Miklós Czóbel

Hypoxia-Induced Generation of Methane in Mitochondria and Eukaryotic Cells - An Alternative Approach to Methanogenesis

Background/Aims: Electrophilic methyl groups bound to positively charged nitrogen moieties may act as electron acceptors, and this mechanism could lead to the generation of methane from choline. The aims were to characterize the methanogenic potential of phosphatidylcholine metabolites, and to define the in vivo relevance of this pathway in hypoxia-induced cellular responses. Methods: The postulated reaction was investigated (1) in model chemical experiments, (2) in rat mitochondrial subfractions and (3) in bovine endothelial cell cultures under hypoxic conditions and in the presence of hydroxyl radical generation. The rate of methane formation was determined by gas chromatography with flame-ionisation detectors. The lucigenin-enhanced chemiluminescence assay was used to determine the reactive oxygen species-scavenging capacity of the choline metabolites. Results: Significant methane generation was demonstrated in all three series of experiments. Phosphatidylcholine metabolites with alcoholic moiety in the molecule (i.e. choline, N,N-dimethylethanolamine and N-methylethanolamine), inhibited oxygen radical production both in vitro and in vivo, and displayed an effectiveness proportional to the amount of methane generated and the number of methyl groups in the compounds. Conclusion: Methane generation occurs in aerobic systems. Phosphatidylcholine metabolites containing both electron donor and acceptor groups may have a function to counteract intracellular oxygen radical production.

Oral phosphatidylcholine pretreatment decreases ischemia-reperfusion induced methane generation and the inflammatory response in the small intestine

We have shown that phosphatidylcholine (PC) metabolites may have a function in counteracting the production of reactive oxygen species (ROS), and that this mechanism can lead to the generation of methane from choline. The aims were to establish whether the dietary administration of PC can protect the reperfused small bowel mucosa by its acting as an anti-inflammatory agent and to investigate this possibility in association with in vivo methane generation. Group 1 (n = 5) of anesthetized dogs served as sham-operated controls, whereas in groups 2 (n = 6) and 3 (n = 6), complete small intestinal ischemia was induced by occluding the superior mesenteric artery for 60 min. Groups 1 and 2 were fed with normal laboratory chow for 1 week before the experiments, whereas the animals in group 3 received a special diet containing 1% soybean PC. The intramucosal pH and the difference of the arterial and local PCO2 (PCO2 gap) were detected by indirect tonometry. Intestinal superoxide production and myeloperoxidase (MPO) activity (a marker of tissue leukocyte infiltration) were ascertained on ileal biopsy samples 180 min after reperfusion. The content of methane in the exhaled air was determined by gas chromatography. I/R was characterized by significant tissue acidosis with ROS generation and elevated MPO activity. These changes were accompanied by increased methane production in the exhaled air during reoxygenation. The PC-enriched diet prevented the decrease in intramucosal pH, diminished the intestinal superoxide generation and the MPO activity, and significantly decreased the exhaled methane concentration. The increased dietary uptake of PC exerts an anti-inflammatory influence in the gastrointestinal tract. Exhaled methane is linked to abnormal ROS generation; a decreased methane production is associated with significantly reduced inflammatory activation during I/R.

Anti-Inflammatory Action of a Phosphatidylcholine, Phosphatidylethanolamine and N-Acylphosphatidylethanolamine-Enriched Diet in Carrageenan-Induced Pleurisy

Background/Aims: Phosphatidylcholine (PC)-derived choline exhibits anti-inflammatory properties in stress conditions. Phosphatidylethanolamine (PE) and N-acylphosphatidylethanolamines (NAPEs) are endogenous bioactive phospholipids linked to the PC and endocannabinoid metabolisms. We hypothesized that an increased dietary input of PC, PE and NAPE may interfere with leukocyte reactions and thus decreases the inflammatory activation. Methods: CFLP mice were fed with a control diet or with a diet supplemented with 1% PC, 0.4% PE and 0.1% NAPE for 7 days before the induction of pleurisy with carrageenan. Pleural leukocyte migration, pulmonary mast cell degranulation (Alcian blue-safranin O staining), and the activities of inducible nitric oxide synthase, xanthine oxidoreductase and myeloperoxidase were determined in lung tissue biopsies. Results: The carrageenan-induced inflammatory response was characterized by pulmonary leukocyte infiltration, mast cell degranulation and significantly increased inducible nitric oxide synthase and xanthine oxidoreductase activities (by 82 and 60%, respectively). Treatment of mice with acetylsalicylic acid or with dietary PC + PE + NAPE supplementation significantly decreased the leukocyte reaction, and suppressed the activity of the pulmonary proinflammatory enzymes. Conclusion: This study confirms a potential for dietary PC + PE + NAPE supplementation to influence events crucial for the remission of acute inflammation. PC + PE + NAPE administration could possibly be a novel preventive or pharmacotherapeutic option in inflammatory pathologies.

Modulation of cardiac contractility through endothelin-1 release and myocardial mast cell degranulation

The aim of this study was to outline the consequences of a hypertonic saline-dextran-40 (HSD) infusion-induced peripheral flow stimulus on the ventricular function in closed-chest, pentobarbital-anesthetized dogs. We hypothesized that HSD-induced elevation in endothelin-1 (ET-1) and nitric oxide (NO) release can have a role in myocardial contractile responses; and that cardiac mast cells (MC) degranulation may be involved in this process. The consequences of disodium cromoglycate (a MC stabilizer) or ETR-p1/fl peptide (an endothelin-A receptor antagonist) treatment were evaluated. A 4 ml/kg iv HSD40 infusion significantly increased cardiac index and myocardial contractility, and resulted in a decreased peripheral resistance. The postinfusion period was characterized by significant plasma NO and ET-1 elevations, these hemodynamic and biochemical changes being accompanied by a decreased myocardial ET-1 content, NO synthase activity and enhanced myocardial MC degranulation. Disodium cromoglycate treatment inhibited the HSD40-induced elevations in myocardial contractility and MC degranulation, and similar hemodynamic changes were noted after treatment with ETR-p1/fl peptide, together with a normalized myocardial myocardial ET-1 content, NO synthesis and a significant reduction in MC degranulation. These results indicate that peripheral NO and ET-1 release modulates the cardiac contractility through myocardial ET-A receptor activation and MC degranulation.

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure-diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg(-1)), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg(-1) iv). Plasma ET-1, nitrite/nitrate (NO(x)) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO(x), significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm(-1)]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.