(full paper is archived in the Miller Library)
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