Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

Abstract

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 Α-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 [ 3 H]methyl- scopolamine ([ 3 H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 Μ M digitonin, the Oxo-M-mediated reduction in [ 3 H]NMS binding was time ( t 1/2 ∼ 5 min) and concentration (EC 50 ∼ 10 Μ 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 [ 3 H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [ 3 H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca 2+ , 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 Μ M ). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is ∼ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [ 3 H]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.