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Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release. [Copyright &y& Elsevier]

The cooperative effect of glucagon-like peptide 1 (GLP-1) and acetylcholine (ACh) was evaluated in a beta cell line model (BRIN BD11). GLP-1 (20 nM) and ACh (100 μM) increased insulin secretion by 24–47%, whereas in combination there was a further 89% enhancement of insulin release. Overnight culture with 100 ng/mL pertussis toxin (PTX) or 10 nM PMA significantly reduced the combined insulinotropic action (P<0.05 and P<0.001, respectively) and the sole stimulatory effects of GLP-1 (PTX treatment; P<0.01) or ACh (PMA treatment; P<0.05). Under control conditions, ACh (50 nM–1 mM) concentration-dependently inhibited by up to 40% (P<0.001) the 10-fold (P<0.001) elevation of cyclic 3′,5′-adenosine monophosphate (cAMP) induced by 20 nM GLP-1. The paradoxical inhibitory action of ACh was abolished by PTX pre-treatment, suggesting involvement of Gi and/or Go G protein alpha subunit. Effects of selective muscarinic receptor antagonists on the concentration-dependent insulinotropic actions of ACh (50 nM–1 mM) on 20 nM GLP-1 induced insulin secretion revealed inhibition by ρ-FHHSiD (M3 antagonist, P<0.05), stimulation with pirenzepine (M1 antagonist, P<0.001) and no significant effects of either methoctramine (M2 antagonist) or MT-3 (M4 antagonist). Antagonism of M2, M3 and M4 muscarinic receptor effects with methoctramine (3–100 nM), ρ-FHHSiD (3–30 nM) or MT-3 (10–300 nM) did not significantly affect the inhibitory action of ACh on GLP-1 stimulated cAMP production. In contrast, M1 receptor antagonism with pirenzepine (3–300 nM) resulted in a concentration-dependent decrease in the inhibitory action of ACh on GLP-1 stimulated cAMP production (P<0.001). These data indicate an important functional cooperation between the cholinergic neurotransmitter ACh and the incretin hormone GLP-1 on insulin secretion mediated through the M3 muscarinic receptor subtype. However, the insulinotropic action of ACh was associated with a paradoxical inhibitory effect on GLP-1 stimulated cAMP production, achieved through a novel PTX- and pirenzepine-sensitive M1 muscarinic receptor activated pathway. An imbalance between these pathways may contribute to dysfunctional insulin secretion. [Copyright &y& Elsevier]