Expression of P-glycoprotein (ABCB1) and Mrp1 (ABCC1) in adult rat brain: focus on astrocytes.

P-glycoprotein (P-gp, ABCB1) and the multidrug resistance-associated protein 1 (Mrp1, ABCC1) are two ATP-driven pumps that mediate the export of organic anions from cells and may confer cellular resistance to many cytotoxic hydrophobic drugs. Immunohistochemistry has shown that P-gp is expressed in rat brain capillary vessels forming the blood-brain barrier (BBB). Mrp1 mRNAs have been detected by RT-PCR in rat brain isolated capillaries. Although many studies have been published in this field, very little information is available on the expression, distribution and physiological functions of the two pumps in rat brain. To characterize the cerebral expression of both P-gp and Mrp1 transporters, we studied immunoreactions of rat brain sections with the two most commonly used antibodies: the monoclonal C219 (anti-P-gp) and the polyclonal 6KQ (anti-Mrp1). Immunological analyses revealed heterogeneity of the P-gp and Mrp1 expressions in rat brain. Indeed, choroidal and ependymal cells expressed Mrp1 rather than P-gp. However, tanycytes lining the third ventricle were strongly immunoreactive with both antibodies, suggesting a particular role for these cells in drug efflux mechanisms. Because of the detection of a 70-kDa component with 6KQ antibodies, immunoreactions obtained in rats were compared with these obtained in wild type and mrp1(-/-) mice. It showed that a positive reaction at the apical surface of the ependymal layer remained obvious, showing that 6KQ antibodies recognize an ependymal molecule, differing from the Mrp1. In addition, a continuous expression of C219-labeled epitopes, similar to endothelial labeling, was detected at the blood-brain barrier, whereas a discontinuous labeling, co-localized with glial fibrillary acidic protein (GFAP) immunostaining, was obtained with 6KQ antibodies. We showed that P-gp was preferentially expressed in the endothelial component and Mrp1 in the astroglial component of the blood-brain barrier. Moreover, Mrp1 was rather expressed than P-gp in parenchyma astrocytes and in glia limitans lining the meninges. These findings provide new insights into the cerebral distribution of two ABC transporters linked to multidrug resistance (MDR).

P-glycoprotein (ABCB1) but not multidrug resistance-associated protein 1 (ABCC1) is induced by doxorubicin in primary cultures of rat astrocytes.

At least two drug efflux pumps involved in multidrug resistance, P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (Mrp1), are expressed in rat astrocyte primary cultures. The aim of this study was to compare the expression of P-gp and Mrp1 in primary cultures exposed to 50 or 500 ng/mL doxorubicin (DOX). Among the two P-gp genes expressed in rodents, mdr1a and mdr1b, a time- and dose-dependent increase in mdr1b mRNA levels was revealed by northern blot analysis. This up-regulation was inhibited by actinomycin D and occurred as early as 2 h after exposure to 50 or 500 ng/mL DOX, whereas mdr1a and mrp1 transcripts were not modified by the DOX exposure. In addition, DOX also strongly enhanced, in a time- and dose-dependent manner, P-gp but not Mrp1 expression. Moreover, DOX raised the cellular efflux of vincristine, a substrate for both P-gp and Mrp1. This efflux was inhibited by the P-gp modulators PSC833 and GW918, but not by the Mrp1 modulator MK571. On the other hand, a 24-h exposure to 500 ng/mL DOX, but not 50 ng/mL DOX, induced apoptosis in primary cultures of rat astrocytes. Fumonisin B1, a ceramide synthase inhibitor, reduced DOX-induced apoptosis, suggesting that de novo synthesis of the ceramide regulatory pathway might be involved in DOX-induced apoptosis. Moreover, western blot analysis showed that fumonisin B1 was not able to decrease the overexpression of P-gp induced by DOX. Our results provide evidence that DOX up-regulates a functional P-gp in primary cultures of rat astrocytes and might cause astrocyte apoptosis via the ceramide pathway.

CSF-contacting epithelia in altered gravity: changes in apical structures and water channels expression.

Apical cytoskeletal structures and water channels are affected in both choroidal and ependymal cells lining the cerebral ventricles. Structural alterations and changes in expression of AQPI and AQP4, evaluated by immuno-cytochemistry and in situ hybridization confirm the impact of variations in gravity in CSF-lining epithelia.