SEPARATE K+ AND CL- TRANSPORT PATHWAYS ARE ACTIVATED FOR REGULATORY VOLUME DECREASE IN JEJUNAL VILLUS CELLS

We assessed ion transport mechanisms operative during regulatory volume decrease (RVD) in jejunal villus enterocytes, isolated in suspension from guinea pig jejunum and examined with electronic cell sizing. Immediately after reduction of osmolarity (153 mosmol/kg medium) enterocytes swelled, but within 5 min they shrank by 50%. This RVD, which was complete by 20 min, was unaffected by Li+ substitution for Na+ or by Na+-free (N-methyl-D-glucose, NMDG+) medium. Passive loss of K+ is required for RVD because both the magnitude and direction of RVD changed when external [K+] varied. Increasing K+ permeability with gramicidin (0.5-mu-M) accelerated RVD in NMDG+ medium (10.0 +/- 0.8 vs. 6.2 +/- 0.4% min-1, P < 0.01) suggesting that K+ loss is rate limiting for RVD. Inhibition of K+- and Ca2+-activated K+ conductance with Ba2+ (5 mM, P < 0.005), quinine (100 mu-M, P < 0.005), or apamin (1-mu-M, P < 0.005) prevented RVD. Inhibition of Cl- conductance with 9-anthracenecarboxylic acid (100-mu-M, P < 0.005) or dipyridamole (75-mu-M, P < 0.005) also prevented RVD. In isotonic HCO3--buffered medium, the addition of gramicidin to cells generated conditions in which anion permeability was rate limiting for cell swelling. This swelling was inhibited 97% by 100-mu-M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In K+-free, HCO3--buffered medium containing DIDS and gramicidin hypotonic swelling resulted in continued (secondary) swelling (rel vol 1.19 +/- 0.01 vs. 1.25 +/- 0.02, P < 0.02). When Cl- was replaced with gluconate secondary swelling was prevented (1.17 +/- 0.02 vs. 1.04 +/- 0.01, P < 0.005); it was also prevented in Cl- medium containing 9-anthracenecarboxylic acid (1.18 +/- 0.01 vs. 1.17 +/- 0.01) demonstrating hypotonic activation of Cl- conductance. Ouabain (0.1 mM) increased volume under isotonic conditions (1.09 +/- 0.01) but had no effect on RVD consistent with a lack of a role for Na+-K+-adenosinetriphosphatase in this process. Evidence from ion substitution, pharmacological sensitivity, and secondary swelling experiments demonstrates that villus enterocyte RVD requires activation of separate K+ and Cl- conductive transport pathways.