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

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 M₁, M₂, and M₅ receptors were not found. Pharmacological studies showed a rank order of potency for muscarinic receptor subtype antagonists to inhibit carba-chol-induced insulin release (half-maximal inhibitory concentration [pIC₅₀] values given in parentheses): atropine (nonselective, 9.0) > 4-diphenyl-acetoxy-iV-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.