Unstirred Water Layers and the Kinetics of Organic Cation Transport.

PURPOSE: Unstirred water layers (UWLs) present an unavoidable complication to the measurement of transport kinetics in cultured cells, and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. METHODS: Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. RESULTS: Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. CONCLUSIONS: Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the apparent affinity of multidrug transporters for substrates.

A mechanical force contributes to the "osmotic swelling" of brush-border membrane vesicles.

Brush-border membrane vesicles and an osmotic swelling assay have been used extensively to monitor the pore-forming activity of Bacillus thuringiensis toxins. After a hypertonic shock, Manduca sexta midgut brush-border membrane vesicles shrink rapidly and reswell partially to a volume that depends on membrane permeability and toxin concentration rather than regaining their original volume as expected from theoretical models. Because efflux of buffer from the vesicles, as they shrink, could contribute to this phenomenon, vesicles were mixed with a hypertonic solution of the buffer with which they were loaded. Under these conditions, they are not expected to reswell, since the same solute is present on both sides of the membrane. Nevertheless, with several buffers, vesicles reswelled readily, an observation that demonstrates the involvement of an additional restoration force. Reswelling also occurred when, in the absence of toxin, the buffers were replaced by glucose, a solute that diffuses readily across the membrane, but did not occur with rat liver microsomes, despite their permeability to glucose. Unexpected swelling was also observed with rabbit jejunum brush-border membrane vesicles, suggesting that the cytoskeleton, present in brush-border membrane vesicles but absent from microsomes, could be responsible for the restoration force.