Identification of Muscarinic Receptor Subtypes in RINm5F Cells by Means of Polymerase Chain Reaction, Subcloning, and DNA Sequencing

Abstract

Carbachol can stimulate insulin release in RINm5F cells by a mechanism that does not involve the elevation of cytosolic free Ca2+ concentrations or the activation of conventional protein kinase Cs (Mol Pharmacol 47:863-870, 1995). Thus, a novel signal transduction pathway links the muscarinic activation of the cells to increased insulin secretion. The question arises as to whether the pathway results from a novel receptor, different from the five established muscarinic receptors, or whether a "normal" receptor in the RINm5F cell activates a novel pathway. To distinguish between these two possibilities, the muscarinic receptors in the RINm5F cell were identified. Using polymerase chain reaction, combined with subcloning and DNA sequencing techniques, the cDNAs that encode the established M3 and M4 receptors were identified. The cDNAs for the Ml, M2, and M5 receptors were not found. Pharmacological studies showed a rank order of potency for muscarinic receptor subtype antagonists to inhibit carbachol-induced insulin release (half-maximal inhibitory concentration [pIC50] values given in parentheses): atropine (nonselective, 9.0) > 4-diphenyl-acetoxy-N-methyl piperidine methiodide (M3/M1, 8.6) > para-fluoro-hexahydrosiladiphenidol (M3, 8.1) > hexahydrosiladiphenidol (M3, 8.0) > tropicamide (M4, 6.4) > pirenzepine (M1, 6.1) > methoctramine (M2, 5.9). This antagonist profile suggests that it is the M3 receptor that mediates carbachol-induced insulin release. In this case, the novel signaling involved in the unusual carbachol response would not be due to a novel receptor but to the well-characterized M3 receptor. It appears, therefore, that the novel portion of the signaling pathway lies downstream of the M3 receptor and may consist of products of phosphatidylinositol hydrolysis, other than inositol triphosphate and diacylglycerol, resulting from the activation of phospholipase C. While a contributory role of the M4 receptor cannot be ruled out, there is no evidence in its favor other than its presence in the cell.

Role of the M3 muscarinic acetylcholine receptor in β‐cell function and glucose homeostasis

“…However, while previous studies reported the expression of additional mAChR subtypes (e.g. M 4 or M 5 ) in islet preparations or in b-cell derived cell lines [19,20], we did not detect M 2 , M 4 or M 5 receptor mRNA in mouse pancreatic islets.…”

“…Receptor localization studies suggest that multiple mAChRs (M 1 , M 3 , M 4 and/or M 5 ) are expressed in pancreatic islets/b-cells or b-cell-derived tumour cell lines [18][19][20]. We initially carried out a series of RT-PCR studies to examine which mAChR subtypes are expressed by mouse pancreatic islets.…”

Muscarinic Stimulation of Pancreatic Insulin and Glucagon Release Is Abolished in M3 Muscarinic Acetylcholine Receptor–Deficient Mice

“…Similar findings were obtained with rat pancreatic islets (23,25). Whereas previous studies (24,25) reported the expression of additional muscarinic receptor subtypes (M 4 or M 5 ) in islet preparations or in ␤-cellderived cell lines, we did not detect M 2 , M 4 , or M 5 receptor mRNA in mouse pancreatic islets. Because the majority of the cells in rat or mouse islets represent ␤-cells, it is highly likely that both M 1 and M 3 receptors are expressed by the insulin-secreting ␤-cells.…”

“…Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic receptor subtypes (M 1 ϪM 5 ) (22). Receptor localization studies suggest that multiple muscarinic receptors (M 1 , M 3 , M 4 , and M 5 ) are expressed in pancreatic islets/␤-cells or ␤-cellϪderived tumor cell lines (23)(24)(25). However, the M 3 muscarinic receptor appears to be the predominant subtype expressed by pancreatic ␤-cells (4 -6,23-25).…”

Recent evidence and potential mechanisms underlying weight gain and insulin resistance due to atypical antipsychotics

“…104 Additionally, the muscarinic receptor type 3 (M3) appears to be the predominant subtype expressed by pancreatic b-cells. [99][100][101][105][106][107] The role of muscarinic receptors in insulin release is well described in studies performed in M3 receptor-deficient mice (M3 -/-). Specifically, the infusion of the muscarinic agonist oxotremorine in islet cells either failed to stimulate insulin release in M3 receptor-deficient mice (M3 -/-) or was strongly inhibited in islets from M3 +/-mice.…”