Functional expression of p-glycoprotein and multidrug resistance-associated protein (mrp1) in primary cultures of rat astrocytes

Abstract

Although it has been well established that the drug efflux pump P-glycoprotein (P-gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial. The aim of this study was to compare the expression of P-gp and of multidrug resistance-associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types. Western blotting analysis revealed higher expression of P-gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P-gp and Mrp1 expression 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 effect of P-gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (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 astrocytes. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P-gp). Taken together, our results provide the first biochemical and functional evidence supporting the expression of P-gp and Mrp1 in rat cultured astrocytes.

Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture

“…The present RT-PCR study demonstrated molecular expression of connexins (Cx32, npg Cx37, Cx43), Px1, MDR1, MRP1 and the P2X7 receptor in both non-swollen and swollen mouse astrocytes. These data are in agreement with those from previous RT-PCR studies that examined MRP1 [30,48] and the P2X7 receptor [49] in mouse astrocytes in normotonic conditions, and Px1 [50], MDR1 [31,45,51] and MRP1 [31,48] in rat astrocytes, also in normotonic conditions. However, the present study failed to detect the CFTR transcript in mouse astrocytes, in contrast to a previous report [30].…”

Evaluation of P-glycoprotein expression in pain relevant tissues: understanding translation of efflux from preclinical species to human

“…P-gp was expressed in the cortical tissues of all species (Figures 3a to 3d) as has been reported previously [24,25]. The localization of P-gp in the brain is controversial with some studies suggesting endothelial expression [24,25], while others reporting expression in the astrocytes or glial cells [26][27][28]. Nonetheless, even by using antibodies against doi: 10.5599/admet.4.3.305 265 distinct P-gp epitopes (C219 and H241), immunoreactivity was exclusively detected in capillary endothelial cells; neuronal expression of P-gp in the brain tissue was not observed in the species evaluated in the present study.…”

Molecular and functional MDR1‐Pgp and MRPs expression in human glioblastoma multiforme cell lines

“…15 Furthermore, astroglial expression of Mrp1 has been already observed in primary cultures of rat astrocytes, suggesting that astrocytes may also express this MDR protein during tumour transformation. 33,34 Our results show that MRP1 was expressed at both mRNA and protein level in the GL15 and 8MG cell lines, with a higher expression in the 8MG cell line. The higher MRP1 expression in the 8MG cells determined by Western blot was also found by immunocytochemistry, which revealed in addition that MRP1 expression occurs predominantly at the cell membrane of 8MG and GL15 cells.…”