The Effects of Bisphosphonates on Taurine Transport in Retinal Capillary Endothelial Cells Under High Glucose Conditions - Advances in Experimental Medicine and Biology - page 58

7.2.2 [ 3 H]Taurine Uptake Study in TR-iBRB Cells

TR-iBRB cells were cultured at 33°C on rat tail collagen type 1-coated 24-well
plates (Iwaki, Tokyo, Japan) for 2 days and washed with 1 mL extracellular fluid
(ECF) buffer consisting of 122 mM NaCl, 25 mM NaHCO 3 , 3 mM KCl, 1.4 mM
CaCl 2 , 1.2 mM MgSO 4 , 0.4 mM K 2 HPO 4 , 10 mM D-glucose, and 10 mM Hepes (pH
7.4) at 37°C. Uptake was initiated by addition of 200 mL ECF buffer containing [ 3 H]
taurine at 37°C. After appropriate time periods, uptake was terminated by removing
the solution and washed with 1 mL ice-cold ECF buffer. To investigate the change
of taurine uptake under high glucose condition, the TR-iBRB cells were pretreated
with 25 mM glucose for 2, 4, 8, 12, 24, and 48 h and the uptake study was performed
as described above. To test the effect of bisphosphonates on the change of taurine
uptake under high glucose condition, TR-iBRB cells were exposed to 10 m M alen-
dronate and 10 mM pamidronate in the presence or the absence of 10 m M gera-
nylgeraniol (GGOH) for 0.5, 4, and 6 h under exposing TR-iBRB cells to 25 mM
glucose for 48 h. Then, the cells were dissolved in 1 N NaOH overnight at room
temperature. An aliquot (50 mL) was taken for protein assay using a DC protein
assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard.
The remaining solution (500 mL) was mixed with 4.5 mL of scintillation cocktail
(Hionic-fluor, Packard, Meriden, CT, USA) for the measurement of radioactivity
using a liquid scintillation counter (LS6500, Beckman Instruments Inc. Fullerton,
CA, USA).

7.2.3 Statistical Analysis

Statistical significance was determined by one-way ANOVA with Dunnett's post
hoc test. Each value was expressed as the mean ± SEM. Differences were consid-
ered statistically significant when the calculated P value was less than 0.05.

7.3 Results

7.3.1 Effect of High Glucose on the [ 3 H]Taurine Uptake

in TR-iBRB Cells

To investigate the effect of excess glucose on taurine transport at the iBRB, [ 3 H]
taurine uptake activity was examined in TR-iBRB cells under 25 mM glucose
pretreatment conditions. By exposing TR-iBRB cells to high glucose for 48 h,
[ 3 H]taurine uptake was decreased continuously (Fig. 7.1 ).