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, streptoiysin-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[H-3]methylscopolamine ([H-3]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 mu M digitonin, the Oxo-M-mediated reduction in [H-3]NMS binding was time (t(1/2) similar to 5 min) and concentration (EC(50) similar to 10 mu M) dependent and was agonist specific (Oxo-M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [H-3]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [H-3]NMS sites observed in the presence of Oro-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 Oro-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 mu M). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is similar to 65-70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [H-3]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide-derived second messengers is not a prerequisite for mAChR sequestration.