Molecular mechanisms of muscarinic acetylcholine receptor–stimulated increase in cytosolic free Ca²⁺ concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line

Muscarinic acetylcholine receptor (mAChR) activation of pancreatic β-cells elevates intracellular Ca²⁺ and potentiates glucose-stimulated insulin secretion. In addition, it activates a number of signaling molecules, including ERK1/2, whose activation has been shown to play an important role in regulating pancreatic β-cell function and mass. The aim of this work was to determine how mAChR activation elevates intracellular Ca²⁺ concentration ([Ca²⁺] _i ) and activates ERK1/2 in the pancreatic β-cell line MIN6. We demonstrate that agonist-stimulated ERK1/2 activation is dependent on the activation of phospholipase C and an elevation in [Ca²⁺] _i , but is independent of the activation of diacylglycerol-dependent protein kinase C isoenzymes. Using a pharmacological approach, we provide evidence that agonist-induced increases in [Ca²⁺] _i and ERK activity require (1) IP₃ receptor-mediated mobilization of Ca²⁺ from the endoplasmic reticulum, (2) influx of extracellular Ca²⁺ through store-operated channels, (3) closure of K_ATP channels, and (4) Ca²⁺ entry via L-type voltage-operated Ca²⁺ channels. Moreover, this Ca²⁺-dependent activation of ERK is mediated via both Ras-dependent and Ras-independent mechanisms. In summary, this study provides important insights into the multifactorial signaling mechanisms linking mAChR activation to increases in [Ca²⁺] _i and ERK activity.