Hopkins Marine Station Student Paper

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Title: Cyclic AMP-dependent [Ca++]i oscillations in HEK-293 cells
Student Author(s): Lynch, Margaret
Faculty Advisor(s): Thompson, Stuart
Pages: 20
Location: Final Papers Biology 176H
Date: June 2005
Abstract: Calcium and cyclic AMP are the two most common cellular “second messengers” (McHenry 1993). The mechanisms by which their levels are regulated in the cell are complex and the proper functioning of such regulatory systems is essential for life. I investigated the effects of cAMP on intracellular calcium concentrations using HEK-293 cells as a study system. Changes in internal Ca++ concentration were monitored using the fluorescent indicators fluo-3 and fura-2 and both conventional and confocal microscopy. First, cAMP was found to potentiate calcium influx through TRPM7 in cells induced to express the channel. Second, a novel cAMP-induced oscillatory response was observed in both M7-expressing and control cells. Treatment with cyclic GMP did not elicit the oscillatory response. The oscillations persisted in the absence of extracellular Ca++ and were blocked by pretreatment with 1uM thapsigargin, indicating they are solely dependent on the release and reuptake of calcium from internal stores. Oscillations continued in the presence of 10uM ryanodine but were blocked by 100uM 2-APB, suggesting a central role for the IP3 receptor and not the ryanodine receptor. Treatment with the broad spectrum kinase inhibitor staurosporine yielded mixed results. Oscillations persisted in some cells but were more prolonged and less frequent. This suggests that the underlying mechanism may involve phosphorylation, but the targets and identity of the phosphorylating enzyme remain unclear