Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells.
Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.
Svoboda
M
M
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
Dupuche
M H
MH
Lambert
M
M
Bui
D
D
Christophe
J
J
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002766:Cholecystokinin / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005755:Gastrins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D010179:Pancreas / Q000378:metabolism*; D010190:Pancreatic Neoplasms; D051381:Rats; D012844:Sincalide / Q000031:analogs & derivatives / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured
D001152:Arsenicals; D004140:Dinitrophenols; D005755:Gastrins; D007457:Iodine Radioisotopes; D013388:Succinimides; C029341:oxophenylarsine; C055121:Bolton Hunter-cholecystokinin nonapeptide; D015638:Cytochalasin D; D002738:Chloroquine; D002766:Cholecystokinin; D012844:Sincalide