No abstract

Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 Ն MRP6 Ͼ MRP4 Ն MRP3 Ͼ MRP1 ϭ MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP an-Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

No abstract

A major focus in gene therapy has been the use of recomand lesions at a young age. After transplantation of the binant viruses to deliver genes in vivo. Although this apoE secreting Pro 175 endothelial cells into apoEapproach shows much promise, there are many safety deficient mice, serum cholesterol levels were measured at concerns associated with the use of viral materials in the 2 week intervals. During the 3 months after the initiation of treatment of human diseases. Our alternative cell-based these experiments, levels of cholesterol in the animals havgene therapy approach utilizes endothelial cells (Pro 175) ing received the apoE secreting endothelial cells were statisolated from the murine embryonic yolk sac. These endoistically lower compared with the levels of age-matched thelial cells were evaluated for their potential use in gene controls having received non-secreting endothelial cells. therapy as a gene delivery platform. As a test model, we Concomitant with cholesterol reduction, atherosclerotic used these cells to deliver apolipoprotein E (apoE) in the aortic plaques were noticeably reduced in the experimental murine apoE knockout atherosclerosis model. The lack of apoE+ animals. These results highlight the potential of apoE protein in these animals results in high levels of these unique endothelial cells as an efficient delivery serum cholesterol and formation of severe aortic plaques platform for somatic gene therapy.