Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

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

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca2+ ([Ca2+]i) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca2+]i in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca2+]i. Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca2+]i. GLP-1 (20 nM) did not affect membrane potential or [Ca2+]i. In contrast, acetylcholine (ACh, 100 μM) induced fast membrane depolarization immediately followed by a modest [Ca2+]i increase. When extracellular Ca2+ was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca2+]i increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP3R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca2+]i and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca2+ through store operated InsP3R

Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity

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

The effects of mid-Holocene fluvio-eolian interplay and coastal dynamics on the formation of dune-dammed lakes in NE Brazil

International audienceWe analyzed the Late Quaternary coastal evolution of the easternmost tip of South America in Brazil in light of fluvial-eolian interactions controlled by relative sea-level, climate, and coastal physiography changes. The chronology obtained by OSL-SAR of 36 samples coupled with sedimentological analysis from stabilized dunes suggest that eolian activity was primarily controlled by episodes of sediment availability because prevailing SE trade winds have been steadily strong throughout the Holocene. Contrary to the most conventional view linking dune activity to aridity, dune buildup occurred in a period of increased humidity in NE Brazil between 11 ka and 6 ka when a rising relative sea level and higher rainfall enhanced sediment delivery benefiting the construction of transgressive dunefields. The interplay of these advancing dunes with the existing drainage pathways is here investigated using a modern regional analog and through the evolution of Boqueirão Lake formed by dune blockage. Analysis of a sediment core from this lake dated between 8.4 and 0.9 ka indicated changes over time in microfossil assemblages, organic geochemistry, and grain size data conforming to fluvial or lacustrine depositional conditions. Between 7.2 and 4.4 ka, during the predominantly regional humid climate, the high abundance of fluvial sponge species correlated with a framework of competent-flow drainage systems diverting from advancing dunes. An abrupt transition from a wetter to a drier climate at 4.4–4.0 ka stimulated episodes of fluvial damming as indicated by sharp changes from sandy to muddy sediments and anomalous concentration of sponge spicules concurrent with significant mortality rates of fluvial adapted species. From 3.9 ka to the present, the disappearance of sponge spicules and peaking diatom concentration attested to a predominant lacustrine environment. Thus, the formation of Boqueirão Lake is mainly a result of the regionally drier climate and not a consequence of increased humidity in coastal NE Brazil