5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Hypoxia

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Topics: Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Disease Models, Animal; Fluorescent Dyes; Free Radical Scavengers; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia; Ion Channels; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Proteins; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Primary Cell Culture; Pulmonary Artery; Rats; Reactive Oxygen Species; RNA, Small Interfering; Uncoupling Protein 2

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

Topics: Apoptosis; Arginine Vasopressin; Benzimidazoles; Bradykinin; Carbocyanines; Cells, Cultured; Chorionic Villi; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Epoprostenol; Female; Formazans; Humans; Hyperoxia; Hypoxia; Membrane Potentials; Mitochondria; Myometrium; Necrosis; Neovascularization, Physiologic; Oxygen; Placenta; Pregnancy; Tetrazolium Salts; Vasodilator Agents

Developing oligodendrocytes (OL precursors, pre-OLs) express alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype glutamate receptors (AMPARs) and are highly vulnerable to hypoxic-ischemic or oxygen-glucose deprivation (OGD)-induced excitotoxic injury, yet the mechanisms of injury remain unclear. Here we investigated the role of glutamate accumulation and mitochondrial function in OGD-induced pre-OL toxicity in vitro. Bulk glutamate concentration in the culture medium did not increase during OGD and OGD-conditioned medium did not transfer toxicity to naïve cells. Facilitation of glutamate diffusion by constant agitation of the culture reduced, while inhibition of glutamate diffusion by increasing medium viscosity with dextran enhanced, OGD-induced pre-OL injury. Depletion of extracellular glutamate by the glutamate scavenging system, glutamate-pyruvate transaminase plus pyruvate, attenuated pre-OL injury during OGD. Together these data suggest that local glutamate accumulation is critical for OGD toxicity. Interestingly, under normoxic conditions, addition of glutamate to pre-OLs did not cause receptor-mediated toxicity, but the toxicity could be unmasked by mitochondrial impairment with mitochondrial toxins. Furthermore, OGD caused mitochondrial potential collapse that was independent of AMPAR activation, and OGD toxicity was enhanced by mitochondrial toxins. These data demonstrate that pre-OL excitotoxicity is exacerbated by mitochondrial dysfunction during OGD. Overall, our results indicate that OGD-induced pre-OL injury is a novel form of excitotoxicity caused by the combination of local glutamate accumulation that occurs without an increase in bulk glutamate concentration and mitochondrial dysfunction. Therapeutic strategies targeting local glutamate concentration and mitochondrial injury during hypoxia-ischemia may be relevant to human disorders associated with pre-OL excitotoxicity.

Topics: Animals; Animals, Newborn; Benzimidazoles; Brain; Calcium; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; In Situ Nick-End Labeling; Ionophores; Mitochondria; Oligodendroglia; Quinoxalines; Rats; Rats, Sprague-Dawley; Time Factors

Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Coloring Agents; Energy Metabolism; Enzyme Inhibitors; Female; Fluorescent Dyes; Hypoxia; Ionophores; Kidney Tubules, Proximal; Membrane Potentials; Mitochondria; Ouabain; Phenazines; Proton-Translocating ATPases; Rabbits

Mitochondrial membrane potential (MMP) regulates the production of high-energy phosphate and apoptotic cascade, both occurring after ischemic impact. The timed profile of MMP differing from grading ischemic impact has to be determined. Primary rat hippocampal cultures were exposed to oxygen-glucose deprivation (OGD) for 30, 60, and 90 min and then were reoxygenated. MMP was expressed as a voltage-dependent dye, JC-1 fluorescence, under confocal microscopy. Cell viability was assessed by calcein AM and ethidium homodimer, each at 3 hours and 24 hours after 30, 60, and 90 min of OGD. The appearance of apoptosis was also evaluated by the TUNEL method at 24 hours. Hyperpolarization of MMP (2.31+/-0.94 normalized JC-1 fluorescence ratio between red and green) was observed during reoxygenation after 30 min OGD, while 60 min OGD induced depolarization (0.66+/-0.22, Valinomycin (potassium ionophore)-induced depolarization: 0.53+/-0.19). The fluorescence of mitochondria became weak after 90 min OGD. Most of the neurons were shrunken after 90 min and neurons were TUNEL-positive 24 hours after 30 min OGD, although most neurons were viable at 3 hours. A longer period of OGD induced necrosis, and most neurons remained viable after only 3 hours. Our data present that the short (30 min) OGD induced hyperpolarization of MMP during reoxygenation, while a longer OGD (60 or 90 min) induced depolarization and acute necrosis. Neurons were still viable even during hyperpolarization of mitochondria, but this hyperpolarization appears to be linked to subsequent apoptotic change.

Topics: Animals; Animals, Newborn; Antigens; Apoptosis; Benzimidazoles; Bromodeoxyuridine; Carbocyanines; CD11b Antigen; Cell Death; Cell Survival; Cells, Cultured; Fluoresceins; Galactosylceramides; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Humans; Hypoxia; In Situ Nick-End Labeling; Membrane Potentials; Mice; Microtubule-Associated Proteins; Mitochondria; Neurons; Oligodendroglia; Oxygen; Rats; Time Factors; von Willebrand Factor

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.

Topics: Acute Disease; Animals; Benzimidazoles; Carbocyanines; Carbon Monoxide; Carotid Body; Fluorescent Dyes; Hypoxia; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Oxygen; Patch-Clamp Techniques; PC12 Cells; Potassium; Rats; Rats, Sprague-Dawley; Thiobarbiturates

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Topics: Adenosine Triphosphate; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Citric Acid Cycle; Fluorescent Dyes; Hypoxia; Ketoglutaric Acids; Kidney Tubules; Membrane Potentials; Mitochondria; Models, Biological; Oxidative Phosphorylation; Oxygen; Rabbits; Time Factors