Temperature dependence of high-affinity CCK receptor binding and CCK internalization in rat pancreatic acini.
125I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperature in isolated rat pancreatic acini. At 37 degrees C, acini readily bound and degraded 125I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (Kd, 18 pM) and low-affinity (Kd, 13 nM) binding sites. At 4 degrees C, acini bound only one-third as much 125I-CCK and degradation was essentially abolished. At 4 degrees C, CCK competition curves were consistent with a single class of low-affinity binding sites (Kd, 19 nM). Internalization of 125I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound 125I-CCK, and this finding was validated for trypsin by electron microscope autoradiography. After 1 h at 37 degrees C, washing showed 67% of bound 125I-CCK to be internalized and autoradiography showed 54% to be internalized. At 4 degrees C, internalization of bound CCK was greatly reduced but not abolished. When internalization of 125I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.
Williams
J A
JA
Cell Biology Laboratory, Mount Zion Hospital and Medical Center, San Francisco 94121.
Bailey
A C
AC
Roach
E
E
D000818:Animals; D002766:Cholecystokinin / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008854:Microscopy, Electron; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D013816:Thermodynamics
D011949:Receptors, Cholecystokinin; D002766:Cholecystokinin