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

Abstract

Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 cells was established by reverse transcriptase-polymerase chain reaction (RT-PCR) a n d q u a n t i fied by RNase protection. Both methods indicated that m3 and m1 receptors were expressed approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, the cell lines, especially RINm5F cells, expressed less of a given receptor subtype than did islets. Immunohistochemistry indicated that m3 receptors were expressed throughout the islet core. Binding studies using the R elease of acetylcholine from vagal efferents on pancreatic islet cells plays an important role in the cephalic phase of insulin secretion. Thus, the neurotransmitter directly evokes a rise in plasma insulin in the immediate postprandial period in rats and humans, before any glycemic elevation (1,2). Acetylcholine also potentiates nutrient-stimulated insulin secretion, an effect best demonstrated in vitro, where confounding postabsorptive influences occurring in vivo are absent (1,2). Thus, potentiation has been demonstrated using perfused pancreas (3) and isolated islets (4-6), and the direct stimulation has been shown in pancreas perfusions (3), insulinsecreting cell lines (7), and cultured (6,8) but not freshly isolated (9,10) islets. The latter preparation might be less responsive because of damage to acetylcholine receptors on the cell surface sustained during collagenase digestion, which is necessary for islet isolation. Acetylcholine has also been shown to prime the -cell in vitro such that a subsequent glucose challenge elicits a larger secretory response than in an unprimed cell (5).The effects of acetylcholine on the -cell are mediated by muscarinic cholinergic, rather than nicotinic, receptors (1,2,5,10). Five subtypes of muscarinic receptors have been d e fined at the molecular level (m1-m5), all of which are members of the G-protein coupled receptor family (11). The m2 and m4 receptor subtypes couple mainly via pertussis toxin-sensitive G-proteins to inhibition of adenylate cyclase. The functional responses of the m1, m3, and m5 subtypes are generally insensitive to pertussis toxin, are mediated via Gq/11, and are associated with activation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) (11). The signaling pathways activated by muscarinic receptor agonists in -cells are insensitive to pertussis toxin (12,13) and are not accompanied by inhibition of adenylate cyclase (6,9), but they do involve Ptd InsP2 hydrolysis (7,8,10,13). Thus, functional islet muscarinic receptors fall into the m1/m3/m5 category, rather than m2/m4. This is broadly consistent with some limited pharmacological analyses suggesting that m3 is the major receptor subtype present in pancreatic -cells (14-17). However, mRNA for other receptor subtypes has also been detected by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of extracts of rat islets (17) and insulin-secreting RINm5F c...

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).…”

Chronic activation of a designer Gq-coupled receptor improves β cell function

“…To explore the mechanism that links enhanced β cell G q signaling to increased IRS2 expression, we used a rat insulinoma cell line (INS1-M3) as a model system (14). This cell line expresses moderate levels of the M 3 muscarinic receptor subtype, a prototypic G q -coupled receptor (15).…”

Role of the autonomic nervous system in the development of hyperinsulinemia by high-carbohydrate formula feeding to neonatal rats

“…␤-cells with ACh or the cholinergic agonist carbachol before a glucose challenge resulted in a higher potentiation of GSIS compared with unprimed ␤-cells (21,42). Furthermore, treatment of HC islets with 4-DAMP in the presence of ACh resulted in significant reductions in cholinergic-induced insulin secretion, suggesting that ACh-induced potentiation of GSIS is mediated through activation of M3R as seen in a study showing that potentiation of GSIS by the cholinergic agonist oxotremorine M is inhibited by 4-DAMP (8).…”