Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets

Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 c ells was established by reverse transcriptase-polymerase chain reaction (RT -PCR) and quantified by RNase protection. Both methods indicated that m3 an d mi receptors were expressed approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, th e cell lines, especially RINm5F cells, expressed less of a given receptor s ubtype than did islets. Immunohistochemistry indicated that m3 receptors we re expressed throughout the islet core. Binding studies using the radiolabe led muscarinic receptor antagonist QNB demonstrated a maximal binding capac ity of INS-1 cells of 23.0 +/- 2.9 fmol/mg protein. Functional analyses wer e undertaken using INS-1 cells stably transfected with either mi or m3 rece ptor cDNAs. Overexpression of either receptor did not affect basal response s but markedly enhanced maximal responses to the muscarinic receptor agonis t carbachol. Although maximal hydrolysis of phosphatidylinositol 4,5-bispho sphate (Ptd InsP2) was twofold greater in ml-transfectants as compared with m3-transfectants, cell lines overexpressing either receptor gave essential ly equivalent secretory responses to a full range of carbachol doses. The r esults demonstrate that both m1 and m3 muscarinic receptors are well expres sed in pancreatic beta-cells, functionally linked to signaling pathways, an d capable of initiating insulin secretion with equal potencies.