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Keywords:

  • Inositol 1,4,5-trisphosphate;
  • Muscarinic;
  • SH-SY5Y cells;
  • Calcium;
  • Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP3) 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 Ca2+ with EGTA or inhibition of Ca2+ channels with Ni2+ showed that the plateau phase was dependent upon Ca2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca2+ from intracellular stores revealed that Ca2+ from this source was capable of supporting the peak phase of the InsP3 response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca2+ concentration (20–100 nM) and suggests that under normal conditions, InsP3 formation is enhanced by increases in cytosolic free Ca2+ 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 InsP3 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 Ca2+ concentration amplify InsP3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP3 accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca2+ or protein kinase C activity.