Sequestration of muscarinic cholinergic receptors in permeabilized neuroblastoma cells.
The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the alpha-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25-30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[3H]methylscopolamine ([3H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 microM digitonin, the Oxo-M-mediated reduction in [3H]NMS binding was time (t1/2 approximately 5 min) and concentration (EC50 approximately 10 microM) dependent and was agonist specific (Oxo-M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [3H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [3H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5'-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Slowiejko
D M
DM
Neuroscience Laboratory, University of Michigan, Ann Arbor 48104-1687.
Levey
A I
AI
Fisher
S K
SK
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019832:N-Methylscopolamine; D009447:Neuroblastoma; D010095:Oxotremorine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006150:Guanine Nucleotides; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D000255:Adenosine Triphosphate; D004072:Digitonin; D002118:Calcium; D019832:N-Methylscopolamine