Relationship between agonist-induced muscarinic receptor loss and desensitization of stimulated phosphoinositide turnover in two neuroblastomas: methodological considerations.
The addition of both full and partial muscarinic agonists to human SK-N-SH neuroblastoma cells resulted in a sustained phosphoinositide (PPI) hydrolysis for up to 2 hr, as monitored either by the accumulation of [3H]inositol phosphates (IP) in the presence of Li+, or alternatively, by an increased labeling of phosphatidate and phosphatidylinositol (PI) from 32Pi. This enhanced PPI hydrolysis was maintained even though 40-50% of cell-surface muscarinic cholinergic receptors (mAChRs) became sequestered. Desensitization of carbachol-stimulated PPI hydrolysis was only detected after a 2-4 hr prior exposure of the cells to the agonist and was accompanied by a comparable reduction in total mAChR number. Prolonged incubation of SK-N-SH cells with the partial agonist bethanechol also resulted in a desensitization of stimulated PPI turnover but at a slower rate than that observed for carbachol and with a loss of fewer mAChR sites. Desensitization of mAChR-stimulated [3H]IP formation appeared to occur more rapidly in mouse NIE-115 neuroblastoma. However, Li+ could not fully prevent the degradation of accumulated [3H]IP in these cells. When an increase in [32P]phosphatidylinositol labeling was used to assess PPI turnover in NIE-115 cells, no desensitization was evident for up to 2 hr. We conclude that 1) in SK-N-SH cells, desensitization more closely parallels the down-regulation than the sequestration of mAChRs, 2) in both neuroblastomas, stimulated PPI hydrolysis desensitizes relatively slowly and 3) the appearance of desensitization can vary as a function of the assay method employed.
Thompson
A K
AK
Neuroscience Laboratory, University of Michigan, Ann Arbor.
Fisher
S K
SK
D006801:Humans; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000378:metabolism; D010277:Parasympathomimetics / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000032:analysis / Q000187:drug effects*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010277:Parasympathomimetics; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D019832:N-Methylscopolamine