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Originally published In Press as doi:10.1074/jbc.M007524200 on November 28, 2000

J. Biol. Chem., Vol. 276, Issue 8, 5613-5621, February 23, 2001
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Signaling Pathways Underlying Muscarinic Receptor-induced [Ca2+]i Oscillations in HEK293 Cells*

Dali LuoDagger , Lisa M. Broad, Gary St. J. Bird, and James W. Putney Jr.§

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 µM) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 µM) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 µM carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 µM Gd3+, as well as 30-100 µM concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 µM 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 µM 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+ entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Pharmacology, Harbin Medical University, Harbin 150086, Peoples Republic of China.

§ To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail: putney@niehs.nih.gov.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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