kinobe RT, Bowers RJ, Brien JF, Nakatsu K, and Szarek WA (2005) Synthesis and evaluation of azalanstat analogues as heme oxygenase inhibitors. Bioorg Med Chem Lett 15: 1457–1461.[CrossRef][Medline]
Vlahakis JZ, Kinobe RT, Bowers RJ, Brien JF, Nakatsu K, and Szarek WA (2006) Imidazole-dioxolane compounds as isozyme-selective heme oxygenase inhibitors. J Med Chem 49: 4437–4441.[CrossRef][Medline]
Vreman HJ and Stevenson DK (1999) Detection of heme oxygenase activity by measurement of CO, in Current Protocols in Toxicology (Maines MD, Costa LG, Reed DJ, Sassa S, and Sipes IG eds) pp 9.2.1–9.2.10, John Wiley and Sons, New York.
Zhou Z, Zhao A, Goping G, and Hirszel P (2000) Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells. Toxicol Sci 54: 194–202.
This article has been cited by other articles:
|
|
 |
|
  L. Di Francesco, L. Totani, M. Dovizio, A. Piccoli, A. Di Francesco, T. Salvatore, A. Pandolfi, V. Evangelista, R. A. Dercho, F. Seta, et al. Induction of Prostacyclin by Steady Laminar Shear Stress Suppresses Tumor Necrosis Factor-{alpha} Biosynthesis via Heme Oxygenase-1 in Human Endothelial Cells Circ. Res., February 27, 2009; 104(4): 506 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. G. Abraham and A. Kappas Pharmacological and Clinical Aspects of Heme Oxygenase Pharmacol. Rev., March 1, 2008; 60(1): 79 - 127. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, significantly higher cell viability compared with gliotoxin-, TNF-
