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

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.