Figure 5.

Hydrogen sulfide donor and glucose-induced alterations in H₂S and its enzymatic machinery in retinal endothelial cells. HRECs, incubated in high glucose, in the absence or presence of homocysteine, and without or with GY for 96 hours were analyzed for (a) extracellular H₂S level in their culture medium using NaHS as a standard, and (b) endogenous H₂S by in situ fluorescent microscopy using azido-4-methylcoumarin fluorescence probe. The accompanying histogram represents mean ± SD from three different experiments, with each measurement performed in six or more cells. (c) Homocysteine levels were quantified using an ELISA kit, and (d, e) the activities of CBS and CSE were measured using spectrophotometric methods. Each measurement was made in duplicate or triplicate in four to five different cell preparations. (f) Concentration curve of GY on extracellular H₂S in the culture medium of HRECs incubated in high glucose. (g) HRECs incubated with 150 µM GY visualized under an Olympus BX50 microscope and imaged at 10 times magnification. NG, cells in 5 mM glucose; HG and HG/GY, cells in 20 mM glucose without and with GY, respectively; HG/H and HG/H + GY, cells in high glucose + homocysteine, in the absence and presence of GY, respectively; L-Glu, cells in 20 mM L-glucose. *P < 0.05 versus NG; #P < 0.05 versus HG, and ^P < 0.05 versus HG/H.