Abstract

The recognition of muscarinic cholinergic receptors (mAChRs) in human SK-N-SH neuroblastoma cells by hydrophilic (quaternary) and lipophilic (tertiary) ligands has been examined. When quiescent cells were incubated at 37 degrees, the same maximum number of mAChRs was revealed by antagonists that possessed either a quaternary nitrogen, e.g., N-methylscopolamine (NMS) and N-methylquinuclidinyl benzilate, or a tertiary nitrogen, e.g., scopolamine and quinuclidinyl benzilate (QNB). If cells were incubated at 0 degree, the quaternary [3H]NMS labeled 15-20% fewer sites than the tertiary [3H]scopolamine; but upon warming to 37 degrees, these inaccessible sites also became labeled. This suggests that mAChRs are present at both cell surface and sequestered sites in this cell, and that an equilibrium exists between the two cellular compartments. In competition studies at 37 degrees, NMS detected a population of [3H]QNB-binding sites which exhibited a very low affinity for the quaternary antagonist. However, the sites were not evident when mAChRs were labeled with [3H]scopolamine, suggesting that factors other than the lipophilic nature of the probe are involved. Although mAChRs were equally accessible to charged and uncharged antagonists at 37 degrees, the quaternary agonist carbamoylcholine competed for the sites labeled by quaternary antagonists with a 10- to 29-fold higher affinity than those labeled by tertiary antagonists, whereas the tertiary agonist OXO-2 displaced all sites with an equal affinity. However, carbamoylcholine competed equally well for [3H]scopolamine-and [3H]NMS-binding sites in either hypotonic cell lysates at 37 degrees or in intact cells maintained at 0 degree. These results suggest that, at 37 degrees, agonists induce the sequestration of cell surface receptors into a lipophilic environment in which receptors become inaccessible to quaternary, but not tertiary, ligands. Addition of NMS inhibited the stimulation of phosphoinositide hydrolysis elicited by either carbamoylcholine or OXO-2. The Ki values were similar for both agonists. It is concluded that mAChRs in SK-N-SH cells cycle between cell surface and sequestered sites. At 37 degrees, this cycling is rapid and all receptors have access to the cell surface compartment, whereas at 0 degree, receptor translocation is prevented and a population of sequestered mAChRs is detected. When cells are exposed to an agonist at 37 degrees, the equilibrium shifts such that more mAChRs are found in a sequestered cell compartment that is inaccessible to quaternary ligands.(ABSTRACT TRUNCATED AT 400 WORDS)