© 2006 The Japanese Biochemical Society.
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Muscarinic Acetylcholine Receptors Stimulate Ca2+ Influx in PC12D Cells Predominantly via Activation of Ca2+ Store–Operated Channels
1 Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033; and 2 The Graduate Program in Molecular, Cellular and Developmental Biology, and the Departments of 3 Pharmacology and 4 Psychiatry, College of Medicine and Public Health, The Ohio State University, 333 West 10th Ave., Columbus, OH 43210, USA
¶ To whom correspondence should be addressed at: Department of Pharmacology, College of Medicine and Public Health, 5072C Graves Hall, 333 West 10th Ave, Columbus, Ohio, USA. Tel: +1-614-688-4573, Fax: +1-614-292-7232, E-mail: saffen.1{at}osu.edu
Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store–operated Ca2+ channel.
* Present address: Neurophysiology Group, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-85566.
Present address: Department of Biology, Pennsylvania State University, 113 Life Science Bldg, University Park, PA 16801, USA.
Present address: Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321, Church St. S.E., Minneapolis, MN 55455, USA.
Present address: Department of Neuroscience/Institute for Cell Engineering, The Johns Hopkins University, BRB 729, 733 N. Broadway, Baltimore, MD 21205, USA.
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  K. Niinomi, Y. Banno, H. Iida, and S. Dohi Nicorandil, an Adenosine Triphosphate-Sensitive Potassium Channel Opener, Inhibits Muscarinic Acetylcholine Receptor-Mediated Activation of Extracellular Signal-Regulated Kinases in PC12 Cells Anesth. Analg., December 1, 2008; 107(6): 1892 - 1898. [Abstract] [Full Text] [PDF]
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