An oral carbon monoxide-releasing molecule protects against acute hyperhemolysis in sickle cell disease.

ABSTRACT: Acute hyperhemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multiorgan failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyperhemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: (1) prevented the upregulation of relevant proinflammatory and proadhesion markers controlled by the NF-κB enhancer of activated B cells, and (2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-κB proinflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyperhemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as patients with SCD with history of DHTR.

Carbon Monoxide Signaling: Examining Its Engagement with Various Molecular Targets in the Context of Binding Affinity, Concentration, and Biologic Response.

Carbon monoxide (CO) has been firmly established as an endogenous signaling molecule with a variety of pathophysiological and pharmacological functions, including immunomodulation, organ protection, and circadian clock regulation, among many others. In terms of its molecular mechanism(s) of action, CO is known to bind to a large number of hemoproteins with at least 25 identified targets, including hemoglobin, myoglobin, neuroglobin, cytochrome c oxidase, cytochrome P450, soluble guanylyl cyclase, myeloperoxidase, and some ion channels with dissociation constant values spanning the range of sub-nM to high µM. Although CO's binding affinity with a large number of targets has been extensively studied and firmly established, there is a pressing need to incorporate such binding information into the analysis of CO's biologic response in the context of affinity and dosage. Especially important is to understand the reservoir role of hemoglobin in CO storage, transport, distribution, and transfer. We critically review the literature and inject a sense of quantitative assessment into our analyses of the various relationships among binding affinity, CO concentration, target occupancy level, and anticipated pharmacological actions. We hope that this review presents a picture of the overall landscape of CO's engagement with various targets, stimulates additional research, and helps to move the CO field in the direction of examining individual targets in the context of all of the targets and the concentration of available CO. We believe that such work will help the further understanding of the relationship of CO concentration and its pathophysiological functions and the eventual development of CO-based therapeutics. SIGNIFICANCE STATEMENT: The further development of carbon monoxide (CO) as a therapeutic agent will significantly rely on the understanding of CO's engagement with therapeutically relevant targets of varying affinity. This review critically examines the literature by quantitatively analyzing the intricate relationships among targets, target affinity for CO, CO level, and the affinity state of carboxyhemoglobin and provide a holistic approach to examining the molecular mechanism(s) of action for CO.

Carbon monoxide is involved in melatonin-enhanced drought resistance in tomato seedlings by enhancing chlorophyll synthesis pathway.

BACKGROUND: Drought is thought to be a major abiotic stress that dramatically limits tomato growth and production. As signal molecule, melatonin (MT) and carbon monoxide (CO) can enhance plant stress resistance. However, the effect and underlying mechanism of CO involving MT-mediated drought resistance in seedling growth remains unknown. In this study, tomato (Solanum lycopersicum L. 'Micro-Tom') seedlings were used to investigate the interaction and mechanism of MT and CO in response to drought stress. RESULTS: The growth of tomato seedlings was inhibited significantly under drought stress. Exogenous MT or CO mitigated the drought-induced impairment in a dose-dependent manner, with the greatest efficiency provided by 100 and 500 µM, respectively. But application of hemoglobin (Hb, a CO scavenger) restrained the positive effects of MT on the growth of tomato seedlings under drought stress. MT and CO treatment promoted chlorophyll a (Chl a) and chlorophyll a (Chl b) accumulations. Under drought stress, the intermediate products of chlorophyll biosynthesis such as protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX), potochlorophyllide (Pchlide) and heme were increased by MT or CO, but uroporphyrinogen III (Uro III) content decreased in MT-treated or CO-treated tomato seedlings. Meanwhile, MT or CO up-regulated the expression of chlorophyll and heme synthetic-related genes SlUROD, SlPPOX, SlMGMT, SlFECH, SlPOR, SlChlS, and SlCAO. However, the effects of MT on chlorophyll biosynthesis were almost reversed by Hb. CONCLUSION: The results suggested that MT and CO can alleviate drought stress and facilitate the synthesis of Chl and heme in tomato seedlings. CO played an essential role in MT-enhanced drought resistance via facilitating chlorophyll biosynthesis pathway.

Recent progress on anti-nociceptive effects of carbon monoxide releasing molecule-2 (CORM-2).

The role of carbon monoxide (CO) has evolved albeit controversial disputes on its toxicity. This biological gasotransmitter participates in the endogenous regulation of neurotransmitters and neuropeptides released in the nervous system. Exogenous CO gas inhalation at a lower concentration has been the subject of investigations, which have revealed its biological homeostatic mechanisms and protective effects against many pathological conditions. This therapeutic procedure of CO is, however, limited due to its immediate release, which favours haemoglobin at a high affinity with the subsequent generation of toxic carboxyhaemoglobin in tissues. In order to address this problem, carbon monoxide releasing molecule-2 (CORM-2) or also known as tricarbonyldichlororuthenium II dimer is developed to liberate a controlled amount of CO in the biological systems. In this review, we examine several potential mechanisms exerted by this therapeutic compound to produce the anti-nociceptive effect that has been demonstrated in previous studies. This review could shed light on the role of CORM-2 to reduce pain, especially in cases of chronic and neuropathic pain.

Type-I Photosensitizer-Triggered Controllable Carbon Monoxide Release for Effective Treatment of Staph Skin Infection.

Staphylococcus aureus (S. aureus) infection is a major infectious skin disease that is highly resistant to conventional antibiotic treatment and host immune defense, leading to recurrence and exacerbation of bacterial infection. Herein, we developed a photoresponsive carbon monoxide (CO)-releasing nanocomposite by integrating anion-π+ type-I photosensitizer (OMeTBP) and organometallic complex (FeCO) for the treatment of planktonic S. aureus and biofilm-associated infections. After optimizing the molar ratio of FeCO and OMeTBP, the prepared nanoparticles, OMeTBP@FeCONPs, not only ensured sufficient loading of CO donors and efficient CO generation but also showed negligible free ROS leakage under light irradiation, which helped to avoid tissue damage caused by excessive ROS. Both in vitro and in vivo results demonstrated that OMeTBP@FeCONPs could effectively inhibit S. aureus methicillin-resistant S. aureus (MRSA), and bacterial biofilm. Our design has the potential to overcome the resistance of conventional antibiotic treatment and provide a more effective option for bacterial infections.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Carbon monoxide (CO) derived from the CO-releasing molecule CORM-2 reduces peritoneal adhesion formation in a rat model.

BACKGROUND: Although low-dose carbon monoxide (CO) administration has been shown to have an anti-fibrotic effect in various fibrotic diseases, its effects on peritoneal adhesion (PA), one of the postoperative complications, are not elucidated. In this study, the effect of CO-releasing tricarbonyldichlororuthenium (II) dimer (CORM-2) administration on the formation of PA and the underlying factors of its potential effect were investigated. METHODS AND RESULTS: After the induction of PA, rats were divided into four groups with 8 rats in each group. The rats received either (i) dimethyl sulfoxide:saline solution (1:10) as a vehicle, (ii) 2.5 mg/kg CORM-2, (iii) 5 mg/kg CORM-2, or (iv) inactive (i) CORM (iCORM) intragastrically every day for a duration of 7 days. PA was not induced in rats (n = 8) designated as sham controls. Gross, histological, immunohistochemical and quantitative real-time polymerase chain reaction analyses were performed to evaluate the effectiveness of CORM-2 administration. Gross analysis showed that CORM-2 administration reduced PA formation compared to rats treated with vehicle. Histological and immunohistochemical examinations showed that increased collagen deposition, myofibroblast accumulation, microvessel density, and M1 macrophage count in the peritoneal fibrosis area of vehicle-treated rats decreased following CORM-2 treatments. PCR analyses showed that CORM-2 treatments decreased hypoxia-induced Hif1a, profibrotic Tgfb1, ECM components Col1a1 and Col3a1, collagen degradation suppressor Timp1, fibrinolysis inhibitor Serpine1, and pro-inflammatory Tnf mRNA expressions, while increasing the M2 macrophage marker Arg1 mRNA expression. CONCLUSIONS: These results suggested that CORM-2 administration reduces PA formation by affecting adhesiogenic processes such as pro-inflammatory response, fibrinolytic system, angiogenesis and fibrogenesis.

Use of Enzymatically Activated Carbon Monoxide Donors for Sensitizing Drug-Resistant Tumor Cells.

The application of gaseous signaling molecules like NO, H2S or CO to overcome the multidrug resistance in cancer treatment has proven to be a viable therapeutic strategy. The development of CO-releasing molecules (CORMs) in a controlled manner and in targeted tissues remains a challenge in medicinal chemistry. In this paper, we describe the design, synthesis and chemical and enzymatic stability of a novel non-metal CORM (1) able to release intracellularly CO and, simultaneously, facilitate fluorescent degradation of products under the action of esterase. The toxicity of 1 against different human cancer cell lines and their drug-resistant counterparts, as well as the putative mechanism of toxicity were investigated. The drug-resistant cancer cell lines efficiently absorbed 1 and 1 was able to restore their sensitivity vs. chemotherapeutic drugs by causing a CO-dependent mitochondrial oxidative stress that culminated in mitochondrial-dependent apoptosis. These results demonstrate the importance of CORMs in cases where conventional chemotherapy fails and thus open the horizons towards new combinatorial strategies to overcome multidrug resistance.

X-Ray-triggered Carbon Monoxide and Manganese Dioxide Generation based on Scintillating Nanoparticles for Cascade Cancer Radiosensitization.

Carbon monoxide (CO) is an endogenous signaling molecule with broad therapeutic effects. Here, a multifunctional X-ray-triggered carbon monoxide (CO) and manganese dioxide (MnO2 ) generation nanoplatform based on metal carbonyl and scintillating nanoparticles (SCNPs) is reported. Attributed to the radioluminescent characteristic of SCNPs, UV-responsive Mn2 (CO)10 is not only indirectly activated to release CO by X-ray but can also be degraded into MnO2 . A high dose of CO can be used as a glycolytic inhibitor for tumor suppression; it will also sensitize tumor cells to radiotherapy. Meanwhile MnO2 , as the photolytic byproduct of Mn2 (CO)10 , has both glutathione (GSH) depletion and Fenton-like Mn2+ delivery properties to produce highly toxic hydroxyl radical (⋅OH) in tumors. Thus, this strategy can realize X-ray-activated CO release, GSH depletion, and ⋅OH generation for cascade cancer radiosensitization. Furthermore, X-ray-activated Mn2+ in vivo demonstrates an MRI contrast effect, making it a potential theranostic nanoplatform.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide.

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

Carbon Monoxide Protects Neural Stem Cells Against Iron Overload by Modulating the Crosstalk Between Nrf2 and NF-κB Signaling.

Although accumulating evidences have demonstrated pro-survival effects of CO against various insults, the precise mechanism explaining how neural stem cells (NSCs) are protected by CO also remains largely unknown. Here we report CO pro-survival effect on NSCs against iron overload was comparable to that obtained with pharmacological inhibitors of reactive oxygen species (ROS). Its pro-survival effect was accompanied by the inhibition of ROS and subsequent inhibition of NF-κB which is mediated through nuclear factor erythroid 2-related factor 2 (Nrf2), in that activation of Nrf2 by CO inhibited ROS via up-regulation of NQO-1 while down-regulation of Nrf2 reversed the pro-survival effect of CO both in vitro and in vivo. CO-mediated preconditioning results in Nrf2 up-regulation and NF-κB inhibition, suggesting that these two pathways act in an inverse manner to maintain redox homeostasis. Our findings revealed CO preconditioning as a promising treatment strategy to improve efficacy of NSCs transplantation after hemorrhagic stroke.

Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

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Comprehensive transcriptome analysis unravels the crucial genes during adventitious root development induced by carbon monoxide in Cucumis sativus L.

BACKGROUND: Carbon monoxide (CO) has been reported to be participated in adventitious rooting. However, knowledge about the interrelationship between CO and phytohormones during rooting is obscure. The molecular mechanism of CO-induced rooting is currently unclear. METHODS AND RESULTS: The roles of CO in adventitious rooting in Cucumis sativus L. at the transcriptional level were investigated. The results show that 10 µM hematin (a CO donor) has a significant positive effect on adventitious rooting in cucumber. A total of 1792 differentially expressed genes (DEGs; 1103 up-regulated and 689 down-regulated) were identified in hematin treatment by RNA sequencing analysis. There were 37, 18 and 19 DEGs significantly enriched in plant hormone signal transduction, sucrose and starch metabolism, and phenylalanine metabolism, respectively. Both transcriptome and real-time quantitative PCR results showed that the expressions of AUX22D, IAA6, SAUR21, SAUR24, GH3.5, CYCD3-3, TIFY10a, TIFY10A and TIF9 promoted the accumulation of IAA, BR, JA and SA in plant hormone signal transduction. The up-regulation of HK3, TPPF, otsB, TPS7, TPS9 and the down-regulation of AGPS1, AGPS3 increased the content of starch and total sugar by mediating the activity of some critical enzymes, including HK, TPS, TPP and AGP. PER47, PER61, PER24, PER66, PER4 and CCR2 increased the lignin content. CONCLUSION: Our results suggest that CO could promote the accumulation of plant hormones, starch, sugar and lignin during adventitious rooting by regulating the expression of some related genes, including AUX22D, IAA6, SAUR21, SAUR24, GH3.5, CYCD3-3, TIFY10a, TIFY10A, TIF9 HK3, otsB, TPS7, TPS9, AGPS1, AGPS3, PER47, PER61, PER24, PER66, PER4, and CCR2. Thus, we provides an interesting candidate gene list for further studies on the molecular mechanisms of adventitious rooting.

Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic ß-cells by activating Phospholipase C signal pathway in diabetic mice.

In early stage of diabetes, insulin secretion from pancreatic ß-cells is increased to deal with the elevated blood glucose. Previous studies have reported that islet-produced carbon monoxide (CO) is associated with increased glucose-stimulated insulin secretion from ß-cells. However, this compensatory mechanism by which CO may act to enhance ß-cell function remain unclear. In this study, we revealed that CO promoted intracellular calcium ([Ca2+]i) elevation and glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells in leptin receptor deficient db/db mice but not in C57 mice. The stimulatory effects of CO on ß-cell function in db/db mice was blocked by inhibition of Phospholipase C (PLC) signaling pathway. We further demonstrated that CO triggered [Ca2+]i transients and enhanced GSIS in C57 islets when ß-cells overexpressed with PLCγ1 and PLCδ1, but not PLCß1. On the other hand, reducing PLCγ1 and PLCδ1 expressions in db/db islets dramatically attenuated the stimulatory effects of CO on ß-cell function, whereas interfering PLCß1 expression had no effects on CO-induced ß-cell function enhancement. Our findings showing that CO elevated [Ca2+]i and enhanced GSIS by activating PLC signaling through PLCγ1 and PLCδ1 isoforms in db/db pancreatic ß-cells may suggest an important mechanism by which CO promotes ß-cell function to prevent hyperglycemia. Our study may also provide new insights into the therapy for type II diabetes and offer a potential target for therapeutic applications of CO.

Application of carbon monoxide in kidney and heart transplantation: A novel pharmacological strategy for a broader use of suboptimal renal and cardiac grafts.

Carbon monoxide (CO) was historically regarded solely as a poisonous gas that binds to hemoglobin and reduces oxygen-carrying capacity of blood at high concentrations. However, recent findings show that it is endogenously produced in mammalian cells as a by-product of heme degradation by heme oxygenase, and has received a significant attention as a medical gas that influences a myriad of physiological and pathological processes. At low physiological concentrations, CO exhibits several therapeutic properties including antioxidant, anti-inflammatory, anti-apoptotic, anti-fibrotic, anti-thrombotic, anti-proliferative and vasodilatory properties, making it a candidate molecule that could protect organs in various pathological conditions including cold ischemia-reperfusion injury (IRI) in kidney and heart transplantation. Cold IRI is a well-recognized and complicated cascade of interconnected pathological pathways that poses a significant barrier to successful outcomes after kidney and heart transplantation. A substantial body of preclinical evidence demonstrates that CO gas and CO-releasing molecules (CO-RMs) prevent cold IRI in renal and cardiac grafts through several molecular and cellular mechanisms. In this review, we discuss recent advances in research involving the use of CO as a novel pharmacological strategy to attenuate cold IRI in preclinical models of kidney and heart transplantation through its administration to the organ donor prior to organ procurement or delivery into organ preservation solution during cold storage and to the organ recipient during reperfusion and after transplantation. We also discuss the underlying molecular mechanisms of cyto- and organ protection by CO during transplantation, and suggest its clinical use in the near future to improve long-term transplantation outcomes.

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD+ and ATP Depletion.

OBJECTIVES: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. CONCLUSIONS: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.

Exogenous carbon monoxide suppresses LPS-Induced platelet SNAREs complex assembly and α-granule exocytosis via integrin αIIbß3-Mediated PKCθ/Munc18a pathway.

Activation of coagulation occurs in sepsis and contributes to the development of thrombosis. Platelet α-granule exocytosis plays an important role in septic coagulation abnormalities. The present study aimed to investigate the effects and the underlying mechanisms of exogenous carbon monoxide, carbon monoxide-releasing molecules II (CORM-2)-liberated CO, on suppressing platelet α-granule exocytosis in sepsis. It was shown that CORM-2 weakened α-granule membrane fusion with platelet plasma membrane and attenuated α-granule contents exocytosis in LPS-Induced platelet. Further studies revealed that CORM-2 suppressed the expression of integrin αIIbß3 in platelets stimulated by LPS. This was accompanied by a decrease in production and phosphorylation of PKCθ and Munc18a, SNARE complex assembly and subsequently platelet α-granule exocytosis. Taken together, we suggested that the potential mechanism of suppressive effect of CORM-2 on LPS-induced platelet SNAREs complex assembly and α-Granule Exocytosis might involve integrin αIIbß3-mediated PKCθ/Munc18a pathway activation.

Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition.

Sickle cell trait (AS) confers partial protection against lethal Plasmodium falciparum malaria. Multiple mechanisms for this have been proposed, with a recent focus on aberrant cytoadherence of parasite-infected red blood cells (RBCs). Here we investigate the mechanistic basis of AS protection through detailed temporal mapping. We find that parasites in AS RBCs maintained at low oxygen concentrations stall at a specific stage in the middle of intracellular growth before DNA replication. We demonstrate that polymerization of sickle hemoglobin (HbS) is responsible for this growth arrest of intraerythrocytic P. falciparum parasites, with normal hemoglobin digestion and growth restored in the presence of carbon monoxide, a gaseous antisickling agent. Modeling of growth inhibition and sequestration revealed that HbS polymerization-induced growth inhibition following cytoadherence is the critical driver of the reduced parasite densities observed in malaria infections of individuals with AS. We conclude that the protective effect of AS derives largely from effective sequestration of infected RBCs into the hypoxic microcirculation.

Role of Methylation in Period2 (PER2) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical-Studies on the Pig Model.

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene (PER2), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

Distinct Pharmacological Properties of Gaseous CO and CO-Releasing Molecule in Human Platelets.

Carbon monoxide (CO)-gaseous or released by CO-RMs-both possess antiplatelet properties; however, it remains uncertain whether the mechanisms involved are the same. Here, we characterise the involvement of soluble guanylate cyclase (sGC) in the effects of CO-delivered by gaseous CO-saturated buffer (COG) and generated by CORM-A1-on platelet aggregation and energy metabolism, as well as on vasodilatation in aorta, using light transmission aggregometry, Seahorse XFe technique, and wire myography, respectively. ODQ completely prevented the inhibitory effect of COG on platelet aggregation, but did not modify antiplatelet effect of CORM-A1. In turn, COG did not affect, whereas CORM-A1 substantially inhibited energy metabolism in platelets. Even though activation of sGC by BAY 41-2272 or BAY 58-2667 inhibited significantly platelet aggregation, their effects on energy metabolism in platelets were absent or weak and could not contribute to antiplatelet effects of sGC activation. In contrast, vasodilatation of murine aortic rings, induced either by COG or CORM-A1, was dependent on sGC. We conclude that the source (COG vs. CORM-A1) and kinetics (rapid vs. slow) of CO delivery represent key determinants of the mechanism of antiplatelet action of CO, involving either impairment of energy metabolism or activation of sGG.