Figure 5.

E2 decreases BCRP transport activity. (A) Representative images of isolated rat brain capillaries incubated for 1 h with 2 μmol/L BODIPY FL prazosin with or without E2. Luminal steady-state accumulation of BODIPY FL prazosin decreased with increasing concentrations of E2. (B) Analysis of confocal images reveals a concentration-dependent decrease of luminal BODIPY FL prazosin fluorescence in capillaries exposed to E2. (C) Nonlinear regression curve for E2 (Hill coefficient=1); EC50: 0.18 nmol/L. (D) Rapid decrease of BCRP transport activity in capillaries exposed to E2. Capillaries were loaded with 2 μmol/L BODIPY FL prazosin for 60 mins to steady state. After adding 10 nmol/L E2 (time 0 min), BCRP transport activity decreased within 15 mins. (E) E2 effects on BCRP transport activity are reversible. Capillaries were loaded for 60 mins to steady state with 2 μmol/L BODIPY FL prazosin. When 10 nmol/L E2 was added to the buffer (time 0 on graph), BCRP activity decreased rapidly; BCRP activity recovered completely when E2 was removed (time point 45 mins). (F) E2 had no effect on luminal accumulation of sulforhodamine 101, an Mrp2-specific substrate, whereas mannitol, a tight junction opener, and the Mrp2 inhibitor, LTC₄, both decreased Mrp2-mediated sulforhodamine 101 accumulation in brain capillaries. These data indicate that E2 did not alter tight junction permeability, but specifically reduced luminal BODIPY FL prazosin fluorescence through decreased BCRP transport activity. Each data point represents the mean±s.e.m. for 7 to 10 capillaries from a single preparation (pooled tissue from 10 rats). Units are arbitrary fluorescence units (scale 0 to 255). Statistical comparison: ^**significantly lower than controls, P<0.01, ^***significantly lower than controls, P<0.001.