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.
Abrahamse
S L
SL
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Bridges
R J
RJ
De Jong
M D
MD
Bindels
R J
RJ
Van Os
C H
CH
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002469:Cell Separation; D003106:Colon / Q000166:cytology / Q000648:ultrastructure*; D004848:Epithelium / Q000648:ultrastructure; D017506:H(+)-K(+)-Exchanging ATPase / Q000032:analysis; D007093:Imidazoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000648:ultrastructure*; D007473:Ion Channels / Q000032:analysis; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis; D010042:Ouabain / Q000494:pharmacology; D011817:Rabbits
D007093:Imidazoles; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; C035235:Sch 28080; D010042:Ouabain; D000469:Alkaline Phosphatase; D017506:H(+)-K(+)-Exchanging ATPase