title: The Role of methane in the regulation of nitrite reductase activities in ischemic mammalian tissues
Creators: name: Stögerer, Tanja
Publisher: name: TU Wien
Year: 2018
Subjects: subject: gasotransmitter
subject: nitrite reductase
subject: hypoxia
subject: nitric oxide
subject: methane
subject: xanthine oxidoreductase

description: During ischemia, the formation of the potent vasodilator and gasotransmitter nitric oxide (NO) mainly takes place via a reduction of nitrite by a family of enzymes and structures called nitrite reductases. In the first part of this project, we have characterized the kinetics of nitrite reduction to NO in different organs without being able to explain the differences in the kinetics of their NO release. The second part of the study was aimed at understanding the mechanisms underlying the kinetics of NO release, particularly the origin of an observed lag-phase. In the last part we identified the major generator of NO in the intestine and its regulation by methane, a gaseous messenger recently discovered in mammalians. The release of NO was monitored using a continuous chemiluminescence NO detection method. We determined the overall NO released from tissue homogenates prepared from the liver, small intestine and heart of Sprague Dawley rats under conditions simulating ischemia. The individual contributions of the previously identified nitrite reductases hemoglobin, myoglobin, xanthine oxidoreductase, and cytochrome bc1, a part of the mitochondrial respiratory chain, were then assigned using different inhibitors. Under severe hypoxia and slightly acidic conditions, xanthine reductase was found to be the main contributor to the NO release from nitrite, compared to hemoglobin, myoglobin and cytochrome bc1. This finding was used to investigate a possible interplay between NO and the recently identified gasotransmitter methane (CH4), which has been shown to decrease the NO-mediated tyrosine nitrosylation in ischemia-reperfusion injury. The ability of CH4 to decrease NO levels has previously been observed in an in vivo study. In this study, rats undergoing ischemia and reperfusion in the intestine were ventilated with air containing 2.2 % CH4, exhibited significantly reduced tissue damage and release of inflammatory mediators, such as NO. This could be replicated in vitro, as the NO release from rat intestine homogenates was significantly decreased in measurements using a mixture of 2.2 % CH4 in nitrogen as a carrier gas, as compared to the control measurements under N2. To clarify the mechanism of this phenomenon, we performed in vitro experiments. First, we examined the effect of CH4 on the NO production from pure xanthine oxidoreductase, supplying only electron donors acting at the molybdenum site of the enzyme, i.e. xanthine and hypoxanthine. In this NO release from xanthine and hypoxanthine conversion by xanthine oxidoreductase, the presence of CH4 had a contrasting effect, increasing rather than decreasing the NO measured. Similarly, xanthine oxidoreductase supplied with a single electron donor, NADH, which acts at the FAD site of the enzyme, gave rise to an increase in NO production in the presence of CH4; however, this effect was counteracted by the addition of the additional electron acceptors and other enzymes using NADH as a cofactor. By boiling the homogenate, we inactivated all enzymes while maintaining physiological concentrations of the substrates NADH, NAD+, xanthine and hypoxanthine. To limit thermal decomposition of the substrates, the duration of boiling was set to 5 minutes, at which point still about 75 % of NAD+, the least stable of the compounds, remains. In these experiments, exogenous xanthine oxidase reproduced the effect observed in vivo, where CH4 inhibited NO generation. DPI, an inhibitor of the FAD site of the xanthine oxidase enzyme, which blocks the interaction with NAD+ and NADH, reversed the effect of CH4 on NO generation. This opposing CH4-induced effect might be attributed to an increased incorporation of NO in tissues (e.g. protein nitrosylation) which results in less free NO in the medium, a process which is represented as a lag-phase in NO release from homogenate samples.