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. 2000 Jun;84(6):631–635. doi: 10.1136/bjo.84.6.631

Basal nitric oxide production is enhanced by hydraulic pressure in cultured human trabecular cells

T Matsuo 1
PMCID: PMC1723520  PMID: 10837391

Abstract

BACKGROUND/AIMS—Nitric oxide donors reduce intraocular pressure. Human trabecular cells in culture were examined for their nitric oxide production in response to hydraulic pressure.
METHODS—Human trabecular cells were cultured from trabeculum tissue fragments excised during trabeculectomy and exposed to hydraulic pressure change in a culture flask connected to a glass syringe. The pressure was exerted by automatic infusion of the piston of the syringe and monitored by a pressure gauge. The intracellular nitric oxide level was measured in real time with a nitric oxide binding fluorescent dye, diaminofluorescein-2.
RESULTS—Intracellular nitric oxide levels in cultured trabecular cells showed spontaneous fluctuation during 400 seconds of observation. Peak levels of intracellular nitric oxide were significantly higher at hydraulic pressure of 30, 40, and 50 mm Hg, compared with 0 and 25 mm Hg (p<0.0001, one way ANOVA, and p<0.05, Tukey-Kramer test). The fluctuation was completely abolished by the presence of N-methyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor. The cultured trabecular cells were shown by immunohistochemistry to express brain nitric oxide synthase (bNOS).
CONCLUSION—Higher levels of hydraulic pressure enhanced basal production of nitric oxide in human trabecular cells. Nitric oxide would be a physiological mediator in the regulation of intraocular pressure.



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Figure 1  .

Figure 1  

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Schematic drawing to show a fluid filled closed system to exert hydraulic pressure on cultured cells.13

Figure 2  .

Figure 2  

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Spontaneous changes in intracellular nitric oxide levels of human trabecular cells at hydraulic pressure of 0, 25, and 50 mm Hg (top to bottom). Nitric oxide levels are higher at the pressure of 50 mm Hg, compared with 0 and 25 mm Hg. Each frame is obtained at 0, 180, and 360 seconds (left to right). Changing intensity of fluorescence, indicative of nitric oxide levels, is shown in pseudocolour. The leftmost column shows phase contrast micrographs of trabecular cells measured for nitric oxide.

Figure 3  .

Figure 3  

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Spontaneous fluctuation of intracellular nitric oxide in each cell at the pressure of 0, 25, and 50 mm Hg during 400 seconds of observation. Fluorescence intensity in each cell of the series of 20 images (shown partly in Fig 2) is quantified. Fluorescence intensity is given in arbitrary units.

Figure 4  .

Figure 4  

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Peak levels of intracellular nitric oxide in human trabecular cells at different levels of hydraulic pressure. Nitric oxide levels are significantly higher at the pressure of 30, 40, and 50 mm Hg, compared with those at the pressure of 0 and 25 mm Hg (p<0.0001, one way ANOVA, asterisks: p<0.05, Tukey-Kramer test, n=20 cells for each pressure level). Fluorescence intensity is normalised by dividing a peak level by a lowest level at the beginning of observation.

Figure 5  .

Figure 5  

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Immunohistochemistry of nitric oxide synthases on human trabecular cells in culture. Trabecular cells are positive for brain nitric oxide synthase (A), while negative for endothelial nitric oxide synthase (B). Bar = 100 µm.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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