title: Kinetic analysis of internalization, recycling and redistribution of atrial natriuretic factor-receptor complex in cultured vascular smooth-muscle cells. Ligand-dependent receptor down-regulation.
abstract: The kinetics of internalization, sequestration and metabolic degradation of atrial natriuretic factor (ANF)-receptor complex were studied in rat thoracic aortic smooth-muscle (RTASM) cells. These parameters were directly determined by measuring 125I-ANF binding to total, intracellular and cell-surface receptors. Pretreatment of cells with the lysosomotropic agent chloroquine and the energy depleter dinitrophenol led to an increase in the intracellular 125I-ANF radioactivity. After 60 min incubation at 37 degrees C, cell-associated 125I-ANF radioactivity fell rapidly in chloroquine-treated cells (> 85%) compared with the controls (< 45%). 125I-ANF radioactivity increased to a peak of 65% of the initial level within 15 min in chloroquine-treated cells compared with only 22% in the control cells. During the initial incubation period at 37 degrees C, chloroquine inhibited the release of both intact and degraded 125I-ANF in a time-dependent manner. However, at later incubation times, the effect of chloroquine was diminished and release of both degraded and intact ligand was resumed. Extracellular unlabelled ANF did not affect the release of degraded 125I-ANF but it accelerated the release of intact ANF by a retroendocytotic mechanism. After the endocytosis, about 30-40% of ANF receptors were restored to the cell surface from the internalized pool of receptors. The restoration was blocked by chloroquine or dinitrophenol but not by cycloheximide. Exposure of RTASM cells to unlabelled ANF resulted in a time- and concentration-dependent loss of ANF receptors. Unlabelled ANF (10 nM) induced a loss of more than 52% of 125I-ANF binding, and a complete loss occurred at micromolar concentrations. It is inferred that ANF-induced down-regulation of its receptor resulted primarily from an increased rate in internalization and metabolic degradation of ligand-receptor complex by receptor-mediated endocytotic mechanisms.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912-2100.
authors.forename: K N
authors.initials: KN
authors.lastname: Pandey
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D015536:Down-Regulation; D004705:Endocytosis; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*
chemical_list: D004140:Dinitrophenols; D009320:Atrial Natriuretic Factor; D002738:Chloroquine; D003513:Cycloheximide; D017461:Receptors, Atrial Natriuretic Factor