Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells.
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 microm) 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 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm 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 microm 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 microm 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.
Luo
D
D
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Broad
L M
LM
Bird
G S
GS
Putney
J W
JW
D016718:Arachidonic Acid / Q000494:pharmacology; D001692:Biological Transport; D001896:Boron Compounds / Q000494:pharmacology; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D004347:Drug Interactions; D005682:Gadolinium / Q000494:pharmacology; D006801:Humans; D011976:Receptors, Muscarinic / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D001896:Boron Compounds; D011976:Receptors, Muscarinic; D016718:Arachidonic Acid; D019284:Thapsigargin; D002217:Carbachol; D005682:Gadolinium; C109986:2-aminoethoxydiphenyl borate