oxalylglycine and ethyl-protocatechuate

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (DeltaPsi(mito)) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, DeltaPsi(mito) is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse DeltaPsi(mito) in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of DeltaPsi(mito) in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F(1)F(0)-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only approximately 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult.

Topics: Adenosine Triphosphate; Amino Acids, Dicarboxylic; Animals; Animals, Newborn; Cell Survival; Cells, Cultured; Cyanides; Cytoprotection; Deoxyglucose; Glycogen; Hydroxybenzoates; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Potential, Mitochondrial; Mice; Mitochondrial Proton-Translocating ATPases; Myocytes, Cardiac; Oxygen; Oxygen Consumption; Procollagen-Proline Dioxygenase; Signal Transduction

Recently a family of O(2)-dependent prolyl hydroxylase domain-containing enzymes (PHD) has been identified as a cellular oxygen-sensing mechanism. Reduced prolyl hydroxylase activity initiates a signalling cascade that includes the accumulation, as well as the activation, of hypoxia-inducible factor (HIF-1alpha). In turn the transcription factor HIF-1alpha, and other targets of the PHD, elicit a myriad of incompletely understood cellular responses. In these studies we have tested: (1) whether a small-molecule prolyl hydroxylase inhibitor (PHI) can effectively activate the oxygen-sensing pathway when administered systemically to mice, and (2) whether the activation of the PHD signalling pathway at the cellular level results in whole-animal hypoxic tolerance.. Mice received daily injections of the PHI, ethyl-3,4 dihydroxybenzoate (EDHB, 100-250 mg kg(-1)) or vehicle. Tissue levels of HIF-1alpha and the serum levels of the HIF-inducible gene, erythropoietin (EPO), were measured to evaluate PHD-pathway activation. To evaluate hypoxic tolerance, the endurance and survival ability of these animals was tested in sublethal (8% O(2)) and lethal hypoxia (5% O(2)) respectively.. Systemic treatment of mice with the PHD inhibitor, EDHB, leads to elevated levels of HIF-1alpha in liver and HIF-inducible EPO in serum, indicating activation of the cellular oxygen-sensing pathway. Animals treated with EDHB display significantly increased viability and enhanced exercise performance in hypoxia.. These results demonstrate a novel pharmacological strategy to induce hypoxic tolerance and are the first to demonstrate that the activation of the PHD oxygen-sensing pathway at the cellular level is sufficient to produce a hypoxic-tolerant phenotype at the physiological level of the whole animal.

Topics: Amino Acids, Dicarboxylic; Animals; Enzyme Inhibitors; Erythropoietin; Hematocrit; Hydroxybenzoates; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Injections, Intraperitoneal; Liver; Male; Mice; Mice, Inbred Strains; Oxygen; Physical Conditioning, Animal; Physical Endurance; Procollagen-Proline Dioxygenase; Signal Transduction

Nerve growth factor (NGF) serves a critical survival-promoting function for developing sympathetic neurons. Following removal of NGF, sympathetic neurons undergo apoptosis characterized by the activation of c-Jun N-terminal kinases (JNKs), up-regulation of BH3-only proteins including BcL-2-interacting mediator of cell death (BIM)(EL), release of cytochrome c from mitochondria, and activation of caspases. Here we show that two small-molecule prolyl hydroxylase inhibitors frequently used to activate hypoxia-inducible factor (HIF) - ethyl 3,4-dihydroxybenzoic acid (DHB) and dimethyloxalylglycine (DMOG) - can inhibit apoptosis caused by trophic factor deprivation. Both DHB and DMOG blocked the release of cytochrome c from mitochondria after NGF withdrawal, whereas only DHB blocked c-Jun up-regulation and phosphorylation. DHB, but not DMOG, also attenuated the induction of BIM(EL) in NGF-deprived neurons, suggesting a possible mechanism whereby DHB could inhibit cytochrome c release. DMOG, on the other hand, was substantially more effective at stabilizing HIF-2alpha and inducing expression of the HIF target gene hexokinase 2 than was DHB. Thus, while HIF prolyl hydroxylase inhibitors can delay cell death in NGF-deprived neurons, they do so through distinct mechanisms that, at least in the case of DHB, are partly independent of HIF stabilization.

Topics: Amino Acids, Dicarboxylic; Animals; Animals, Newborn; Cell Death; Cells, Cultured; Cytochromes c; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Hydroxybenzoates; Mitochondria; Nerve Growth Factor; Neurons; Procollagen-Proline Dioxygenase; Proto-Oncogene Proteins c-jun; Rats; Superior Cervical Ganglion