Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

Heme oxygenase modulates selectin expression in different regional vascular beds

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, iron, and CO. The inducible isoform (HO-1) has been implicated as a modulator of the inflammatory response. HO-1 activity can be induced by hemin and inhibited with zinc protoporphyrin IX (ZnPP). Using these reagents, we assessed the possibility that HO-1 modulates the inflammatory response by altering the expression of endothelial cell adhesion molecules. Endotoxin (lipopolysaccharide, LPS)-induced expression of P- and E-selectin expression was quantified in different vascular beds of the rat using the dual radiolabeled monoclonal antibody technique. Pretreatment with hemin attenuated, whereas ZnPP treatment exacerbated, the increased selectin expression normally elicited by LPS. Biliverdin, at an equimolar dosage, was as effective as hemin in attenuating LPS-induced selectin expression in the lung, kidneys, liver, and intestines. These findings indicate that the anti-inflammatory properties of HO-1 may be related to an inhibitory action of P- and E-selectin expression in the vasculature. Biliverdin (or its metabolite, bilirubin), rather than CO, may account for this action of HO-1 on endothelial cell adhesion molecule expression.     

Pegylated zinc protoporphyrin: a water-soluble heme oxygenase inhibitor with tumor-targeting capacity

Heme oxygenase (HO) is a key enzyme in heme metabolism; it oxidatively degrades heme to biliverdin, accompanied by formation of free iron and carbon monoxide. Biliverdin is subsequently reduced by cytosolic biliverdin reductase to form bilirubin, a potent antioxidant. We recently found that tumor cells utilize HO to protect themselves from oxidative stress by producing the antioxidant bilirubin. This result suggested an important potential therapeutic strategy: suppression of bilirubin production with the use of HO inhibitors; hence, cancer cells become vulnerable to oxidative stress induced by anticancer drugs or leukocytes of the host. This concept was validated by using the intraarterial administration of an HO inhibitor, zinc protoporphyrin, in nonphysiological solution. In the present study, zinc protoporphyrin (ZnPP) was conjugated with poly(ethylene glycol) (PEG) with molecular weight of 5000, to make ZnPP, a water-soluble compound (PEG-ZnPP), and to improve its tumor-targeting efficiency. PEG was conjugated to ZnPP through newly introduced amino groups, where ethylenediamine residues were added at C6 and C7 of protoporphyrin. The divalent zinc cation was chelated into the protoporphyrin ring to obtain PEG-ZnPP. PEG-ZnPP did become highly water-soluble, and it formed multimolecular associations with molecules larger than 70 kDa in aqueous media. PEG-ZnPP inhibited splenic microsomal HO activity in vitro in a competitive manner in the presence of hemin, with an apparent inhibitory constant of 0.12 microM. Most important, PEG-ZnPP injected intravenously significantly suppressed intratumor HO activity in a murine solid tumor model, which suggests that tumor-targeted inhibition of HO is possible with the use of PEG-ZnPP.     

Backbone assignments of the apo and Zn(II) protoporphyrin IX-bound states of the soluble form of rat heme oxygenase-1

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.     

Inhibition of endogenous CO by ZnPP protects against stress-induced gastric lesion in adult male albino rats

Carbon monoxide (CO) has been found to be produced in every living cell in a biochemical reaction catalyzed by heme-oxygenase (HO) enzyme which degrades heme into biliverdin, CO, and iron. Endogenous CO is not a waste product, but acts as a chemical messenger mediating and modulating many intracellular biochemical reactions that regulate physiological functions. This study was designed to investigate the effect of inhibition of endogenous CO production by zinc protoporphyrin (ZnPP), an HO inhibitor, on the gastric secretion and ulceration induced by cold-restraint stress (CRS) in adult male albino rats. Rats were pylorically ligated and divided randomly into the following groups (six rats each): control, ZnPP treated (50 μmol/kg/day, s.c. for 10 days), CRS, and stressed ZnPP treated groups. Blood samples were collected from the retro-orbital sinus of anesthetized rats for determination of CO concentration. We found that ZnPP pretreatment significantly decreased HO-1 level, CO level, and volume of gastric juice as compared to the control non-stressed rats. In the present study, ZnPP pretreatment proved to be protective against development of ulcerative lesions in CRS model as evidenced by reduction of the ulcer index, and this could be mediated through reduction of free and total acidity of gastric secretion and decreased lipid peroxidation but with significantly decreased gastric protective nitric oxide and prostaglandin E(2) levels. In conclusion and according to our results, the protective effect of ZnPP on CRS-induced gastric ulcers despite of inhibition of endogenous CO could be attributed to the presence of zinc which is known to have a protective anti-ulcer effect.     

内源性一氧化碳减轻大鼠双侧后肢缺血再灌注所致的肺损伤

通过观察血红素氧化酶（HO）阻断剂－－锌原卟啉（ZnPP)对肺组织、肺泡间质多形核白细胞数目肺组织丙二醛含量和湿重干重之比的影响,并对肺组织HO活性和血内碳氧血红蛋白水平（COHb）进行检测,以探讨内源性HO／一氧化碳（CO）在肢体缺血再灌注（I／R）所致肺损伤中的作用。结果发现,大鼠双侧后肢I／R可导致急性肺损伤,同时使肺组织中HO活性和血内COHb水平显著升高;应用ZnPP预处理可使HO活性和COHb水平显著降低,但肺损伤却进一步加重。上述实验结果表明,肢体I／R致肺损伤时,肺组织中HO活性和内源性CO生成增多或减轻大鼠肢体I／R所致的肺损伤。     

Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait.

BACKGROUND: Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. MATERIALS AND METHODS: Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. RESULTS: The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. CONCLUSIONS: The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.     

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

Zinc Protoporphyrin Regulates Cyclin D1 Expression Independent of Heme Oxygenase Inhibition

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis.     

Purification and properties of heme-deficient hepatic soluble guanylate cyclase: effects of heme and other factors on enzyme activation by NO, NO-heme, and protoporphyrin IX

Purified hepatic soluble guanylate cyclase (EC 4.6.1.2) had maximal specific activities in the unactivated state of 0.4 and 1 μmol cyclic GMP min^?1 mg protein^?1, when MgGTP and MnGTP, respectively, were used as substrates. The apparent K_m for GTP was 85 or 10 μm in the presence of excess Mg²⁺ or Mn²⁺, respectively. Guanylate cyclase purified as described was deficient in heme but could be readily reconstituted with heme by reacting enzyme with hematin and excess dithiothreitol at 4 °C and pH 7.8. Unpurified guanylate cyclase was activated 20- to 84-fold by NO, nitroso compounds, NO-heme, and protoporphyrin IX. The purified enzyme was only slightly (2- to 3-fold) activated by NO and nitroso compounds but was markedly (50-fold) activated by NO-heme and protoporphyrin IX, achieving maximal specific activities of 10 μmol cyclic GMP min^?1 mg protein^?1. Enzyme activation by NO and nitroso compounds was restored by addition of hematin or by reconstitution of guanylate cyclase with heme. Excess hematin, however, inhibited enzyme activity. A partially purified heat-stable factor (activation-enhancing factor) was found to enhance (2- to 35-fold) enzyme activation without directly stimulating guanylate cyclase. In the presence of optimal concentrations of hematin, enzyme activation was still increased (2-fold) by the activation-enhancing factor but not by bovine serum albumin. Guanylate cyclase was markedly inhibited by SH reactive agents such as cystine, o-iodosobenzoic acid, periodate, and 5,5′-dithiobis (2-nitrobenzoic acid). In addition, CN^? and FMN inhibited enzyme activation by NO-heme, but not by protoporphyrin IX, and did not affect basal enzymatic activity. Hepatic soluble guanylate cyclase appears to possess SH groups required for catalysis and to require heme and/or other unknown factors for the full expression of enzyme activation by NO and nitroso compounds.     

Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron‐restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual‐wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual‐wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (r_s = 0.943, p < 0.0001; range 37–689 μmol ZnPP/mol heme, 84–1238 nmol/L) and for PPIX (r_s = 0.959, p < 0.0001; range 42–4212 μmol PPIX/mol heme, 93–5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single‐wavelength excitation and with commercial front‐face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual‐wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Characterization of the heme of cytochrome P-450 using gas chromatography-mass spectrometry

The heme moieties of cytochromes P-450 and P₁-450 (P-448) have been characterized. CO-binding particles, devoid of cytochrome b₅, were prepared from normal or 3-methylcholanthrene-treated animals. Heme was removed by acid-acetone treatment of the CO-binding particles and crystallized. Heme isolated from hemoglobin of the corresponding animals served as a control. Reductive degradation (hydriodic acid) followed by gas chromatography/mass spectrometry analysis indicated the presence of opso-, crypto-, hemo-, and phyllopyrrole. Visible spectra of the iron-free tetrapyrroles isolated from microsomal heme and hemoglobin were identical and showed typical aetioporphyrin spectra. Finally, the mass spectra of the tetrapyrrole dimethyl esters isolated from microsomal heme and hemoglobin were identical to authentic protoporphyrin IX dimethyl ester. These data strongly suggest that the heme of cytochrome P-450 and P₁-450 are identical and are the same the same as that of hemoglobin, namely protoporphyrin IX.     

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO-), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (DeltaPsim), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2) became tolerant to the depolarization of DeltaPsim and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.     

Endothelin-1 release from the lamb ductus arteriosus: are carbon monoxide and nitric oxide regulatory agents?

We have proposed that endothelin-1 (ET-1), formed through the activation of a cytochrome P450 (CYP450)-based monooxygenase reaction, is important for generation of contractile tone in the ductus arteriosus and, consequently, for closure of the vessel at birth. The present investigation was undertaken to ascertain, using an isolated ductus preparation from near-term fetal lambs, whether carbon monoxide (CO) and nitric oxide (NO) qualify as regulators of the CYP450/ET-1 system. Preparations released ET-1 at rest and its amount showed no significant reduction following removal of the endothelium. Basal release was not changed by the NO synthesis inhibitor, N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), nor by agents altering cyclic GMP content (i.e. increase; ONO-1505, 1 microM) and action (i.e. decrease; LY-83583, 10 microM). These findings extend previous work showing no effect of the CO synthesis inhibitor zinc protoporphyrin IX (ZnPP, 10 microM) under the same conditions (10). Conversely, both CO (65 microM) and the NO donor, sodium nitroprusside (SNP, 10 microM), curtailed ET-1 release. ET-1 release was increased by oxygen and reduced by pyrogens (endotoxin and IL-1, both at 100 ng mL(-1)). The endotoxin effect tended to be reversed by L-NAME and ZnPP, used singly or in combination. We conclude that ET-1 is formed naturally in the ductus and that its formation may change in response to physiological (oxygen) and pathophysiological (pyrogens) stimuli. Endogenous CO and NO, however, appear to have little or no role as ET-1 regulators.     

Expression of Heme Oxygenase Isozyme mRNAs in the Human Brain and Induction of Heme Oxygenase-1 by Nitric Oxide Donors

Abstract: Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase-1 and heme oxygenase-2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors—sodium nitroprusside, 3-morpholinosydnonimine, and S -nitroso- l -glutathione—caused a significant increase in the levels of heme oxygenase-1 mRNA but not in the levels of heme oxygenase-2 and heat-shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase-1 protein but not the levels of heat-shock protein 70 in T98G cells. The increase in content of heme oxygenase-1 mRNA caused by sodium nitroprusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8-bromo cyclic GMP, sodium nitrite, or various neuropeptides, such as calcitonin gene-related peptide, endothelin-1, and corticotropin-releasing hormone. The present study has shown the expression profiles of heme oxygenase-1 and -2 mRNAs in the human brain and the induction of heme oxygenase-1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.