AJP - Gastrointestinal and Liver Physiology, Vol 258, Issue 5 665-G674, Copyright © 1990 by American Physiological Society

ARTICLES

Regulatory volume increase in mammalian jejunal villus cells is due to bumetanide-sensitive NaKCl2 cotransport

R. J. MacLeod and J. R. Hamilton
Department of Pediatrics, McGill University-Montreal Children's Hospital, Research Institute, Quebec, Canada.

We assessed ion transport mechanisms operative during regulatory volume increase (RVI) in villus enterocytes isolated in suspension from guinea pig jejunum and examined with electronic cell sizing and 86Rb influx. After validation of the electronic-sizing technique with direct measurements of cell water, the response of cell volume to hypertonic media was evaluated in detail. When shrunk by exposure to hyperosmotic media (455 mosmol/kg medium) cells demonstrated a RVI that was complete in 20 min. RVI required extracellular Na+, K+, and Cl-; this cell swelling showed the following ion sensitivity; Na+ greater than Li+ greater than choline, K+ = Rb+, and Cl- greater than or equal to Br- greater than NO3- = acetate = gluconate. Bumetanide inhibition of villus cell swelling was concentration dependent from 10(-10) to 10(-5) M (7.0 +/- 4.5% vs. 87.8 +/- 0.3%); furosemide (10(-3)M) inhibited RVI (74.1 +/- 9.5%), but amiloride (10(-4) M) had little effect on cell swelling. Disulfonic stilbenes, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10(-4) M), generated the same inhibition of RVI in either nominally HCO3(-)-free phosphate-buffered saline (PBS) or HCO3(-)-buffered PBS, suggesting anion exchange was not involved. Ouabain (10(-4) M) stimulated cell swelling. Hypertonic shrinkage increased the initial rate of bumetanide-sensitive 86Rb influx (80 +/- 38 vs. 1,011 +/- 241 pmol.mg protein-1.min-1; P less than 0.005) and required extracellular Na+ and Cl- (11 +/- 16 vs. 28 +/- 61 pmol.mg protein-1.min-1). RVI was prevented in low-K+ media (0.2 mM), but the addition of KCl initiated cell swelling. Our data strongly suggest that RVI in jejunal villus enterocytes occurs because of the hypertonic activation of NaKCl2 cotransport.

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