Mechanism of agonist-induced down-regulation and subsequent recovery of muscarinic acetylcholine receptors in a clonal neuroblastoma x glioma hybrid cell line.
The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.
Ray
P
P
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Middleton
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Berman
J D
JD
D000818:Animals; D001414:Bacitracin / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005910:Glioma / Q000378:metabolism*; D006822:Hybrid Cells / Q000378:metabolism*; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000378:metabolism*; D015739:Nocodazole; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D011503:Transglutaminases / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D008744:Methylamines; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D001414:Bacitracin; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; C027451:methylamine; D011503:Transglutaminases; D015739:Nocodazole; D003078:Colchicine; D019832:N-Methylscopolamine