Zinc protoporphyrin-IX Research Articles

The role of heme oxygenase-1 in non-alcoholic steatohepatitis

To investigate the potential role of heme oxygenase-1 on preventing non-alcoholic steatohepatitis (NASH) in mice. Experimental models of NASH were established by feeding male C57BL/6J mice with choline-methionine deficient diet (MCD) for four weeks. Control animals were fed with choline-methionine supplemented diet. The treatment groups were fed with MCD diet combined with HO-1 inducer hemin or inhibitor zinc protoporphyrin IX (ZnPP-IX). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by enzymic method with automatic biochemistry analyzer. The degree of hepatic steatosis, inflammation and fibrosis were examined under HE staining. The hepatic mRNA and protein expressions of HO-1, TNFalpha and IL-6 were analyzed by RT-PCR and Western blot respectively. MCD fed mice showed increased serum ALT and AST levels and moderate to severe hepatic steatosis with inflammatory infiltration, hepatic spot or focal necrosis, light portal and sinus hepaticus fibrosis in the liver sections, which associated with enhanced expression of HO-1, TNFalpha and IL-6 mRNA and protein (1.13+/-0.11, 1.74+/-0.05; 0.20+/-0.01, 1.92+/-0.10; 0.58+/-0.02, 2.06+/-0.05 vs 0.43+/-0.02, 0.75+/-0.05; 0.08+/-0.00, 0.59+/-0.02; 0.22+/-0.01, 0.91+/-0.02). Administration of hemin significantly decreased serum ALT and AST levels and attenuated hepatic steatosis and necroinflammation which associated with up-regulation of antioxidative gene HO-1 and down-regulation of pro-inflammatory cytokines TNFalpha and IL-6 (P < 0.01). A contrary effect on serum aminotransferase levels and liver histopathology was observed in mice injected with ZnPP-IX (P < 0.01). The effect was associated with suppressed HO-1 expression and increased TNFaLPHA and IL-6 expression. The data provided a biochemical, morphological and molecular biological evidence for the protective role of HO-1 in ameliorating hepatic steatosis, necroinflammation in experimental nutritional steatohepatitis.

Haem oxygenase delays programmed cell death in wheat aleurone layers by modulation of hydrogen peroxide metabolism.

Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H2O2) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H2O2 either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H2O2 production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H2O2 production.

Enhanced Hemeoxygenase‐1 Signaling in the Rostral Ventrolateral Medulla Mediates a Critical Protective Role Against Genetically Elevated Blood Pressure in SHR Rats

Heme oxygenase (HO)‐derived carbon monoxide (CO) contributes to central regulation of blood pressure (BP). However, the few reported studies demonstrating reductions in BP following microinjection into the nucleus tractus solitarius of the HO substrate hemin, were carried out in anesthetized normotensive rats. We hypothesized that HO‐CO signaling is enhanced in the RVLM of conscious SHRs to prevent exacerbation of hypertension in this genetic model of hypertension. To test this hypothesis, we investigated the effects of intra‐RVLM hemin on BP, HR and RVLM neuronal norepinephrine (index of sympathetic activity; measured in real‐time by in vivo electrochemistry) in SHRs and their normotensive control WKY rats. Furthermore, the type of the specific HO isoenzyme implicated in the hypotensive response was investigated by administering the selective HO‐1 inhibitor zinc protoporphyrin IX (ZNPPIX) 30 minutes prior to hemin. Intra‐RVLM hemin elicited greater and longer‐lasting reductions in BP and RVLM NE in SHRs. These hemodynamic and electrochemical response, which suggest enhanced HO‐CO signaling in the RVLM of SHRs, were confirmed by the greater enzymatic activity of HO in SHRs than in WKY rats, Moreover, ZNPPIX, not only abolished the hemin‐evoked responses in either strain, albeit to a greater extent in SHRs, but also caused a marked increase in baseline pressure in SHRs. The findings suggest an exaggerated HO‐CO signaling, due to higher HO‐1 activity, in the RVLM of SHR, a brainstem area that controls sympathetic outflow. Such enhancement of HO‐CO signaling seems to serve as a protective mechanism that opposes further increases in BP in the SHR.

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells

Heme oxygenase-1 (HO-1) catalyses the rate-limiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP(+)) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP(+) and whether HO-1 has a protective role against the MPP(+)-induced cytotoxicity in PC-12 cells. MPP(+) triggered a relatively rapid induction of HO-1. The MPP(+)-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP(+)-induced cytotoxicity.

Morphine-Induced Ocular Hypotension Is Modulated by Nitric Oxide and Carbon Monoxide: Role of μ3Receptors

Recent findings generated from our laboratory have demonstrated the involvement of nitric oxide (NO) in morphine-induced reduction of intraocular pressure (IOP). The present study was designed to investigate the possible involvement of carbon monoxide (CO) in morphine-induced reduction of IOP and the role of mu(3) opioid receptors. New Zealand rabbits were used in this study. They were pretreated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 1%, 30 microL), or an inhibitor of heme oxygenase (HO), zinc protoporphyrin-IX (ZnPP-IX; 0.1 mg/kg; i.v.). The same animals were then treated with morphine (100 microg/30 microL) with or without NO or CO donors administration, sodium nitroprusside (SNP) and tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), respectively. A separate set of animals were pretreated with the nonselective opioid receptor antagonist, naloxone (100 microg/30 microL), or the micro(3) opioid receptor inhibitor, L-glutathione (GSH, 1%, 30 microL), in the presence of SNP or CORM-3 followed by morphine administration. IOP measurements were taken at different times after monolateral instillation of morphine. Morphine induced a significant decrease in IOP and pretreatment with ZnPP-IX or L-NAME significantly prevented this effect whereas administration of NO or CO donors amplified morphine-induced decrease in IOP. This effect was partially abrogated both by pretreatment with ZnPP-IX or L-NAME, and by pretreatment with naloxone and GSH suggesting that the decrease in IOP relies on exogenous NO and CO liberated from SNP and CORM-3, respectively. We conclude that the endogenous NO/CO system and micro(3) receptors contribute to morphine-induced ocular hypotension and that the reduction of IOP elicited by morphine can be augmented by exogenous NO and CO.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

ObjectiveHeme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice.MethodsC57BL/6J mice were fed with methionine-choline deficient (MCD) diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin), HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX) and/or adenovirus carrying HO-1 gene (Ad-HO-1) were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot.ResultsHepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1), inhibited cytochrome c (Cyt-c) release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes.ConclusionsThe present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

PurposeCarbon monoxide (CO) may mediate smooth muscle relaxation in the rat corpus cavernosum smooth muscle (CCSM). We hypothesized that CO plays a role in neurally derived, frequency-dependent relaxation of rat CCSM.Materials and MethodsTo study the effect of CO on CCSM relaxation induced by electrical field stimulation (EFS), a CCSM bundle was mounted on a force transducer and perfused with Hanks' balanced salt solution at 37℃ with 95% O2 and 5% CO2. After 1 hour equilibration with -500 mg of passive tension, contraction of the CCSM bundle was elicited by 10-5 M phenylephrine, which was continuously added with different concentrations of CO (1%, 2%, and 5%). Frequency-dependent relaxation was induced by EFS trains (0.2 ms at 0.5-32 Hz, for 10 s) repeated at 2 min intervals over 15 min in the presence of adrenergic and muscarinic receptor blocking agents (guanethidine and atropine, respectively). To study the distribution of heme oxygenase-2 (HO-2) in the rat CCSM, we performed immunohistochemical evaluation.ResultsCO produced a dose-dependent enhancement of EFS-induced relaxation. Pretreatment with NG-nitro-L-arginine (a nitric oxide synthase blocker) greatly reduced the EFS-induced relaxation in the presence of CO (-45%). Pretreatment with zinc protoporphyrin-IX (ZnPP-9, a heme oxygenase inhibitor) had no significant effect on EFS-induced relaxation in the absence or the presence of CO. We found immunoreactivity for HO-2 in CCSM and immunoreactivity for protein gene product 9.5 (PGP 9.5) in nerve fibers.ConclusionsWe conclude that CO produced a dose-dependent enhancement of EFS-induced relaxation in rat CCSM bundles, but neurally derived, frequency-dependent relaxation in the rat CCSM depended mostly on nitric oxide in response to nonadrenergic noncholinergic neurotransmission. Immunoreactivity for HO-2 was found in rat CCSM but not nerve fibers.

C-terminal mechano growth factor protects dopamine neurons: A novel peptide that induces heme oxygenase-1

To assess potential efficacy of mechano growth factor (MGF) for chronic neurodegenerative disorders, we studied whether MGF protects dopamine (DA) neurons subjected to neurotoxic stress. We show that a short 24-amino acid C-terminal peptide of MGF (MGF24) upregulates heme oxygenase-1 (HO-1) expression and protects SH-SY5Y cells against apoptosis and cell loss induced by three DA cell-specific neurotoxins: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP +), and rotenone. MGF24 maintains the mitochondrial membrane potential and blocks the release of mitochondrial apoptotic-inducing factor into the cytoplasm induced by 6-OHDA, MPP +, and rotenone. Chemical inhibition of HO-1 with zinc protoporphyrin-IX prevents neuroprotection by MGF24 against the three neurotoxins. MGF24 does not activate Akt signaling nor does Akt inhibition block MGF24 protection of SH-SY5Y cells. In 6-OHDA-lesioned rats, central or peripheral MGF24 administration protects against the development of contralateral forelimb under-utilization, reduces ipsilateral nigral DA cell body loss, and attenuates tyrosine hydroxylase fiber loss in the ipsilateral striatum, independent of IGF-1 receptor activation. Peripheral MGF24 administration upregulates HO-1 expression in striatal and midbrain tissue. This report is the first to demonstrate that a small peptide, MGF24, upregulates HO-1, an important cell defense mediator, and protects DA cells, suggesting new strategies for neuroprotection in Parkinson's disease.

The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin

Hemin, which is toxic to brain cells, has been reported to be taken up by cultured astrocytes; however, the mechanism of uptake is currently unknown. The present study investigated the mechanism of hemin uptake by rat primary astrocyte cultures. In medium containing 10% fetal calf serum, cultured astrocytes failed to accumulate significant amounts of heme-iron, while in serum-free medium the accumulation of heme-iron was found to be time- and concentration-dependent. After 6 h of incubation with 24 muM hemin, cells contained 36.2 +/- 2.4 nmol heme-iron/mg protein, which was 21% of the applied hemin. These results suggest that the accumulation of hemin in astrocytes does not require serum proteins such as hemopexin. A potential mechanism of hemin uptake in astrocytes involves the heme carrier protein 1 (HCP1), which is reported to mediate hemin uptake into intestinal cells. RT-PCR analysis revealed that astrocyte cultures contained HCP1 mRNA, and immunocytochemical staining and Western blot analysis confirmed the expression of HCP1 protein in cultured astrocytes. The functionality of HCP1 in astrocytes was demonstrated by incubating cells with zinc protoporphyrin IX (ZnPPIX), which is known to be transported into cells via HCP1, and ZnPPIX autofluorescence was detected in HCP1-positive astrocytes. In addition, ZnPPIX was found to attenuate the accumulation of heme-iron by astrocytes. These results are the first to demonstrate that cultured astrocytes contain functional HCP1 and that this transporter contributes to hemin uptake by astrocytes. HCP1 may therefore provide a new target for reducing hemin-related toxicity in brain cells.

Carbon monoxide produced by intrasinusoidally located haem‐oxygenase‐1 regulates the vascular tone in cirrhotic rat liver

Carbon monoxide (CO) produced by haem-oxygenase isoforms (HO-1 & HO-2) is involved in the regulation of systemic vascular tone. We aimed to elucidate the vasoregulatory role of CO in the microcirculation in normal and thioacetamide cirrhotic rat livers. Haem-oxygenase expression was examined by Western blot. Total HO enzymatic activity was measured spectrophotometrically. Sensitivity of hepatic stellate cells (HSCs) to CO-mediated relaxation was studied by a stress-relaxed-collagen-lattice model. To define the relative role of CO, the CO-releasing molecule CORM-2, the HO-inhibitor zinc protoporphyrin-IX and the HO-1 inducer hemin were added to an in situ liver perfusion set-up. The topography of vasoactive CO production was evaluated by applying different CO- and nitric oxide-trapping reagents in the liver perfusion set-up and by immunohistochemistry. Western blot showed decreased expression of both HO isoenzymes (P<0.036 for HO-1; P<0.001 for HO-2) in cirrhotic vs normal rat livers, confirmed by the HO-activity assay (P=0.004). HSCs relaxed on exposure to CORM-2 (P=0.013). The increased intrahepatic vascular resistance (IHVR) of cirrhotic rats was attenuated by perfusion with CORM-2 (P=0.016) and pretreatment with hemin (P<0.001). Inhibition of HO caused a dose-related increase in IHVR in normal and cirrhotic liver. In normal liver, the haemodynamically relevant CO production occurred extrasinusoidally, while intrasinusoidally HO-1 predominantly regulated the microcirculation in cirrhotic livers. We demonstrate a role for CO and HO in the regulation of normal and cirrhotic microcirculation. These findings are of importance in the pathophysiology of portal hypertension and establish CO/HO as novel treatment targets.

Heme Oxygenase Induced Endothelial BKCa Channel Activity Following Chronic Hypoxia

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist‐induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reversed by either removal of the endothelium or by blockade of the CO producing enzyme heme oxygenase (HO). Interestingly, endothelial cell (EC) specific blockade of large conductance Ca2+‐activated K+ (BKCa) channels also restores vasoreactivity in arteries from CH rats. Patch clamp studies demonstrate EC BKCa activity following CH, but not in ECs from control rats, although channels are observed by immunofluorescence (IF) in cells from both groups. Since HO‐derived CO is an endogenous activator of BKCa channels, we hypothesized that HO and BKCa form a functional unit that results in enhanced channel activity following CH. In support of the hypothesis, the HO inhibitor zinc protoporphyrin IX (ZnPPIX) significantly decreased K+ currents in ECs from CH rats, but was without effect in EC from control rats. In addition, the HO substrate hemin increased K+ currents only in the CH group. HO‐2, but not HO‐1, was found to co‐localize with BKCa in cells from each group. These results suggest that although HO and BKCa channels are expressed in EC from each group, HO‐derived CO only stimulates channel activity following CH.

Protective effect of hemin against cadmium-induced testicular damage in rats

The protective effect of hemin, the heme oxygenase-1 inducer, was investigated in rats with cadmium induced-testicular injury, in which oxidative stress and inflammation play a major role. Testicular damage was induced by a single i.p. injection of cadmium chloride (2 mg/kg). Hemin was given for three consecutive days (40 μmol/kg/day, s.c.), starting 1 day before cadmium administration. Hemin treatment significantly increased serum testosterone level that was reduced by cadmium. Hemin compensated deficits in the antioxidant defense mechanisms (reduced glutathione, and catalase and superoxide dismutase activities), and suppressed lipid peroxidation in testicular tissue resulted from cadmium administration. Also, hemin attenuated the cadmium-induced elevations in testicular tumor necrosis factor-α and nitric oxide levels, and caspase-3 activity. Additionally, hemin ameliorated cadmium-induced testicular tissue damage observed by light and electron microscopic examinations. The protective effect afforded by hemin was abolished by prior administration of zinc protoporphyrin-IX, the heme oxygenase-1 inhibitor. It was concluded that hemin, through its antioxidant, anti-inflammatory and antiapoptotic effects, represents a potential therapeutic option to protect the testicular tissue from the detrimental effects of cadmium.

Therapeutic potential of hemin in acetaminophen nephrotoxicity in rats

The therapeutic potential of hemin, the heme oxygenase-1 inducer, was investigated against renal damage induced by acute acetaminophen overdose in rats. Nephrotoxicity was induced by a single oral dose of acetaminophen (2.5 g/kg). Hemin was given as a single s.c. injection (40 μmol/kg), 1 h following acetaminophen administration. Hemin treatment restored blood urea nitrogen and serum creatinine levels that were elevated by acetaminophen. Hemin also compensated deficits in the antioxidant defense mechanisms (reduced glutathione, and catalase and superoxide dismutase activities), and suppressed lipid peroxidation in renal tissue resulted from acetaminophen administration. Hemin attenuated the acetaminophen-induced elevations in renal tumor necrosis factor-α and nitric oxide levels, and caspase-3 activity. Additionally, hemin ameliorated acetaminophen-induced renal damage observed by light microscopic examination. The therapeutic effect afforded by hemin was abolished by prior administration of zinc protoporphyrin-IX, the heme oxygenase-1 inhibitor. It was concluded that hemin represents a potential therapeutic option to protect renal tissue from the detrimental effects of acute acetaminophen overdose.

Overexpression of Heme Oxygenase-1 Protects Dopaminergic Neurons against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP(+)). Seven days after injection of MPP(+) and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP(+); and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP(+) treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP(+)-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP(+)-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

The Heme Oxygenase/Carbon Monoxide System Is Involved in the Auxin-Induced Cucumber Adventitious Rooting Process

Indole acetic acid (IAA) is an important regulator of adventitious rooting via the activation of complex signaling cascades. In animals, carbon monoxide (CO), mainly generated by heme oxygenases (HOs), is a significant modulator of inflammatory reactions, affecting cell proliferation and the production of growth factors. In this report, we show that treatment with the auxin transport inhibitor naphthylphthalamic acid prevented auxin-mediated induction of adventitious rooting and also decreased the activity of HO and its by-product CO content. The application of IAA, HO-1 activator/CO donor hematin, or CO aqueous solution was able to alleviate the IAA depletion-induced inhibition of adventitious root formation. Meanwhile, IAA or hematin treatment rapidly activated HO activity or HO-1 protein expression, and CO content was also enhanced. The application of the HO-1-specific inhibitor zinc protoporphyrin IX (ZnPPIX) could inhibit the above IAA and hematin responses. CO aqueous solution treatment was able to ameliorate the ZnPPIX-induced inhibition of adventitious rooting. Molecular evidence further showed that ZnPPIX mimicked the effects of naphthylphthalamic acid on the inhibition of adventitious rooting, the down-regulation of one DnaJ-like gene (CSDNAJ-1), and two calcium-dependent protein kinase genes (CSCDPK1 and CSCDPK5). Application of CO aqueous solution not only dose-dependently blocked IAA depletion-induced inhibition of adventitious rooting but also enhanced endogenous CO content and up-regulated CSDNAJ-1 and CSCDPK1/5 transcripts. Together, we provided pharmacological, physiological, and molecular evidence that auxin rapidly activates HO activity and that the product of HO action, CO, then triggers the signal transduction events that lead to the auxin responses of adventitious root formation in cucumber (Cucumis sativus).

Role of nitric oxide and carbon monoxide in Nω-Nitro- l-arginine methyl ester-resistant acetylcholine-induced relaxation in chicken carotid artery

The current study examined the hypothesis that acetylcholine-induced N ω-Nitro- l-arginine methyl ester ( l-NAME)-resistant endothelium-dependent relaxations in the chicken carotid artery are mediated by nitric oxide and carbon monoxide. Acetylcholine (1 nM–3 µM) caused a concentration-dependent relaxation (pD 2 6.81 ± 0.05, R max 115 ± 3%) of the artery segments precontracted with phenylephrine (3 µM). l-NAME (1 mM) decreased the sensitivity (pD 2 6.44 ± 0.06), but not the efficacy ( R max 108 ± 3%) of acetylcholine. It also partially decreased the acetylcholine (3 µM)-stimulated nitrite release. While treatment with N ω-Nitro- l-arginine ( l-NNA; 1 mM) plus l-NAME (1 mM) decreased the acetylcholine-stimulated nitrite release to the basal level, it moderately inhibited ( R max 77 ± 3%) the maximal relaxation elicited with the muscarinic agonist. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 µM) a specific scavenger of nitric oxide (NO) plus the two NOS inhibitors further decreased the acetylcholine-evoked relaxation ( R max 34 ± 2%). Although soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) markedly inhibited the acetylcholine-stimulated increase in tissue cGMP to less than the basal levels, it only decreased the sensitivity, but not the efficacy of the agonist either in the presence or absence of l-NAME (1 mM). Zinc Protoporphyrin-IX (ZnPP; 10 µM), a hemeoxygenase (HO) inhibitor, partially inhibited ( R max 72 ± 3%) the l-NAME-resistant acetylcholine-induced relaxations. A combined treatment of the arterial rings with l-NAME, l-NNA, PTIO and ZnPP nearly abolished ( R max 7 ± 0.9%) the vasodilator responses to acetylcholine. Endothelium removal abolished the relaxation response to acetylcholine. In conclusion, it is suggested that the acetylcholine-induced l-NAME-resistant relaxation is primarily, mediated by NO with a small but significant contribution from endothelium-derived carbon monoxide in the chicken carotid artery.

Protective Effects of a Heme Oxygenase-1-Secreting Lactococcus lactis on Mucosal Injury Induced by Hemorrhagic Shock in Rats

To investigate the protective effects of a heme oxygenase-1 (HO-1)-secreting Lactococcus lactis (LL-HO-1) on mucosal injury induced by hemorrhagic shock in rats. The ability of recombinant LL-HO-1 to secrete biological active HO-1 in the rat intestine was determined in situ after 3 d of daily intragastric administration. The therapeutic potential of LL-HO-1 strain was then evaluated on mucosal injury induced by hemorrhagic shock in rats. After successful resuscitation, mean arterial blood pressure was recorded at 5, 10, 20, and 30 min. One hour after resuscitation, the ileum was harvested for evaluation of mucosal injury by blinded microscopic inflammatory score (Chiu's grade 0-5), myeloperoxidase activity, bacterial translocation, and by the secretion of pro- and anti-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-10, respectively). Intragastric administration of HO-1-secreting L. lactis strain led to bioactive delivery of HO-1 at intestinal mucosa and significantly enhanced mean arterial blood pressure and interleukin-10 levels. Moreover, intragastric administration of LL-HO-1 significantly decreased Chiu's score, myeloperoxidase activity, bacterial translocation, and tumor necrosis factor-alpha levels when compared with rats treated with the wild-type strain. The protective effect of recombinant LL-HO-1 could be abolished by co-administration of a HO-1 inhibitor, the zinc protoporphyrin-IX. These results suggest that intragastric administration with HO-1-secreting L. lactis reduces mucosal injury induced by hemorrhagic shock.

Intragastric administration with recombinant Lactococcus lactis producing heme oxygenase-1 prevents lipopolysaccharide-induced endotoxemia in rats

Gut injury is a pivotal initiating event in the dysfunctional inflammatory response that causes postinjury multiple organ failure. Heme oxygenase-1 (HO-1) is an important enzyme that provides cellular protection against oxidative stress in different in vitro and in vivo systems. In this study, we evaluated the protective effects of intragastrically administered live Lactococcus lactis secreting bioactive HO-1 to treat intestinal mucosal injury induced by lipopolysaccharide in rats. Intragastric administration with this recombinant L. lactis strain led to active delivery of HO-1 at the mucosa and significantly decreased morbidity and mortality of lipopolysaccharide -induced endotoxemia as confirmed by blinded macroscopic and microscopic inflammatory scores (Chiu's grade), myeloperoxidase activity, mortality, and tumor necrosis factor-alpha and IL-10 cytokine stimulation. This protective effect could be abolished by an HO-1 inhibitor, the zinc protoporphyrin-IX. Our results suggest that a food-grade bacterium genetically modified to deliver bioactive HO-1 in situ exerts a protective effect against intestinal mucosal injury in rats with endotoxemia via modulation of the immune system. This novel approach may be beneficial for the maintenance of the intestinal barrier and anti-inflammatory response of the lower intestine.

Effects of exogenous and endogenous carbon monoxide on dorsal aorta pressure and cardiac output in the rainbow trout

Carbon monoxide (CO) is an important regulator of vasomotor tone in mammals. Its endogenous effects in non‐mammals, however, are unknown. Therefore, surgeries were performed on rainbow trout (Oncorhynchus mykiss), during which the dorsal aorta and nares were canulated and a flow probe was placed around the ventral aorta. Dorsal aorta pressure, stroke volume, heart rate, and respiration rate were compared on a resting trout, trout treated with inhibitors of the endogenous CO production, and trout treated with exogenous CO (delivered via a soluble ruthenium compound, CORM‐3). Zinc protoporphyrin‐IX, and inhibitor of heme‐oxygenase, elicited a dose‐dependent (at 5, 10, and 20 μmol/kg) increase in dorsal aorta pressure (approximately 20, 40, and 50% respectively). QC‐10, a non‐specific HO inhibitor, and QC‐15, a specific inhibitor of the HO‐1 isoform, both elicited a 25% increase in dorsal aorta pressure at 3 mg/kg. QC‐15 elicited a maximal response in approximately 5 minutes, while QC‐10 responses did not reach maximum until 1 hour. Both QC‐10 and QC‐15 caused small but significant increases in cardiac output. Unexpectedly, 30 μmol/kg CORM‐3 caused dorsal aorta pressure to approximately double in minutes. The results of the inhibitors suggest that CO is constitutively produced in trout to modulate vascular resistance. The surprising result with exogenous CO delivered via CORM‐3 requires further study.

Propofol attenuation of hydrogen peroxide-mediated oxidative stress and apoptosis in cultured cardiomyocytes involves haeme oxygenase-1

Our aim was to investigate the cytoprotective effect of propofol against hydrogen peroxide (H2O2)-mediated injury and the effects on the haeme oxygenase-1 system, which is a possible new cytoprotective pathway of propofol. Primary cultured newborn rat cardiomyocytes were divided into five groups: (1) untreated (Group control); (2) treated with 200 micromol L(-1) H2O2 (Group H) and treated with 200 micromol L(-1) H2O2 in the presence of propofol (25, 50 and 100 micromol L(-1), (3) Group 25P + H, (4) Group 50P + H and (5) Group 100P + H, respectively); added with zinc protoporphyrin IX (ZnPPIX) (10 micromol L(-1)), a potent inhibitor of haeme oxygenase activity, or SC-3060 (0.2 micromol L(-1)), a specific synthetic inhibitor of nuclear factor kappaB. All were incubated for 6 h. The protective effects of propofol were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cytotoxicity assay, the concentration of malondialdehyde, superoxide dismutase activity and cell apoptosis by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to detect haeme oxygenase-1 expression. Compared with H2O2, propofol concentrations (ranging from 50 to 100 micromol L(-1)) significantly increased haeme oxygenase-1 expression and decreased cardiomyocytes apoptosis, accompanied with a decrease in malondialdehyde, but with an increase in superoxide dismutase activity and cell activity (P < 0.05 and P < 0.01, respectively). The protective effects of propofol were mitigated by the addition of ZnPPIX. The addition of SC-3060 reversed propofol-induced haeme oxygenase-1 expression. Propofol can protect cardiomyocytes against H2O2-mediated cytotoxicity in a dose-dependent manner and increase haeme oxygenase-1 expression, which may partly mediate the cytoprotective effects of propofol.