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Muscarinic cholinergic receptors of two cell lines that regulate cyclic AMP metabolism by different molecular
Abstract:
The attenuation of cyclic AMP accumulation occurs by different mechanisms in 1321N1 astrocytoma cells and NG108-15 neuroblastoma X glioma cells. In 1321N1 cells, cholinergic agonists reduce cyclic AMP accumulation through a Ca2+-dependent activation of phosphodiesterase; in NG108-15 cells, muscarinic receptor-mediated effects on cyclic AMP metabolism occur through inhibition of adenylate cyclase. The goal of the current study was to determine whether different pharmacological specificities were expressed by the muscarinic receptor populations of these two cell lines. The affinity of muscarinic receptors for [3H]quinuclidinyl benzilate (6 pM), [3H]N-methylscopolamine (50 pM), and atropine (80 pM) was similar in membrane preparations from each cell line. The affinity of the antagonist, pirenzepine, which has been proposed to be a selective ligand for a muscarinic receptor subtype, was 3-fold higher in competition binding assays carried out with membranes of 1321N1 cells, than with NG108-15 cells. The Hill coefficients of pirenzepine competition curves were not significantly different from unity in both cell lines. This selectivity of pirenzepine was also apparent in studies of the competitive inhibition of carbachol-induced attenuation of cyclic AMP accumulation in intact cells. Differences in the relative affinities of agonists were observed in competition binding analyses carried out with membranes in the presence of GTP and absence of Mg2+. The Ki values of bethanechol and carbachol were 5- and 12-fold lower for receptors of NG108-15 cells than those of 1321N1 cells and the Ki of methacholine was 3.5-fold lower for 1321N1 cells than for NG108-15 cells. The affinities of oxotremorine and arecoline were similar between the two cell lines. These differences in agonist affinities between the two cell lines were much smaller in analyses of muscarinic receptor-mediated effects on cyclic AMP metabolism in intact cells. Taken together, these data suggest that muscarinic receptors of differing pharmacological specificities regulate cyclic AMP metabolism by different mechanisms in 1321N1 and NG108-15 cells.
Insights
This study reveals distinct muscarinic receptor profiles in astrocytoma and neuroblastoma X glioma cells. Pirenzepine shows higher affinity for astrocytoma cell receptors, indicating subtype differences that influence cyclic AMP regulation.
Area of Science:
- Pharmacology
- Neuroscience
- Cell Biology
Background:
- Cyclic AMP (cAMP) accumulation is modulated by different mechanisms in 1321N1 astrocytoma and NG108-15 neuroblastoma X glioma cells.
- 1321N1 cells utilize Ca2+-dependent phosphodiesterase activation, while NG108-15 cells use adenylate cyclase inhibition for muscarinic receptor-mediated cAMP changes.
Purpose of the Study:
- To investigate the pharmacological specificities of muscarinic receptor populations in 1321N1 and NG108-15 cells.
- To determine if differences in receptor subtypes correlate with the distinct cAMP regulation mechanisms observed in these cell lines.
Main Methods:
- Radioligand binding assays using [3H]quinuclidinyl benzilate, [3H]N-methylscopolamine, and atropine to determine muscarinic receptor affinity.
- Competition binding assays with pirenzepine and various agonists (bethanechol, carbachol, methacholine, oxotremorine, arecoline) in membrane preparations.
- Studies on the competitive inhibition of carbachol-induced cAMP accumulation in intact cells.
Main Results:
- Muscarinic receptor affinity for [3H]quinuclidinyl benzilate, [3H]N-methylscopolamine, and atropine was similar in both cell lines.
- Pirenzepine exhibited a 3-fold higher affinity for 1321N1 cell receptors compared to NG108-15 cells.
- Agonist affinities varied, with lower Ki values for bethanechol and carbachol in NG108-15 cells, and for methacholine in 1321N1 cells, though these differences were less pronounced in intact cell assays.
Conclusions:
- The study suggests that 1321N1 and NG108-15 cells possess muscarinic receptors with differing pharmacological specificities.
- These receptor differences likely contribute to the distinct mechanisms by which muscarinic agonists regulate cyclic AMP metabolism in these two cell lines.