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 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 μmGd3+, 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.