Zinc protoporphyrin-IX Research Articles

Hemin, a heme oxygenase-1 inducer, improves aortic endothelial dysfunction in insulin resistant rats

Under an insulin resistance (IR) state, overproduction of reactive oxygen species (ROS) may be playing a major role in the pathogenesis of endothelial dysfunction, hypertension and atherosclerosis. Recently, increasing attention has been drawn to the beneficial effects of heme oxygenase-1 (HO-1) in the cardiovascular system. This study aimed to investigate the effects of HO-1 on vascular function of thoracic aorta in IR rats and demonstrate the probable mechanisms of HO-1 against endothelial dysfunction in IR states. Sprague-Dawley (SD) rats fed with high-fat diet for 6 weeks and the IR models were validated with hyperinsulinemic-euglycemic clamp test. Then the IR rat models (n = 44) were further randomized into 3 subgroups, namely, the IR control group (n = 26, in which 12 were sacrificed immediately and evaluated for all study measures), a hemin treated IR group (n = 10) and a zinc protoporphyrin-IX (ZnPP-IX) treated IR group (n = 8) that were fed with a high-fat diet. Rats with standardized chow diet were used as the normal control group (n = 12). The rats in IR control group, hemin treated IR group and ZnPP-IX treated IR group were subsequently treated every other day with an intraperitoneal injection of normal saline, hemin (inducer of HO-1, 30 micromol/kg) or ZnPP-IX (inhibitor of HO-1, 10 micromol/kg) for 4 weeks. Rats in the normal control group remained on a standardized chow diet and were treated with intraperitoneal injections of normal saline every other day for 4 weeks. Systolic arterial blood pressure (SABP) was measured by tail-cuffed microphotoelectric plethysmography. The blood carbon monoxide (CO) was measured by blood gas analysis. The levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), blood glucose (BG), insulin, total cholesterol (TC) and triglyceride (TG) in serum, and the levels of total antioxidant capacity (TAOC), malondialdehyde (MDA) and superoxide dismutase (SOD) in the aorta were measured. The expression of HO-1 mRNA and HO-1 protein in aortal tissue were detected by semi-quantitative RT-PCR and Western blot. The vasoreactive tensometry was performed with thoracic aortic rings (TARs). Compared with the normal control group, the levels of SABP, BG, insulin, TC, TG, NO, iNOS and MDA were higher, while the levels of CO, TAOC, SOD and eNOS were lower in IR control rats. After treatment of IR rats for 4 weeks a more intensive expression of HO-1 mRNA and HO-1 protein were observed in hemin treated IR group compared with the normal control group. And compared with 4-week IR control rats, the levels of CO, TAOC, SOD and eNOS were increased, while the levels of SABP and iNOS activity were lower in the hemin treated IR group. Administration of hemin in IR rats appeared to improve the disordered vasorelaxation of TARs to acetylcholine (ACh). Alternatively, the reverse results of SABP, CO, TAOC, SOD, iNOS and vasorelaxation responses to ACh were observed in IR rats with administration of ZnPP-IX. The endothelial dysfunction in the aorta is present in the IR state. The protective effects of HO-1 against aortic endothelial dysfunction may be due to its antioxidation and regulative effect of vasoactive substances. It is proposed that hemin, inducer of HO-1, could be a potential therapeutic option for vascular dysfunction in IR states.

YS 49, 1-(α-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1

Overexpression of the gene for heme oxygenase (HO)-1 leads to a reduction in pressor responsiveness to angiotensin II (Ang II) in experimental animals. Using rat vascular smooth muscle cells (VSMCs), we tested whether YS 49 [1-(α-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] inhibits Ang II-stimulated proliferation of VSMCs via induction of HO-1. YS 49 induced HO-1 protein production in a dose-and time-dependent manner in VSMCs. Treatment with YS 49 significantly and dose-dependently inhibited Ang II-induced VSMC proliferation, ROS production, and phosphorylation of JNK, but not P38 MAP kinase or ERK1/2. The antiproliferation effect of YS 49 was reversed by pretreatment with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), or with hemoglobin, a carbon monoxide (CO) scavenger. Similarly, VSMC proliferation, ROS production and phosphorylation of JNK by Ang II were significantly inhibited in VSMCs transfected with the HO-1 gene. Thus, HO-1 and the HO-1 product CO play, at least in part, a crucial role in Ang II-stimulated VSMC proliferation through the regulation of ROS production and JNK phosphorylation. Therefore, YS 49 has potential as a therapeutic strategy for the pathogenesis of Ang II-related vascular diseases such as hypertension and atherosclerosis, via the induction of HO-1 gene activity.

Carbon monoxide‐induced stomatal closure in Vicia faba is dependent on nitric oxide synthesis

Recently, in animals, carbon monoxide (CO), like nitric oxide (NO), was implicated as another important physiological messenger or bioactive molecule. Previous researches indicate that heme oxygenase (HO)-1 (EC 1.14.99.3) catalyzes the oxidative conversion of heme to CO and biliverdin IXa (BV) with the concomitant release of iron. However, little is known about the physiological roles of CO in plant, especially in stomatal movement of guard cells. In the present paper, the regulatory role of CO during stomatal movement in Vicia faba was surveyed. Results indicated that, like sodium nitroprusside (SNP), CO donor hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proved by the addition of gaseous CO aqueous solution with different concentrations, showing for the first time that CO and NO exhibit similar regulation role in the stomatal movement. Moreover, our data showed that 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)/N(G)-nitro-L-arginine-methyl ester (L-NAME) not only reversed stomatal closure by CO, but also suppressed the NO fluorescence induced by CO, implying that CO-induced stomatal closure probably involves NO/nitric oxide synthase (NOS) signal system. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX), NO scavenger cPTIO and NOS inhibitor L-NAME reversed the darkness-induced stomatal closure and NO fluorescence. These results show that, maybe like NO, the levels of CO in guard cells of V. faba is higher in dark than that in light, HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness, respectively, and that CO being from HO-1 mediates darkness-induced NO synthesis in guard cells' stomatal closure of V. faba.

Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1

Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200 mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression.

Hemin, an Inducer of Heme Oxygenase-1, Lowers Intraocular Pressure in Rabbits

Carbon monoxide (CO) generated from heme may induce vasodilation and exert cyto-protective properties in the eye. This study was undertaken to investigate the effects of hemin, a potent inducer of heme oxygenase-1 (HO-1), on models of ocular hypertension in rabbits. Ocular hypertension was induced by injecting alpha-chymotrypsin in both eyes under local anesthesia. Only rabbits with an intraocular pressure (IOP) of 25 mmHg or more were used. The dose-response study of the hemin effect on IOP was made by an intravenous injection of the drug (50, 75, and 100 mg/kg) and subsequent IOP monitoring every 6 h. A separate set of animals was pretreated with the HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX, 0.1 mg/kg) 6 h before the vehicle or a 100-mg/kg hemin injection. Ocular hypertension was also obtained by the subconjunctival injection of betamethasone 21-phosphate disodium (4 mg/mL) in both eyes every week for 4 weeks. Only animals with an IOP of 30 mmHg or more were included in the experimental session. A group of these animals was pretreated with ZnPP-IX (0.1 mg/kg) 6 h before the vehicle or a 100-mg/kg hemin injection, and IOP was assessed every 6 hours. Hemin caused a significant dose-related reduction of IOP in rabbits with alpha-chymotrypsin-induced ocular hypertension. No significant effect was observed in the normotensive eyes of the control animals or on pupil diameter. Pretreatment with the HO-1 inhibitor, ZnPP-IX, abolished the decrease of IOP that was induced by the maximum dose of hemin (100 mg/kg). A similar reduction in IOP was observed in those rabbits with betamethasone-induced ocular hypertension who were treated with 100 mg/kg of hemin. Furthermore, pretreatment with ZnPP-IX prevented the hemin effect on IOP. The induction of HO-1 by hemin leads to a reduction of IOP in the alpha-chymotrypsin and betamethasone models of ocular hypertension. These results suggest an involvement of CO in the regulation of ocular pressure in rabbits.

Induction of growth elongation in wheat root segments by heme molecules: a regulatory role of carbon monoxide in plants?

Recent studies suggest that carbon monoxide (CO), which is mainly produced by heme oxygenase (HO EC 1.14.99.3), may function as a physiological messenger or bioactive molecule by interacting with nitric oxide (NO) in animal cells. In this study, we report that application of the hematin and hemin, two heme molecules cleaved by HO to yield CO in animals, dose-dependently induced the significant increase in wheat root elongation as well as the actions of IAA and NO donor sodium nitroprusside (SNP). These responses were mimicked by the application of aqueous solution of CO with different saturation. Also, above heme molecule-induced effect is specific for CO since the potent inhibitor of HO-1, zinc protoporphyrin-IX (ZnPPIX) or CO scavenger hemoglobin (Hb) blocked the action of hematin and hemin, respectively. Further results proved that treatment with hematin or IAA could result in either the potent induction of HO-1 transcript or CO releasing in wheat root segments, both of which were reversed by the addition of ZnPPIX. ZnPPIX with lower concentration could prevent the elongation induced by IAA, while in the SNP-treatment the prevention of root growth occurred solely at higher concentrations. Also, wheat root segments elongation induced by IAA, SNP or hematin, was blocked by the specific NO scavenger, inhibitors of NO synthase (NOS) and guanylate cyclase (GC), respectively. Meanwhile, production of reactive oxygen species (ROS) could be demonstrated in the growing zone of wheat root segments treated by hematin or SNP using specific histochemical assay combined with the inhibitor investigation. Taken together, above results suggested that CO produced by HO might mediate the induction of growth elongation of wheat root segments by IAA, which might be also related to NO/cGMP- and even ROS-dependent pathways.

Expression and Protective Role of Heme Oxygenase‐1 in Delayed Myocardial Preconditioning

In the study the authors aimed to demonstrate the expression and protective effect of heme oxygenase-1 (HO-1) in the delayed preconditioning (PC) on cultured myocardiac cells. Neonatal rat cardiac myocytes were exposed to ischemic (ischemic medium [IM] for 20 min) and pharmacological (adenosine, epinephrine, opioid) PC. Twenty-four hours later cells were subjected to a simulated ischemia (SI)--culturing for 3 h in IM, followed by 2-h reperfusion in normal medium--and then lactate dehydrogenase (LDH), live/death ratio, and apoptosis were measured. For demonstrating the protective role of HO-1, its enzymatic activity was competitively inhibited by administration of zinc protoporphyrin IX (ZnPPIX), and HO-1 synthesis was blocked with HO-1 siRNA. Cells in control group were cultured under normoxic conditions. In SI group, cells underwent only an SI without PC. HO-1 expression in all of the groups was demonstrated with immunostaining. Our results showed a significant decrease of LDH release, apoptosis, and cell death in PC groups versus SI group, which has been risen in ZnPPIX- and HO-1 siRNA-treated groups. HO-1 immunostaining showed an appreciable HO-1 expression in PC groups, which was abolished with HO-1 siRNA administration, but not in ZnPPIX group. The results therefore suggest that HO-1 expression increases in both ischemic and pharmacological PC, and HO-1 has cellular protective effect against cell death and apoptosis in ischemia-reperfusion-induced oxidative injury.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea.

Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

Nitric oxide modulates hypoxic pulmonary smooth muscle cell proliferation and apoptosis by regulating carbon monoxide pathway

To explore the role of carbon monoxide (CO) in the regulation of hypoxic pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis by nitric oxide (NO). PASMC of Wistar rats was cultured in vitro in the presence of a NO donor, sodium nitroprusside, or an inhibitor of heme oxygenase (HO), zinc protoporphyrin-IX, or under both normoxic and hypoxic conditions. Nitrite and carboxyhemoglobin in PASMC medium were detected with spectrophotometry. The proliferating and apoptotic percentage of PASMC was measured by flow cytometry. The expression of HO-1 mRNA in PASMC was analyzed by fluorescent real-time quantitative PCR, and the proliferating cell nuclear antigen and caspase-3 were examined by immunocytochemical analysis. The results showed that hypoxia suppressed NO generation from PASMC, which promoted hypoxic PASMC proliferation and induced apoptosis. Meanwhile, hypoxia induced HO-1 expression in PASMC and promoted CO production from PASMC, which inhibited PASMC proliferation and regulated PASMC apoptosis. NO upregulated the expression of HO-1 mRNA in hypoxic PASMC; NO also inhibited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regulating the production of CO. The results indicated that CO could inhibit proliferation and regulate apoptosis of PASMC, and NO inhibited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regulating the production of CO.

Carbon monoxide counteracts the inhibition of seed germination and alleviates oxidative damage caused by salt stress in Oryza sativa

Although carbon monoxide (CO) has always been regarded as a toxic gas, recent reports suggested that it is one of the products of heme oxygenase (HO; EC 1.14.99.3) catalysis in animals, and could confer beneficial cytoprotection against oxidative damage. Here, we investigated the effects of exogenous putative CO donor hematin and CO aqueous solution on rice seed germination under 100 mM NaCl salt stress. Both hematin and CO aqueous solution dose-dependently attenuated the inhibition of seed germination and seedling growth caused by salinity. Moreover, 1.0 μM hematin and 5% CO-saturated aqueous solution activated amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar. Furthermore, hematin and CO aqueous solution induced catalase (CAT) and superoxide dismutase (SOD) activities, thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Such above CO-induced effects were also confirmed by using semi-quantitative RT-PCR, including the up-regulation of expression patterns of α-amylase, CAT and Cu/ Zn-SOD genes. Also, the similar function of hematin might contribute to endogenous HO-derived CO, since addition of the CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX) and CO scavenger hemoglobin (Hb) reversed above effects. Meanwhile, further results also proved that treatment with hematin for 36 h could result in the potent induction of HO-1 transcript, HO activity and CO content in the germinating seeds. Collectively, these results indicated that CO performed an advantageous effect on attenuation of inhibition of seed germination and seedling growth induced by salt stress, and alleviated oxidative damage via activating anti-oxidant enzymes.

Ondamtanggamibang protects neurons from oxidative stress with induction of heme oxygenase-1

Ondamtanggamibang (ODG) has been used as a remedy to treat psychological anxiety and depression in Oriental medicine. In this study, we found the protective effects of ODG against oxidative stress by induction of the antioxidative enzyme, heme oxygenase (HO)-1 in neuronal PC12 cells. Pretreatment with ODG extract protected neuronal cells from damage induced by H 2O 2 and 6-hydroxydopamine (6-OHDA), but simultaneous treatment with ODG extract did not. ODG also inhibited the intracellular reactive oxygen species elevation by H 2O 2 and 6-OHDA. ODG stimulation strongly induced the expression of HO-1 in PC12 cells. The protective effect of ODG extract on oxidative stress-induced damage was suppressed by HO inhibitor, zinc protoporphyrin IX (ZnPP-IX). Taken together, these data suggest that ODG treatment has potential protective effects in neuronal cells under oxidative stress.

Effect of Plasmodium falciparum Parasitemia on Erythrocyte Zinc Protoporphyrin

In regions holoendemic for malaria, both iron deficiency and asymptomatic malaria parasitemia are common (1). Measurements of transferrin receptor and erythrocyte zinc protoporphyrin IX (ZnPPIX) concentrations have been suggested as screening tools to detect iron deficiency (1)(2). Chronic iron-deficient erythropoiesis leads to a relative increase in the insertion of zinc rather than iron into erythrocyte PPIX. Anemia of chronic disease and hyperbilirubinemia can decrease the specificity of an iron deficiency diagnosis based on hematofluorometry-determined increases in the ratio of ZnPPIX to heme. The influence of malaria parasitemia on front-face hematofluorometric ZnPPIX determinations is controversial. Increased ZnPPIX has been statistically associated with malaria, and interference by increased bilirubin from hemolysis has been postulated (3). In a study by Asobayire et al.(4), malaria did not increase ZnPPIX in washed blood, but in that study only 5%–8% of malaria-positive persons had >5000 …

Induction of Heme Oxygenase-1 Is Involved in Carbon Monoxide-Mediated Central Cardiovascular Regulation

Carbon monoxide (CO) has been identified as an endogenous biological messenger in the brain. Heme oxygenase (HO) catalyzes the metabolism of heme to CO and biliverdin. Previously, we have shown the involvement of CO in central cardiovascular regulation, baroreflex modulation, and glutaminergic neuro-transmission in the nucleus tractus solitarii (NTS) of rats. In this study, we examined which HO isoform could be induced after hemin injection in the NTS. We also investigated their in situ distributions in the NTS after induction. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure was monitored intra-arterially. Unilateral microinjection of hemin (1 nmol), a heme molecule cleaved by HO to yield CO, produced significant decrease in blood pressure and heart rate. These cardiovascular effects of hemin were attenuated by prior administration of HO inhibitor zinc protoporphyrin IX (ZnPPIX). Microinjection of hemin into NTS resulted in significant induction of HO-1 protein expression in situ. Pretreatment of ZnPPIX significantly inhibited the HO-1 induction after hemin injection. No significant changes of HO-2 expression were found after hemin injection and ZnPPIX pretreatment. The in situ inductions of the HO-1 protein expression were further confirmed to be in glial cells and neurons after hemin injections into the NTS. These results indicated HO-1 but not HO-2 might be responsible for the generation of CO and contribute to central control of cardiovascular effects.

Protective effects of Korean herbal remedy against oxidative stress in cardiomyocytes

Ondamtanggagambang (ODG) has been used as a prescription for psychological anxiety and depression in Korean medicine. In this study, we found that ODG have protective effects against oxidative stress in cardiomyocytes. Pretreatment with ODG extract prevented H2O2 and ZnCl2-induced cell damage in H9c2 cardiomyocytes, whereas simultaneous treatment of ODG extract did not. The protective effect of ODG extract on oxidative stress-induced damage was suppressed significantly by heme oxygenase (HO) inhibitors, zinc protoporphyrin-IX (ZnPP-IX, p < 0.01) and tin protoporphyrin-IX (SnPP-IX, p < 0.01) in H9c2 cells. ODG stimulation of cells strongly induced the expression of HO-1 protein. Taken together, it is suggested that ODG-induced expression of HO-1 may have a beneficial role in cardiomyocytes under oxidative stress.

Heme oxygenase‐1 is essential for and promotes tolerance to transplanted organs

This investigation focused on obtaining a further understanding of the role of heme oxygenase-1 (HO-1) in tolerance induction. Hearts from C57BL/6 (H-2b) mice survived long-term when transplanted into BALB/c (H-2d) recipients treated with the tolerance-inducing regimen of anti-CD40L antibody (MR-1) plus donor-specific transfusion (DST). Grafts did not, however, survive long-term in (HO-1-/-) recipients given the same treatment. Similarly, long-term survival induced by DST was ablated when HO-1 activity was blocked by zinc protoporphyrin IX (ZnPPIX). We further asked whether modulation of HO-1 expression/activity could be used to promote the induction of graft tolerance. DST alone (day 0) failed to promote any prolongation of survival of DBA/2 (H-2d) hearts transplanted into B6AF1 (H-2(b,k/d)) recipients. However, long-term survival and (dominant peripheral) tolerance were readily induced when DST was combined with induction of HO-1 expression by cobalt protoporphyrin IX (CoPPIX). HO-1 induction plus DST led to a significant up-regulation of Foxp3, TGF-beta, IL-10, and CTLA4, which suggests a prominent role for CD4+CD25+ regulatory T cells (Tregs). In fact, the tolerogenic effect of HO-1 plus DST was dependent on CD4+CD25+ Tregs as suggested by adoptively transferring these cells into irradiated recipients under various regimens. Taken together, these findings show that expression of HO-1 in a graft recipient can be essential for long-term graft survival and for induction of tolerance and that modulation of HO-1 expression/activity can be used therapeutically to synergize in the generation of graft tolerance.

Metalloporphyrins inactivate caspase‐3 and ‐8

Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc(II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1.

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3-5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). nN^{omega } n-Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.

Hyperglycemia in Streptozotocin-Induced Diabetic Rat Increases Infarct Size Associated With Low Levels of Myocardial HO-1 During Ischemia/Reperfusion

This study investigated the role of heme oxygenase (HO)-1 in the cardiac tissue injury of acute ischemia/reperfusion (I/R) in diabetic streptozotocin (STZ)-induced hyperglycemic rats. The effects of 1) hemin, an inducer of HO expression and activity, and 2) zinc protoporphyrin IX (ZnPP-IX), an inhibitor of HO activity, have also been investigated on the tissue injury by I/R and some mediators released in these circumstances. STZ hyperglycemic rats had impaired levels of HO-1 within the cardiac tissue and increased myocardial infarct size (IS) following I/R, as compared with the nondiabetic rats. In these rats, administration of hemin 4 mg/kg 18 h before I/R increases the levels of HO-1 within the tissue. However, the values of HO-1 assayed in these circumstances were significantly lower (P < 0.01) than those assayed in nondiabetic animals subjected to the same procedures; IS was much more extended (P < 0.01) than in the parent nondiabetic group. STZ hyperglycemic rats also predisposed the heart to produce high levels of the cytokines interleukin (IL)-1beta and CXCL8. Subsequent I/R further increased (P < 0.01) the cytokine production, an effect partly prevented by hemin treatment. This recovered the huge number of infiltrated polymorphonuclear (PMN) leukocytes within the cardiac tissue associated with the STZ hyperglycemic state and I/R damage.

The role and interactions of nitric oxide (NO), carbon monoxide (CO), and prostanoids in the pathogenesis of postoperative ileus in rats.

The effects of heme oxygenase (HO) inhibitors, zinc-protoporphyrin-IX (ZnPP-IX), and tin protoporphyrin-IX (SnPP-IX) and their interactions with L-arginine/nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways were investigated in postoperative ileus in rats. Intestinal transit was measured as Evans blue migration after skin incision, laparotomy or laparotomy plus gut evisceration and handling. Laparotomy and small intestinal manipulations increased blood plasma nitrites/nitrates level 1.88-fold. N(omega)-nitro-L-arginine methyl ester, indomethacin, a selective COX-1 blocker (resveratrol) and COX-2 antagonists (nimesulide, DuP-697, NS-398) reversed the additional inhibitory effects of gut manipulation subsequent to laparotomy. In contrast, N-(3-(aminomethyl)benzyl)acetamidine or S-methylisothiourea, highly selective inducible NOS blockers, remained ineffective. ZnPP-IX and SnPP-IX overturned the effects of laparotomy on dye propulsion, but were only partially effective after laparotomy and gut handling attenuating the additional inhibitory influences of gut manipulation, the intestinal transit reaching 89.21%, 92.87%, 53.46%, and 48.56% of respective controls transit. Salutary effects of L-NAME, ZnPP-IX, and SnPP-IX were dose-dependent, L-arginine or hemin (HO substrate) sensitive. Administration of indomethacin and resveratrol subsequent to SnPP-IX reversed the inhibitory effects of laparotomy and manipulation, amounting to 93.91% and 87.43% of controls. On the other hand, L-NAME injected after SnPP-IX abolished the salutary effects of the latter, study dye migration reached 25.18% of control rat. Therefore we demonstrated that nitric oxide, carbon monoxide, and prostanoids play a role in the pathogenesis of postoperative ileus albeit in different mechanisms. Laparotomy stimulated HO activity, whereas gut manipulation led to an excessive constitutive NOS stimulation accompanied by augmented prostanoid synthesis by COX-1. Unaffected synthesis of either NO or CO enables a return of gastrointestinal transit during postoperative period, whereas a pharmacological blockade of two complementary metabolic pathways provides a most effective measure against postoperative ileus development.

Effects of endogenous carbon monoxide on collagen synthesis in pulmonary artery in rats under hypoxia

To study the role of endogenous carbon monoxide (CO) in collagen metabolism during hypoxic pulmonary vascular remodeling, a total of 18 Wistar rats were used in the study and they were randomly divided into three groups: hypoxia group (n = 6), hypoxia with zinc protoporphyrin-IX (ZnPP-IX) group (n = 6) and control group (n = 6). The measurement of mean pulmonary artery pressure (mPAP) and carboxyhemoglobin (HbCO) formation in lung tissue homogenates was measured. A morphometric analysis of pulmonary vessels was performed, in which the percentage of muscularized arteries (MA); partially muscularized arteries (PMA) and nonmuscularized arteries (NMV) in small and median pulmonary vessels, relative medial thickness (RMT) and relative medial area (RMA) of pulmonary arteries were analyzed. Collagen type I and III and transforming growth factor-β 3 (TGF-β 3) expressions were detected by immunohistochemical assay. The expressions of procollagen type I and III and TGF-β 3 mRNA were detected by in situ hybridization. The results showed that ZnPP-IX significantly increased mPAP and markedly decreased HbCO formation in lung tissue homogenates in rats under hypoxia ( P<0.01). In the hypoxia rats treated with ZnPP-IX, the percentage of muscularized arteries of small and median pulmonary vessels was obviously increased, and RMT and RMA of intra-acinar muscularized pulmonary arteries were markedly increased compared with hypoxic rats. Ultrastructural changes, such as hyperplasia and hypertrophy of endothelial cells (ECs) and smooth muscle cells (SMCs) and the increased number of SMCs in synthetic phenotype were found in intra-acinar pulmonary muscularized arteries of hypoxic rats treated with ZnPP-IX. Meanwhile, ZnPP-IX promoted the expression of collagen type I and III and TGF-β 3 protein in pulmonary arteries of rats under hypoxia ( P<0.01). Furthermore, ZnPP-IX elevated obviously the expressions of procollagen type I and III mRNA, and TGF-β 3 mRNA in pulmonary arteries of rats under hypoxia ( P<0.01). The results of this study suggested that ZnPP-IX played an important role in promoting collagen synthesis in pulmonary arteries of rats with hypoxic pulmonary structural remodeling by increasing the expression of TGF-β 3. The above findings also suggested a possible role of endogenous CO in the pathogenesis of chronic hypoxic pulmonary hypertension.