Creatine Protects against Excitoxicity in an In Vitro Model of Neurodegeneration

Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H2O2. In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H2O2-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

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Determination of endogenous levels of cyclic ADP-ribose in rat tissues

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10°C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 ± 0.64 pmol/mg, in heart is 1.04 ± 0.08 pmol/mg and in brain is 2.75 ± 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.link_to_subscribed_fulltex

Dynamic Phosphometabolomic Profiling of Human Tissues and Transgenic Models By O-18-Assisted P-31 Nmr and Mass Spectrometry

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope O-18-assisted P-31 nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The O-18 labeling procedure is based on the incorporation of one O-18 into Pi from [O-18]H2O with each act of ATP hydrolysis and the distribution of O-18-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded O-18-labeling procedures has revealed significant differences in the dynamics of G-6-P[O-18] (glycolysis), G-3-P[O-18] (substrate shuttle), and G-1-P[O-18] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[O-18], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1-/-), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[O-18] and decreased G-1-P[O-18] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit-/--deficient hearts is characterized by a higher G-6-[O-18]P turnover rate, G-6-P levels, glycolytic capacity, gamma/beta-phosphoryl of GTP[O-18] turnover, as well as beta-[O-18]ATP and beta-[O-18]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus, O-18-assisted gas chromatography-mass spectrometry and P-31 NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.Wo

Electron Spray Ionization Mass Spectrometry And 2D P-31 Nmr For Monitoring O-18/O-16 Isotope Exchange And Turnover Rates Of Metabolic Oligophosphates

A new method was here developed for the determination of O-18-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (alpha-, beta-, and gamma-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of O-18/O-16 exchange was validated with gas chromatography-mass spectrometry and 2D P-31 NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D P-31 NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, O-18-labeling kinetics and turnover rates of alpha-, beta-, and gamma-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.Wo