Abstract
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.
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|