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Muscarinic receptor activation down-regulates the type I inositol 1,4,5-trisphosphate receptor by accelerating its
R J Wojcikiewicz1, T Furuichi, S Nakade
1Department of Cell Physiology and Pharmacology, University of Leicester.
Abstract:
Stimulation of SH-SY5Y human neuroblastoma cells with carbachol, a muscarinic agonist, down-regulates the type I inositol 1,4,5-trisphosphate (InsP3) receptor by > 90% with maximal and half-maximal effects after approximately 6 h and approximately 1 h, respectively. Examination of the mechanistic basis of this down-regulation revealed that carbachol increased the rate of type I InsP3 receptor degradation (radiolabeled immunoprecipitable receptor was lost from cells with half-times of > 8 h and approximately 1 h in the absence and presence of carbachol, respectively) and that the concentration of type I InsP3 receptor mRNA, despite a transient decrease after 3 h, did not correlate with levels of the receptor. Only those muscarinic receptor subtypes coupled to stimulation of phosphoinositide hydrolysis were capable of causing type I InsP3 receptor down-regulation. Ca2+ mobilization was pivotal to the mechanisms of receptor down-regulation, since perturbation of Ca2+ homeostasis with either EGTA or thapsigargin blocked the ability of carbachol to accelerate receptor degradation. Studies with thapsigargin also revealed that both functional InsP3-sensitive Ca2+ stores and persistent elevation of InsP3 concentration were required for down-regulation to occur. In conclusion, phosphoinositidase C-linked muscarinic receptors down-regulate the type I InsP3 receptor by accelerating its degradation. It appears that this process is initiated by persistent discharge of intracellular Ca2+ stores via the channels formed by tetramerically complexed type I InsP3 receptors.
Insights
Carbachol stimulation accelerates the degradation of the type I inositol 1,4,5-trisphosphate (InsP3) receptor in neuroblastoma cells. This process requires calcium mobilization and is linked to muscarinic receptors stimulating phosphoinositide hydrolysis.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- The type I inositol 1,4,5-trisphosphate (InsP3) receptor plays a crucial role in intracellular calcium signaling.
- Muscarinic receptor activation can modulate the expression and function of various cellular components, including ion channels and receptors.
Purpose of the Study:
- To investigate the mechanism by which carbachol, a muscarinic agonist, down-regulates the type I InsP3 receptor in SH-SY5Y human neuroblastoma cells.
- To elucidate the role of calcium (Ca2+) mobilization and specific muscarinic receptor subtypes in this down-regulatory process.
Main Methods:
- Stimulation of SH-SY5Y cells with carbachol.
- Measurement of type I InsP3 receptor degradation rates using radiolabeled immunoprecipitation.
- Analysis of type I InsP3 receptor mRNA levels.
- Perturbation of Ca2+ homeostasis using EGTA and thapsigargin.
- Investigation of muscarinic receptor subtype involvement.
Main Results:
- Carbachol significantly down-regulated the type I InsP3 receptor (>90% reduction) primarily by increasing its degradation rate.
- The down-regulation was dependent on muscarinic receptor subtypes coupled to phosphoinositide hydrolysis.
- Calcium (Ca2+) mobilization was essential, as EGTA and thapsigargin blocked carbachol-induced receptor degradation.
- Functional InsP3-sensitive Ca2+ stores and sustained InsP3 elevation were necessary for the down-regulation process.
Conclusions:
- Phosphoinositidase C-linked muscarinic receptors down-regulate the type I InsP3 receptor by accelerating its degradation.
- The process appears to be initiated by the persistent discharge of intracellular Ca2+ stores through type I InsP3 receptors.