Limits...
Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs after Exposure to Hyperoxia.

Sepehr R, Audi SH, Staniszewski KS, Haworth ST, Jacobs ER, Ranji M - IEEE J Transl Eng Health Med (2013)

Bottom Line: ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity.The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats).Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs.

View Article: PubMed Central - PubMed

ABSTRACT
Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of subunits of mitochondrial oxidative phosphorylation complexes. Hydrogen ions are transported from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space by complexes I, III, and IV. The movement of hydrogen ions down the electrochemical gradient is coupled to the phosphorylation of adenosine diphosphate (ADP) to form adenosine triphosphate (ATP) by complex V. Electrons from the autofluorescent reducing agent, nicotine adenine dinucleotide (NADH), move from complex I through ubiquinone to complex III and then complex IV via cytochrome c (Cyt c). Electrons from succinate, another reducing agent, enter the respiratory chain through flavin adenine dinucleotide (FAD), which is covalently linked to complex II of the respiratory chain. Like NADH, the reduced form of FAD (FADH) is autofluorescent. Rotenone (ROT) and potassium cyanide (KCN) inhibit complex I and IV, respectively. Pentachlorophenol (PCP) is a protonophore which increases membrane proton conductivity, disrupts the proton gradient across the membrane, and as a result uncouples mitochondrial electron transport chain from phosphorylation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4219590&req=5

fig2: Schematic representation of subunits of mitochondrial oxidative phosphorylation complexes. Hydrogen ions are transported from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space by complexes I, III, and IV. The movement of hydrogen ions down the electrochemical gradient is coupled to the phosphorylation of adenosine diphosphate (ADP) to form adenosine triphosphate (ATP) by complex V. Electrons from the autofluorescent reducing agent, nicotine adenine dinucleotide (NADH), move from complex I through ubiquinone to complex III and then complex IV via cytochrome c (Cyt c). Electrons from succinate, another reducing agent, enter the respiratory chain through flavin adenine dinucleotide (FAD), which is covalently linked to complex II of the respiratory chain. Like NADH, the reduced form of FAD (FADH) is autofluorescent. Rotenone (ROT) and potassium cyanide (KCN) inhibit complex I and IV, respectively. Pentachlorophenol (PCP) is a protonophore which increases membrane proton conductivity, disrupts the proton gradient across the membrane, and as a result uncouples mitochondrial electron transport chain from phosphorylation.

Mentions: Recently, we demonstrated the utility of optical fluorometry (Fig. 1) to detect a change in the redox status of lung mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide ), in isolated perfused rat lungs [18]. NADH and (Flavin Adenine Dinucleotide) are mitochondrial metabolic coenzymes, and are the primary electron carriers in oxidative phosphorylation. The oxidation of these two via the mitochondrial electron transport chain involves the transport of protons from mitochondrial complexes I, III, and IV into the mitochondrial intermembrane space (Fig. 2). This creates a proton gradient, which, along with the presence of adenosine diphosphate (ADP), yields the production of the cell's basic unit of energy, adenosine triphosphate (ATP). This process accounts for approximately 85% of ATP production in lung tissue [19]. Therefore, a change in the redox state of the electron transport chain, and thus NADH and , is a quantitative marker of lung tissue mitochondrial bioenergetics, and hence mitochondrial function [10], [20].Fig. 1.


Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs after Exposure to Hyperoxia.

Sepehr R, Audi SH, Staniszewski KS, Haworth ST, Jacobs ER, Ranji M - IEEE J Transl Eng Health Med (2013)

Schematic representation of subunits of mitochondrial oxidative phosphorylation complexes. Hydrogen ions are transported from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space by complexes I, III, and IV. The movement of hydrogen ions down the electrochemical gradient is coupled to the phosphorylation of adenosine diphosphate (ADP) to form adenosine triphosphate (ATP) by complex V. Electrons from the autofluorescent reducing agent, nicotine adenine dinucleotide (NADH), move from complex I through ubiquinone to complex III and then complex IV via cytochrome c (Cyt c). Electrons from succinate, another reducing agent, enter the respiratory chain through flavin adenine dinucleotide (FAD), which is covalently linked to complex II of the respiratory chain. Like NADH, the reduced form of FAD (FADH) is autofluorescent. Rotenone (ROT) and potassium cyanide (KCN) inhibit complex I and IV, respectively. Pentachlorophenol (PCP) is a protonophore which increases membrane proton conductivity, disrupts the proton gradient across the membrane, and as a result uncouples mitochondrial electron transport chain from phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4219590&req=5

fig2: Schematic representation of subunits of mitochondrial oxidative phosphorylation complexes. Hydrogen ions are transported from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space by complexes I, III, and IV. The movement of hydrogen ions down the electrochemical gradient is coupled to the phosphorylation of adenosine diphosphate (ADP) to form adenosine triphosphate (ATP) by complex V. Electrons from the autofluorescent reducing agent, nicotine adenine dinucleotide (NADH), move from complex I through ubiquinone to complex III and then complex IV via cytochrome c (Cyt c). Electrons from succinate, another reducing agent, enter the respiratory chain through flavin adenine dinucleotide (FAD), which is covalently linked to complex II of the respiratory chain. Like NADH, the reduced form of FAD (FADH) is autofluorescent. Rotenone (ROT) and potassium cyanide (KCN) inhibit complex I and IV, respectively. Pentachlorophenol (PCP) is a protonophore which increases membrane proton conductivity, disrupts the proton gradient across the membrane, and as a result uncouples mitochondrial electron transport chain from phosphorylation.
Mentions: Recently, we demonstrated the utility of optical fluorometry (Fig. 1) to detect a change in the redox status of lung mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide ), in isolated perfused rat lungs [18]. NADH and (Flavin Adenine Dinucleotide) are mitochondrial metabolic coenzymes, and are the primary electron carriers in oxidative phosphorylation. The oxidation of these two via the mitochondrial electron transport chain involves the transport of protons from mitochondrial complexes I, III, and IV into the mitochondrial intermembrane space (Fig. 2). This creates a proton gradient, which, along with the presence of adenosine diphosphate (ADP), yields the production of the cell's basic unit of energy, adenosine triphosphate (ATP). This process accounts for approximately 85% of ATP production in lung tissue [19]. Therefore, a change in the redox state of the electron transport chain, and thus NADH and , is a quantitative marker of lung tissue mitochondrial bioenergetics, and hence mitochondrial function [10], [20].Fig. 1.

Bottom Line: ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity.The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats).Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs.

View Article: PubMed Central - PubMed

ABSTRACT
Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia.

No MeSH data available.


Related in: MedlinePlus