Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes

Although it has been well established that the drug efflux pump P-glycoprot ein (P-gp) protects the brain against the entry of cytotoxic drugs, its rea l in situ localization, i.e., at brain capillary endothelial cells or on as trocyte foot processes, is still controversial. The aim of this study was t o compare the expression of P-gp and of multidrug resistance-associated pro tein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astro cytes and in cultured brain capillary endothelial cells. Reverse transcript ase-polymerase chain reaction (RT-PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mR NA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp 1 was expressed in both cell types. Western blotting analysis revealed high er expression of P-gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P-gp and Mrp1 expressio n was not modified in more differentiated astrocytes obtained when cultured with db-cAMP for 48 hr. Our functional analysis of P-gp showed a modest ef fect of P-gp modulators (CsA, verapamil, PSC 833) on the uptake of colchici ne (a substrate of P-gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P-gp and MRP) by astrocyt es. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modi fy the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and su lfinpyrazone increased the uptake of fluorescein (a substrate of MRP but no t of P-gp). Taken together, our results provide the first biochemical and f unctional evidence supporting the expression of P-gp and Mrp1 in rat cultur ed astrocytes. (C) 2000 Wiley-Liss, Inc.