Phenytoin Transport by RLIP76. A) Requirement of both ATP and RLIP76 for increased liposomal uptake of 14C-PHE. Experiments in a-e were performed four times and triplicate determinations were performed for each data point. B) Time dependent uptake of 14C-PHE and 14C-CBZ by RLIP76-proteoliposomes in the presence of ATP. Data were fitted by y = y0+A1e(-x/t). C) Saturable kinetics of 14C-PHE and 14C-CBZ transport by purified recombinant human RLIP76 with respect to ATP concentrations. Data were fitted by a single exponential. D) Transport of PHE and CBZ by RLIP76-proteoliposomes. Radiolabeled drugs were incubated in the absence or presence of 4 mM ATP with RLIP76 liposomes containing variable amounts of RLIP76. Unless both ATP and RLIP76 were present, drug-uptake was close to the detection limit. Data points were fitted by a Sigmoid y = A2 + (A1-A2)/(1 + exp(x-x0)/dx). E) Determination of Km values for RLIP76-mediated transport of 14C-PHE and 14C-CBZ. F) RLIP76 mRNA expression in normal (aneurysm, HUVEC) and "epileptic" endothelial cells isolated from tissue resections [31,34]. Note that RLIP76 was significantly (p < 0.05) increased in endothelial cells isolated from multiple drug resistant patients (n = 6) compared to control tissue (n = 8). G) Analysis of specimens by Western-blot analysis confirmed these findings. We compared protein expression in dysplastic or normal cortex within the same patient [42,43]. Note that the actively epileptic cortex was characterized by gross abnormality and increased expression of RLIP76. The Coomassie stained band shows the migration pattern of purified human RLIP76 to emphasize the increased levels of signal in both bands. The arrows point to histological sections from the same regions used to isolate protein. Note that abnormal clustering of cells is evident on H&E stained sections and in neighboring samples stained with DAPI. The bars indicate 100 μm; the dotted and dashed lines show the extent of abnormal nuclear clustering while the asterisks refer to abnormal vascular structure present in these grossly malformed cortices. H) RLIP76 is up-regulated in epileptic brain and RLIP76 protein expression levels correlate with transport activity in inside-out membrane vesicles prepared from the same tissues. Data were fitted linearly (R value of 0.99).
Awasthi et al. BMC Neuroscience 2005 6:61 doi:10.1186/1471-2202-6-61