Apical membrane isolation of surface and crypt cells from rabbit distal colon

Surface and crypt cells of rabbit distal colon were separately isolated, and amiloride-sensitive 22Na+ uptake could only be demonstrated in a crude membrane fraction derived from surface cells. For purification of apical membranes of surface and crypt cells (H(+)-K+)-ATPase and alkaline phosphatase were used as putative apical membrane markers. Apical membranes of surface cells were isolated after mild homogenization, low speed centrifugation, and subsequent fractionation on a Percoll density gradient. Apical membranes of crypt cells were collected after more vigorous homogenization, followed by high speed centrifugation, and fractionation on a Percoll gradient. In surface and crypt cells, (H(+)-K+)-ATPase and alkaline phosphatase activity accumulated in a low and a high density Percoll band. Further fractionation of the low density Percoll band from crypt cells on a discontinuous sucrose gradient yielded a vesicle fraction with 7- to 10-fold enrichment in (H(+)-K+)-ATPase activities. To demonstrate the usefulness of the isolated fractions in studying transport mechanisms, vesicle volume was determined and planar lipid bilayer studies were performed. In the latter studies, a 83-pS 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive Cl(-)-channel, resembling the outward rectifying intermediate conductance (ORIC) Cl(-)-channel of secretory epithelia, was encountered most frequently. This channel was present in fractions of surface and crypt cells.