Serial permeability barriers to water transport in human placental vesicles |
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Authors: | Nicholas P Illsley A S Verkman |
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Institution: | (1) Division of Nephrology, Cardiovascular Research Institute, University of California, 94143 San Francisco, California |
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Abstract: | Summary 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 (P
f
) permeability.P
f
was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes.P
f
decreased from 3.0×10–3 to 0.6×10–3 cm/sec with increasing gradient size (65 to 730mm; 23°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 ofP
f
on gradient size and the effect of the channel-forming ionophore gramicidin onP
f
fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects onP
f
of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (E
a) forP
f
. The decreasedP
f
resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane.E
a
measured using a 250-mm inwardly directed sucrose gradient was 5.4±0.6 kcal/mol (T>27°C) and 10.0±0.6 kcal/mol (T<27°C).E
a above 27°C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited byp-chloromercuribenzenesulfonate. |
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Keywords: | human placenta water transport microvillous membrane vesicles light scattering urea transport |
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