Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels

Tatsuhiko Ebihara, Feifan Guo, Lei Zhang, Ju Young Kim, David Saffen

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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 PC 12. 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.

Original languageEnglish (US)
Pages (from-to)449-458
Number of pages10
JournalJournal of Biochemistry
Volume139
Issue number3
DOIs
StatePublished - Mar 2006

Bibliographical note

Funding Information:
This work was supported by Grants-in-Aid (#07279107) for Scientific Research in Priority Areas on ‘‘Functional Development of Neural Circuits,’’ the Ministry of Education, Science, Sports and Culture of Japan, the Japan Science and Technology Corporation (CREST), funds from the Department of Pharmacology, Ohio State University, and a grant from the Psychiatric Research Foundation, Columbus, Ohio. We would like to thank Tatsuya Haga (Gakushuin University) and Tomoyuki Takahashi (University of Tokyo) for critical comments and valuable discussions.

Keywords

  • Calcium
  • Carbachol
  • Muscarinic acetylcholine receptor
  • PC12D
  • Thapsigargin

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