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RR Harris, D Boyle, K Powell, D Takemoto; Effects of Galactosemia on Rat Lens PKC- and Gap Junctions . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2350.
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Purpose: To determine and measure the effects of galactosemia on PKC-γ levels and activity, diacylglycerol levels, and gap junction activity. Methods: 6 week old Sprague Dawley rats (126-150 grams) were used for these experiments. The first group was made galactosemic by feeding them a diet of 50% galactose for up to 10 days. The second group was fed a control diet consisting of 50% fiber. Sample rats from the two groups were sacrificed on Days 1, 2, 3, 5, 7, and 10, and lenses were removed. PKC-γ protein levels were measured using western blotting, while PKC-γ activity was measured by phosphorylation assays. Diacylglycerol levels were measured by converting diacylglycerol into radioactive [32P] phosphatidic acid using the sn-1,2 Diacylglycerol (DAG) Reagents System kit by Amersham Pharmacia Biotech. Gap junction activity was analyzed by Lucifer yellow permeability in the lens for 30 minutes using a Zeiss LSM 410 confocal microscope. Dextran red was added as a control. Results: PKC-γ protein levels decreased at day 7-10. PKC-γ activity reflected an increase in diacylglycerol. This resulted in early decreases in gap junction activity (day 3). Conclusions: The changes in PKC-γ and initial decrease in gap junction activity indicate a possible down regulation of PKC-γ which may be mediated by increased diacylglycerol levels.
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