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Received October 12, 2004
Revised October 22, 2004
Accepted after revision October 22, 2004
1 NIEHS, NIH, MD F2-02
2 NIEHS, NIH
* To whom correspondence should be addressed. E-mail: bird{at}niehs.nih.gov.
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 maneuvers 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.
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