Figure 7.

Cholesterol repletion inhibits ABCG1-dependent ATP release. HEK cells were transfected with ABCG1 or RFP; 48 h later, the cells were incubated with a mixture of 10 mm cholesterol/MβCD for cholesterol repletion or vehicle for 3 h and stimulated with hypotonic solution (final concentration, 250 mmol/kg) before the calcium response or ATP release was assayed. A, the experimental overview. B, cholesterol repletion suppressed hypotonicity-induced calcium responses from ABCG1-transfected HEK cells. Traces and quantifications of peak calcium responses (ΔF/F) with error bars are shown (n = 4). C, acetylcholine (ACh)–induced (final concentration, 100 μm) calcium responses from endogenous (Endo.) mAChRs were unaltered by cholesterol/MβCD treatment in the RFP-transfected HEK cells. Traces and quantifications of peak calcium responses (ΔF/F) with error bars are shown (n = 4). D, cholesterol repletion suppressed ABCG1-dependent ATP release. ABCG1 transfection increased hypotonicity-induced ATP release. Cholesterol repletion reduced extracellular ATP substantially in ABCG1-transfected HEK cells (n = 8) (two-way ANOVA; F(1,28) = 33.59; p < 0.001 for cholesterol comparison and F(1,28) = 18.25, p < 0.001 for ABCG1 comparison; interaction, F(1,28) = 11.56, p = 0.002). The data are means ± standard deviation (unpaired t test (B and C) and two-way ANOVA followed by Tukey's test (D)). ***, p < 0.001. RLU, relative luminescent unit.