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Distribution of the muscarinic acetylcholine receptor on human fibroblasts was determined by immunofluorescence and immunoperoxidase staining using the anti-muscarinic receptor antibody M-35b. The receptor appeared to be randomly distributed on the cell surface in 1- or 2-day nonconfluent cultures. Brief exposure to 0.1 mM carbamylcholine (15 min to 1 hr) induced receptor accumulation in several restricted domains of the cell surface. This process was associated with sequestration into uncoated vesicles. Random receptor distribution was restored by incubation in ligand-free medium for 4 hr after carbamylcholine treatment, and vesicular profiles were no longer detectable. When incubation with the agonist was prolonged (3 hr at 37 degrees C), endocytotic 'smooth vesicles' fused and formed multivesicular structures presumably implicated in receptor down-regulation. Conversely, when nonconfluent cells were exposed to the muscarinic antagonist atropine, receptor redistribution was revealed, leading to the formation of clusters where receptor accumulated. Muscarinic receptor redistribution induced by atropine therefore does not involve the sequestration process seen in carbamylcholine-treated cells.
Raposo et al. (Thu,) studied this question.