Enterocyte isolation procedure specifically effects ATP-dependent Ca2+-transport in small intestinal plasma membranes.
Epithelial villus cells from rat small intestine were isolated either in citrate buffers or by vibration in EDTA containing solutions. Basolateral plasmamembrane vesicles (BLMV) were isolated and active Ca2+-transport studied. The rate of ATP-dependent Ca2+-transport in BLMV was 11.2, 1.2 and 0.8 nmol Ca2+/min.mg protein in duodenum, mid-jejunum and terminal ileum when enterocytes had been isolated in citrate buffers. These transport rates were 10.5, 4.8 and 5.8 respectively when cells were isolated by vibration. The specific activities of various marker enzymes were not influenced by the cell isolation procedure. The enrichment factors for (Na+-K+)-ATPase and the latency of this enzyme activity, the mannitol spaces and the half-times for mannitol equilibration among the three BLMV preparations were independent of the cell isolation method. It was demonstrated that active Ca2+-transport in BLMV from jejunum and ileum could be destroyed when cells isolated by vibration were incubated with low proteolytic activity (1 microgram/ml trypsin). Therefore, Ca2+-ATPase in jejunum and ileum is far more susceptible to extracellular proteolytic activity than Ca2+-ATPase more proximally located. Addition of various protease inhibitors to the citrate buffer only partly prevented the selective damage of Ca2+-transport in basolateral membranes.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D002951:Citrates; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000494:pharmacology; D014732:Vibration
D002021:Buffers; D002951:Citrates; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium