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Alterations in receptor-coupled second messenger systems at up-regulated muscarinic receptors: analysis using primary
1Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan.
Abstract:
The effect of a long-term exposure (5 days) to atropine on muscarinic acetylcholine receptors and receptor-coupled second messenger systems was investigated using mouse cerebral cortical neurons in primary culture. The long-term exposure of neurons to atropine (10 nM) induced increases in both the Bmax and Kd values of [3H]quinuclidinyl benzilate (QNB) binding to muscarinic acetylcholine receptors. Alterations in muscarinic receptor-coupled second messenger systems, such as phosphoinositide (PI) hydrolysis and cyclic GMP (cGMP) formation following a long-term exposure to atropine, were also examined. Carbachol-stimulated PI hydrolysis was found to be decreased by the exposure to atropine in spite of the increase of muscarinic receptors. In addition, a long-term exposure to atropine had no effect on carbachol-stimulated cGMP formation as well as on the rightward shift of the carbachol competition curve of [3H]QNB binding in the presence of GTP. These results suggest that the up-regulation in muscarinic cholinergic receptors induced by long-term exposure to atropine may involve not only the increase in number of muscarinic receptors but also the decreased responsiveness in muscarinic receptor-coupled second messenger systems.
Insights
Long-term atropine exposure up-regulates muscarinic acetylcholine receptors in mouse brain cells. This involves increased receptor numbers and reduced signaling pathway responsiveness, impacting neuronal function.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Muscarinic acetylcholine receptors (mAChRs) are crucial for neuronal function.
- Understanding receptor regulation is key to developing targeted therapeutics.
- Atropine is a known muscarinic antagonist with complex cellular effects.
Purpose of the Study:
- To investigate the long-term effects of atropine on mAChRs and associated signaling pathways.
- To determine if atropine induces receptor up-regulation and alters second messenger system function.
- To elucidate the mechanisms underlying atropine-induced changes in neuronal excitability.
Main Methods:
- Primary culture of mouse cerebral cortical neurons.
- Long-term exposure (5 days) to atropine (10 nM).
- Radioligand binding assays using [3H]quinuclidinyl benzilate (QNB) to quantify mAChRs.
- Measurement of phosphoinositide (PI) hydrolysis and cyclic GMP (cGMP) formation.
- Analysis of receptor competition curves in the presence of GTP.
Main Results:
- Long-term atropine exposure increased both the Bmax and Kd values of [3H]QNB binding, indicating mAChR up-regulation.
- Carbachol-stimulated PI hydrolysis decreased despite the increase in mAChRs.
- No significant effect on carbachol-stimulated cGMP formation was observed.
- GTP did not alter the carbachol competition curve, suggesting no change in receptor-G protein coupling.
Conclusions:
- Long-term atropine exposure leads to mAChR up-regulation in mouse cortical neurons.
- This up-regulation is associated with a decreased responsiveness in PI hydrolysis, a key second messenger system.
- The findings suggest a complex regulatory mechanism involving both receptor number and signal transduction efficiency.