34] Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes

Abstract

Hepatocytes represent highly polarized secretory cells that exhibit eificient transport of a wide variety of endogenous and exogenous compounds from blood into bile. While sinusoidal and lateral surfaces (i.e., the "basolateral" pole of hepatocytes) provide for etficient exchange of various ions, organic solutes, and proteins with blood plasma, the bile canalicular or apical pole of the cell is separated from the plasma space by tight junctions and is highly specialized for the primary secretion of bile. To be able to separately study basolateral and canalicular membrane transport processes without interference of intracellular metabolic events, it is necessary to selectively isolate basolateral (blLPM) and/or canalicular (cLPM) liver plasma membrane vesicles. Hence, for in vitro transport studies isolation procedures are still required that result in both high yields and high purification of basolateral and canalicular membrane subfractions within reasonable working hours. The procedure described here represents an extensive modification of the Evan's procedure using a combination of rate zonal flotation and high speed discontinuous sucrose gradient centrifugation techniques. The method permits the simultaneous isolation of bILPM and cLPM vesicles from the same homogenate in a yield sufficient for a large number of individual transport studies to be carried out in both plasma membrane subfractions.