guanosine-triphosphate and Reperfusion-Injury

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) it was investigated the changes of biochemical processes in the different areas of heart (intact, risk and necrotic zone) during intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible cardioprotective mechanisms of flocalin action at ischemia-reperfusion conditions: the preservation of sufficient levels of de novo (by cNOS) NO synthesis, an inhibition of de novo (by iNOS) and salvage (by NADH-dependent nitratreductase) NO synthesis, an inhibition of L-arginine degradation by arginase, an inhibition of oxidizing metabolism due to limitation of ROS and RNS generation, inhibition of free arachidonic acid and eicosanoids synthesis, inhibition of ATP and GTP degradations and, possibly, stimulation of protective haem degradation. These changes may prevent formation of toxic peroxynitrite and suggest the possibility of participating in flocalin-mediated cardioprotective effects of warning a mitochondrial permeability transition pore (MPTP) opening and inhibition of apoptosis and/or necrosis of cardiomyocytes induced by it.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Arachidonic Acid; Arginase; Arginine; Dogs; Eicosanoids; Guanosine Triphosphate; Heme; KATP Channels; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardium; Nitrate Reductase (NADH); Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Pinacidil; Reactive Nitrogen Species; Reactive Oxygen Species; Reperfusion Injury

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. GTP depletion was recently identified as an important inducer of apoptosis during chemical anoxia in vitro and ischemic injury in vivo. It has also been shown that GTP salvage with guanosine prevented apoptosis and protected function. This study investigates the role of p53 in mediating the apoptotic response to GTP depletion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion. p53 protein levels increased significantly in the medulla over 24 h post-ischemia. The provision of guanosine inhibited the increase in p53. Pifithrin-alpha, a specific inhibitor of p53, mimicked the effects of guanosine. It had no effect on necrosis, yet it prevented apoptosis and protected renal function. Pifithrin-alpha was protective when given up to 14 h after the ischemic insult. The effects of pifithrin-alpha on p53 included inhibition of transcriptional activation of downstream p53 targets like p21 and Bax and inhibition of p53 translocation to the mitochondria. Similar results were obtained in cultured renal tubular cells. It is concluded that p53 is an important mediator of apoptosis during states of GTP depletion. Inhibitors of p53 should be considered in the treatment of ischemic renal injury.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Guanosine; Guanosine Triphosphate; Kidney; Kidney Cortex; Kidney Medulla; LLC-PK1 Cells; Male; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Swine; Thiazoles; Tissue Distribution; Toluene; Tumor Suppressor Protein p53

Topics: Actins; Animals; Flavonoids; Gene Expression Regulation; Gluconates; Guanosine Triphosphate; Hydroxyethyl Starch Derivatives; Lung; Male; Organ Preservation; Organ Preservation Solutions; Phenols; Phosphates; Polymers; Polyphenols; Pulmonary Circulation; Pulmonary Edema; Rats; Rats, Inbred Lew; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Tea; Transcription, Genetic; Trehalose; Tumor Necrosis Factor-alpha

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. Recently, we linked anoxia-induced apoptosis in renal cell cultures specifically to the depletion of GTP. We therefore hypothesized that enhancing GTP repletion in vivo might protect function by reducing apoptosis in postischemic tubules. Male C57 black mice (the "I" group of animals) underwent bilateral renal artery clamp for 32 minutes to induce ischemia and then received either normal saline ("NS") or guanosine ("G"). After 1 hour of reperfusion, renal GTP levels in NS/I were reduced to nearly half of those in sham operated mice, whereas these levels were nearly unchanged in G/I mice. Morphologic examination of tubular injury revealed no significant differences between the two groups. However, there was a significant reduction in the number of apoptotic tubular cells in the medulla in the G/I group as compared with the NS/I group. At 24 hours, creatinine was significantly elevated in the NS/I group, compared to the G/I group. We conclude that guanosine protects against renal ischemic injury by replenishing GTP stores and preventing tubular apoptosis.

Topics: Animals; Apoptosis; Cell Death; Cell Line; Guanosine; Guanosine Triphosphate; Hypoxia; In Situ Nick-End Labeling; Ischemia; Kidney; Male; Mice; Mice, Inbred C57BL; Phenotype; Protein Binding; Reperfusion Injury; Swine; Time Factors

Isolated rat hepatocytes were used for the evaluation of nucleotide depletion and oxidative stress as two causal components of postischemic injury following oxygen deficiency. The ATP and GTP loss during anoxia was accompanied by temporary increases of nucleotide degradation products. The critical duration of anoxia for a complete ATP restoration during reoxygenation was between 30 and 60 min. The oxidative stress during reoxygenation was demonstrated by decrease of GSH concentration and increase of TBA-RS level. The tremendous GSH loss could not be balanced by the slight GSSG increase during reoxygenation. Prevention of GSH decrease and TBA-RS increase in parallel to prevention of viability loss in presence of oxipurinol in contrast to lacking improvement of ATP and GTP restoration by this drug speak in favor for the oxidative stress as major causal component for postischemic injury of hepatocytes in comparison with depletion of energy-rich purine nucleotides. The inhibition of formation of reactive oxygen species via xanthine oxidase reactions was found to be the dominant protective effect of oxipurinol against postischemic injury of hepatocytes in comparison with lacking influence on nucleotide salvage and ATP/GTP regeneration and with radical scavenging.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Cell Hypoxia; Cell Survival; Cells, Cultured; Energy Metabolism; Glutathione; Guanosine Triphosphate; Liver; Male; Nucleotides; Oxidation-Reduction; Oxygen; Oxygen Consumption; Oxypurinol; Rats; Rats, Wistar; Reperfusion Injury; Stress, Physiological; Thiobarbituric Acid Reactive Substances

Ischemia (one hour) and following reperfusion (up to one hour) of the small intestine induce biochemical changes which are indices for the formation and action of oxygen free radicals and which occur predominantly during the reperfusion period. But the villi and the epithelial cells show different patterns of damage, occurring both at the end of the ischemic period and during the reperfusion period. Although the quantitative morphological changes are increased during the reperfusion in comparison with the ischemic phase the quality of the pattern of structural damage is the same in both periods of the experiment. This pattern of the damage includes: 1. the neighbourhood of groups of villi with total ischemic-lytic dissolution of the villi, of villi with damage of the epithelial cells at the tip and at the lateral area and of normal villi; 2. the different degree of structural damage of neighbouring epithelial cells within one villus whose cells are either of regular structural or damaged at subcellular organelles including the plasma membrane or of those being necrotically destroyed and on the way of release into the luminal space; 3. a differentiation of the structural changes of the microvilli and other organelles within single and neighbouring epithelial cells. The biochemical findings on purine nucleotide metabolism and on the formation of oxygen free radicals as "mean values" of a homogenate from a large group of cells cannot reflect the morphological-ultrastructural changes of single villi or even single epithelial cells. The possible reasons for the mosaicism of the morphological changes during ischemia and reperfusion are discussed.

Topics: Adenosine Triphosphate; Aldehydes; Animals; Glutathione; Guanosine Triphosphate; Intestine, Small; Male; Microscopy, Electron; Rats; Rats, Wistar; Reperfusion Injury

The small intestine of rats was prepared according to a procedure which is taken for preservation and transplantation in clinical practice. The blood supply of the rat intestine was completely interrupted for 30 min in situ. During this period the lumen of the intestine was rinsed with Ringer-lactate solution. This ischaemic period was followed by 10 min of reperfusion. As a result a decrease in ATP, and GTP concentrations, and of the total adenine nucleotide content during the preservation period occurred. In animals pretreated with superoxide dismutase (i.v. application; superoxide dismutase preparation from human erythrocytes) an accelerated restoration of nucleotide concentrations during the reperfusion period was observed. From the beneficial effect of superoxide dismutase it can be concluded, that there is a considerable formation of active species of oxygen which disturb the energy generation by the mitochondrial respiratory chain during ischaemia/re-oxygenation.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Energy Metabolism; Guanosine Triphosphate; Intestine, Small; Ischemia; Male; Mesenteric Arteries; Perfusion; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

This study was designed to clarify the mechanism of a reversal of the ischemia-induced decrease in adenosine triphosphate (ATP) in relation to the changes in liver blood flow. All vessels to the left and median lobes were occluded for 15 or 30 min and then reperfused for 15 or 30 min, respectively. Ischemia led to a significant decrease in the ATP level. ATP levels recovered fully after 30 min of reperfusion following 15 min of occlusion. However, a significantly low ATP level was observed even after 30 min of reperfusion following 30 min of occlusion. Premedication with CV-4151 (5 mg/kg, i.v.), a thromboxane A2 (TXA2) synthetase inhibitor, significantly improved the recovery of ATP levels after 30 min of reperfusion following 30 min of occlusion. Liver blood flow was restored fully immediately after reperfusion following 15 min of occlusion. In contrast a significantly low liver blood flow was observed after 30 min of reperfusion following 30 min of occlusion. Premedication with CV-4151 accelerated the recovery of liver blood flow after reperfusion. Morphological studies revealed that microthrombi were formed during ischemia, and CV-4151 mitigated the formation of microthrombi. These results indicate that the formation of microthrombi, which might be associated with TXA2 synthesis during ischemia, inhibited the restoration of liver blood flow, which might be responsible for the obstruction of the recovery of ATP.

Topics: Adenosine Triphosphate; Animals; Fatty Acids, Monounsaturated; Guanosine Triphosphate; Liver; Male; Pyridines; Rats; Rats, Inbred Strains; Reperfusion Injury; Thrombosis; Thromboxane-A Synthase