Serial permeability barriers to water transport in human placental vesicles

Microvillous vesicles were prepared from term human placenta by shearing, differential centrifugation and Mg2+ precipitation. Vesicles were purified further on a sucrose density gradient producing two bands with densities of 1.16 to 1.18 g/ml (C1) and 1.13 to 1.15 g/ml (C2). The C2 fraction, which had a 24-fold enrichment of alkaline phosphatase and a three-fold reduction in Na+, K+-ATPase activity compared to homogenates, was used to measure osmotic water (Pf) permeability. Pf was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes. Pf decreased from 3.0 X 10(-3) to 0.6 X 10(-3) cm/sec with increasing gradient size (65 to 730 mM; 23 degrees C). Four possible causes of this behavior were examined theoretically and experimentally: an unstirred layer, saturation of water transport, large changes in the vesicle surface area with changes in volume and a structural restriction to vesicle volume change. The measured dependence of Pf on gradient size and the effect of the channel-forming ionophore gramicidin on Pf fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects on Pf of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (Ea) for Pf. The decreased Pf resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane. Ea measured using a 250-mM inwardly directed sucrose gradient was 5.4 +/- 0.6 kcal/mol (T greater than 27 degrees C) and 10.0 +/- 0.6 kcal/mol (T less than 27 degrees C). Ea above 27 degrees C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited by p-chloromercuribenzenesulfonate.