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

Abstract

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 KATP 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.