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Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

ABSTRACT: The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H(2)O(2)) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H(2)O(2) on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H(2)O(2)-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H(2)O(2) solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H(2)O(2) (-29%) and 100 mM H(2)O(2) (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H(2)O(2) caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H(2)O(2) than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult.

SUBMITTER: Masihzadeh O

PROVIDER: S-EPMC5042686 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

Masihzadeh Omid O Ammar David A DA Lei Tim C TC Gibson Emily A EA Kahook Malik Y MY

Experimental eye research 20110224 3

The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H(2)O(2)) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H(2)O(2) on the biochemistry of the TM cells and the initial respo ...[more]

PMID: 21354135

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Dataset Information

Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

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Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

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