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Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

Abstract
The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.
AuthorsR J Wojcikiewicz, S R Nahorski
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 266 Issue 33 Pg. 22234-41 (Nov 25 1991) ISSN: 0021-9258 [Print] United States
PMID1657992 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Calcium Channels
  • ITPR1 protein, human
  • Inositol 1,4,5-Trisphosphate Receptors
  • Inositol Phosphates
  • Plant Extracts
  • Receptors, Cell Surface
  • Receptors, Cytoplasmic and Nuclear
  • Terpenes
  • Colforsin
  • Phorbol 12,13-Dibutyrate
  • Tretinoin
  • Thapsigargin
  • Inositol 1,4,5-Trisphosphate
  • Carbachol
  • Calcium
Topics
  • Calcium (metabolism)
  • Calcium Channels
  • Carbachol (pharmacology)
  • Cell Line
  • Cell Membrane (metabolism)
  • Cell Membrane Permeability
  • Colforsin (pharmacology)
  • Humans
  • Inositol 1,4,5-Trisphosphate (metabolism, pharmacology)
  • Inositol 1,4,5-Trisphosphate Receptors
  • Inositol Phosphates (metabolism)
  • Kinetics
  • Neuroblastoma
  • Phorbol 12,13-Dibutyrate (pharmacology)
  • Plant Extracts (pharmacology)
  • Receptors, Cell Surface (drug effects, isolation & purification, metabolism)
  • Receptors, Cytoplasmic and Nuclear
  • Terpenes (pharmacology)
  • Thapsigargin
  • Tretinoin (pharmacology)

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