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1 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, PO Box 12233, Research Triangle Park, NC 27709, USA
Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca2+]i), known as [Ca2+]i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca2+]i spikes does not. The oscillations do not persist in the absence of extracellular Ca2+, leading to the conclusion that entry of Ca2+ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl3, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca2+]i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca2+]i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca2+. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca2+]i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca2+ entry that supports Ca2+ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca2+ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.
(Received 4 August 2004;
accepted after revision 22 October 2004;
first published online 28 October 2004)
Corresponding author G. St. J. Bird: NIEHS, PO Box 12233, Research Triangle Park, NC 27709, USA. Email: bird{at}niehs.nih.gov
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