Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes.
Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist-induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell-surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell-surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50-70% less [(125)I]-alpha-bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse-chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell-surface nAChRs.
St  John
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Department of Cell Biology & Anatomy, The University of Arizona College of Medicine, Tucson, AZ 85724, USA. StJohn@email.arizona.edu
Gordon
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002038:Bungarotoxins / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000187:drug effects / Q000294:innervation / Q000502:physiology*; D009469:Neuromuscular Junction / Q000502:physiology; D009473:Neuronal Plasticity; D018722:Nicotinic Agonists / Q000494:pharmacology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects; D011978:Receptors, Nicotinic / Q000187:drug effects*
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D018722:Nicotinic Agonists; D011956:Receptors, Cell Surface; D011978:Receptors, Nicotinic; D002217:Carbachol