Role of Mitochondrial Injury During Oxidative Injury to Hepatocytes: Evidence of a Mitochondrial Permeability Transition by Laser Scanning Confocal Microscopy
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
In freshly isolated rat hepatocytes, cyanide caused progressive cell killing over 2½ hours (Figure 1). Fructose protected completely against this cell killing. To test the hypothesis that protection was mediated by glycolytic ATP generation, we examined the effect of the mitochondrial uncoupler, CCCP, on fructose protection. CCCP collapses the mitochondrial proton electrochemical gradient and stimulates the mitochondrial F1F0-ATPase. Thus, if fructose protection is mediated through intracellular ATP generation, accelerated ATP hydrolysis induced by CCCP should block fructose protection. This is what was found experimentally (Figure 1). To further test the hypothesis that cell killing is ATP-linked, we evaluated the effect of oligomycin. Oligomycin is an inhibitor of the mitochondrial F1F0- ATPase. By itself, oligomycin is cytotoxic,3 but in the presence of fructose and CCCP, oligomycin should block accelerated ATP hydrolysis and prevent cell killing. Again, the experiments confirmed this expectation (Figure 1). In parallel experiments, we measured cellular ATP levels. Cyanide caused ATP to fall below measurable levels (Figure 2). Fructose partially restored ATP, but CCCP blocked the fructose-induced restoration of ATP, an effect reversed by oligomycin. In every case a positive correlation was observed between preservation of cell viability and ATP.
The Toxicology and Biological Properties of Organotin Compounds*
Nate F. Cardarelli in Tin as a Vital Nutrient:, 2019
The action of trialkyltins directly on the energy conservation apparatus (ATP synthase system) has many points in common with oligomycin. Respiration of mitochondria stimulated by the uncouplers such as 2,4-dinitrophenol is not inhibited either by oligomycin or trialkyltins. In contrast ATP hydrolysis stimulated by 2,4-dinitrophenol is inhibited by both oligomycin and trialkyltins. These findings which are obtained in a chloride-free medium indicate that the action of trialkyltins is on the ATP synthase system.52 Other observations showing differences between the action of trialkyltins and oligomycin and the observation that yeast mutants can be obtained that are resistant to the action of trialkyltins, but not to oligomycin, probably indicate that the binding site for these two compounds are not identical.110–112 A binding site for trimethyl- and triethyl-tin has been established with the correct affinity constant to explain the inhibitory effect.107,116 Recent work has provided some evidence that this high affinity site is a proteolipid or is closely associated with a proteolipid.108
Ultraviolet and Light Absorption Spectrometry
Adorjan Aszalos in Modern Analysis of Antibiotics, 2020
The antibiotic oligomycin A (26-methylrutamycin) contains a 26-membered lactone ring. Its UV spectrum shows two characteristic absorptions [236] at 225 nm (ε = 2020 m2/mol; conjugated diene, π ⃗ π*) and at 232 nm (ε= 1820 m2/mol; α,βunsaturated lactone,π ⃗ π*). In oligomycins A, B, and C there are differences in the intensity of the two bands. Primicin contains a 36-membered macrocycle substituted with a side chain bearing a guanidine group. Its UV spectrum displays a high-intensity absorbance (ε= 3777 m2/mol) at 209 nm.
Cold stored platelets in the management of bleeding: is it about bioenergetics?
Published in Platelets, 2023
Chloe E. George, Christine V. Saunders, Alex Morrison, Tom Scorer, Sarah Jones, Nina C. Dempsey
The use of modulators of the ETC in the XF analyzer has the potential to provide a more precise understanding of platelet concentrate metabolism during storage through the isolation of individual parts of the ETC. The use of the inhibitor oligomycin inhibits ATP synthase, resulting in a reduction in the OCR that reflects the fraction of oxygen consumption as a result of ATP synthesis via OXPHOS. The subsequent addition of rotenone (a complex I inhibitor) and antimycin A (a complex III inhibitor) shuts down mitochondrial respiration and, with the simultaneous measurement of the acidification rate, enables the calculation of glycolysis-driven ATP production [87]. The respiratory reserve of mitochondria can be measured with the addition of a proton uncoupler, which allows protons to bypass the ATP synthase and consume oxygen at the maximum potential rate. By subtracting the basal OCR from this maximal rate, it is possible to quantify the ability of the cells to respond to a stressor with increased ATP production [88].
Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus
Published in Chronobiology International, 2023
Prabha Rajput, Dhanananajay Kumar, Sairam Krishnamurthy
Mitochondrial respiration was assessed as previously described by (Rajput and Krishnamurthy 2022; Samaiya and Krishnamurthy 2015), with a Clark-type electrode in a sealed, thermostatically controlled chamber at 37°C. Briefly, the mitochondria were added to the respiratory chamber states were evaluated with suitable substrates and inhibitors. Purified mitochondrial protein was suspended in a respiration buffer in a final volume of 250 μL. State II respiration was initiated by adding pyruvate/malate; (P/M) shows a basal respiration rate. The addition of adenosine diphosphate-initiated state III respiration; (ADP); the high level of oxygen utilization indicates that ADP is converted into ATP. The addition of oligomycin measured state IV. The addition of FCCP measured state V. This causes uncoupling of the ETC to ATP synthesis and represents the maximum respiration rate. Rotenone was then added to shut down complex I-mediated respiration. The addition of succinate determined state V. This is the maximum respiration rate via complex II since FCCP is present. The RCR was calculated by dividing state III respiration (presence of ADP) by state IV respiration (absence of ADP).
Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions
Published in Platelets, 2022
Noriko Tamura, Shinichi Goto, Hideo Yokota, Shinya Goto
The contributing roles of mitochondrial function in platelet adhesion, activation, and thrombus formation were shown by three of mitochondrial function inhibitors with different mechanisms of action. The FCCP is an uncoupler of mitochondrial oxidative phosphorylation [29]. Antimycin A blocks the function of cytochrome-c reductase in mitochondrial complex III [5,30,31]. Oligomycin is a specific inhibitor of F1F0-ATP synthase at mitochondria. All three inhibitors worked at the dose tested because the glucose consumption rates increased in their presence. Thus, our results of inhibited rapid increase in [Ca2+]i upon adhering VWF is likely dependent on the inhibition of mitochondrial function. Mitochondrial function is important for cell signaling and death [32]; one may argue that the reduced rise in [Ca2+]i in platelets adhering to VWF may be based on the vital status of platelets in the presence of mitochondrial functional blockers. To avoid potential cell death induced by the presence of mitochondrial functional blockers, we conducted all experiments within 2 h after drawing blood. Moreover, our experimental results that the addition of mitochondrial functional blockers did not influence platelet adhesion and thrombus formation support the notion that a substantial number of platelets are still alive even in the presence of the three mitochondrial functional blockers we used in our experiments. However, further investigations are necessary for a precise understanding of the role of mitochondria and the rapid increase in [Ca2+]i.
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