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Muscarinic Receptor‐Mediated Inositol 1,4,5‐Trisphosphate Formation in SH‐SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca 2+ and by Rapid Desensitization
Author(s) -
Wojcikiewicz Richard J. H.,
Tobin Andrew B.,
Nahorski Stefan R.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.63010177.x
Subject(s) - inositol , thapsigargin , egta , muscarinic acetylcholine receptor , inositol phosphate , carbachol , endocrinology , receptor , medicine , phospholipase c , phosphatidylinositol , chemistry , inositol trisphosphate , biology , biophysics , biochemistry , extracellular , calcium , signal transduction
Stimulation of muscarinic receptors expressed in SH‐SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5‐trisphosphate (InsP 3 ) accumulation, with a dramatic increase (six‐ to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ∼60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca 2+ with EGTA or inhibition of Ca 2+ channels with Ni 2+ showed that the plateau phase was dependent upon Ca 2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca 2+ from intracellular stores revealed that Ca 2+ from this source was capable of supporting the peak phase of the InsP 3 response. Carbachol‐stimulated phosphoinositidase C activity in permeabilized SH‐SY5Y cells was also shown to be highly dependent on free Ca 2+ concentration (20–100 n M ) and suggests that under normal conditions, InsP 3 formation is enhanced by increases in cytosolic free Ca 2+ concentration that accompany muscarinic receptor activation. Measurement of carbachol‐stimulated total inositol phosphate accumulation in the presence of Li + indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor‐mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP 3 accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca 2+ concentration amplify InsP 3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP 3 accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca 2+ or protein kinase C activity.