Studies on the fate of receptor-bound 125I-interleukin 1 beta in porcine synovial fibroblasts.
Binding of porcine interleukin 1, radiolabeled with Bolton-Hunter reagent (125I IL 1), to monolayers of porcine synovial fibroblasts (PSF) was found to be a temperature-dependent process. The rate of uptake and the amount of cell-associated ligand was higher at 37 degrees C than at 4 degrees C or 19 degrees C, and exceeded the apparent equilibrium binding capacity. The amount of bound 125I IL 1 that was removed by brief treatment with acidic buffers decreased from 80% at 4 degrees C to 35% for PSF incubated at 37 degrees C; this procedure was used to distinguish surface-bound from internalized ligand. In untreated PSF, surface binding was maximal at 1 hr and was maintained for at least 5 hr during which time the internal pool continued to increase. The lysosomotropic agent methylamine (20 mM) decreased surface binding by 50%; monensin (20 microM) decreased the rate and extent of internalization. Cycloheximide (10 micrograms/ml) did not affect ligand uptake, hence, continual expression of surface receptors could not be ascribed to their de novo synthesis. 40% of the radioactivity taken up by PSF during incubation at 37 degrees C subsequently appeared in the culture medium upon prolonged postincubation (5 hr) in the absence of added 125I IL 1: 60% of this fraction was trichloroacetic acid-soluble in untreated cultures, but the extent of degradation was halved by treatment with methylamine or monensin. Direct measurement of the rate of internalization of prebound 125I IL 1 was obtained by monitoring the formation of covalently cross-linked ligand-receptor complexes after warming PSF monolayers to 37 degrees C. By using gel electrophoresis we observed a decrease (t1/2 = 9 to 11 min) in labeling of the major cross-linkable species.
D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007375:Interleukin-1 / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism*; D017472:Receptors, Interleukin-1; D013552:Swine; D013583:Synovial Membrane / Q000378:metabolism*; D013696:Temperature
D007375:Interleukin-1; D008565:Membrane Proteins; D008744:Methylamines; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D008985:Monensin; D003513:Cycloheximide; C027451:methylamine