Abstract
Purpose::
Nitric oxide (NO) is responsible for a variety of physiological functions throughout the body. In the eye, several tissues are capable of generating NO and utilizing it for regulation of IOP and ocular blood flow. Alterations of this pathway may play a role in the development of glaucoma. Due to the transient nature of NO, it is difficult to assess its levels in real time. Therefore, the purpose of this study was to: 1) evaluate a method of detecting NO levels in real time in ocular cells; 2) establish a method of detecting NO and cGMP (a marker of NO activity) in rabbit ocular tissue; and 3) assess the release kinetics of nitric oxide from NO Donors using these two methods.
Methods::
In vitro: Human primary ocular cells were incubated with NO donors after loading with diaminofluorescein (DAF) and nitric oxide release was detected via fluorescence microscopy and quantified using ImagePro. In vivo: Sodium nitroprusside (SNP) and S-Nitroso-N-penicillamine (SNAP), NO-donating compounds were administered topically to the eyes of normotensive rabbits. NO levels were assessed in the aqueous humor and iris/ ciliary body (ICB) at various time points for each compound, using a Nitrate/Nitrite Fluorometric assay kit. Levels of cGMP were also measured using an EIA kit.
Results::
In vitro: SNP and SNAP produced a time and concentration-dependent increase in fluorescence intensity in human primary ocular cells. In vivo: A slight increase in NO levels was detected in the rabbit aqueous humor and ICB after topical administration of the NO donors. However, cGMP levels increased more dramatically in the aqueous humor compared to NO levels indicating that determination of cGMP levels may be more suitable for in vivo assessment of NO activity.
Conclusions::
The in vitro cell based assay is an effective method of visualizing the real-time NO release from the donor compound and assessment of its intracellular levels. The Nitrite/Nitrate fluorometric assay is capable of detecting NO in rabbit samples but the cGMP EIA kit provides a better window from control in a time course study. Therefore, both the in vitro and in vivo assays provide valuable information when studying nitric oxide release from NO donors in the eye.
Keywords: nitric oxide • ciliary muscle • aqueous