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 M-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 luM thapsigargin, indicating they are
solely dependent on the release and reuptake of calcium from internal stores. Oscillations
continued in the presence of 1OuM ryanodine but were blocked by 10OuM 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.
Introduction:
Life has in many ways evolved around calcium. The Cat ion is highly energetic ion and
common in the natural environment; in fact, it is the fifth most common element in the earth’s
crust and most prevalent mineral in the human body (McHenry 1993). In response to the
ubiquity and biological activity of calcium, cells have developed a complex system of regulating
their intracellular calcium concentrations, [Ca, in response to changes in the internal and
external environments. The precise functioning of this regulatory system is critical for life; overly
high calcium levels are cytotoxic and can trigger apoptotic cell death. Nor could life continue in
the absence of calcium; the element plays a critical role in fertilization, muscle contraction and the
transmission of nerve impulses, and is a key second messenger in a wide variety of signaling
pathways in both neuronal and non-neuronal cells.
One critical way in which intercellular calcium concentrations are controlled is through the
regulation of plasma membrane influx and efflux channels. Human embryonic kidney cells
(HEK-293), which served as the study system for these experiments, have been shown to have
four types of calcium influx channels: highly selective LoAc channels, Imp Imx and nonselective
INs channels (Bujag et al 2005). In addition, some of the cells used in this experiment were
induced to express a murine version of the ion channel TRPM, a novel member of the growing
family of transient receptor potential (TRP) channels. Also known as LTRPG, TRPM is
bifunctional, consisting of both ion channel and kinase domains (Runnels et al 2001). It is
ubiquitously expressed in all vertebrate cell types studied to date and seems to be essential for life
cellular death occurs when the DT4O LTRPC gene is removed (Nadler et al 2001).
The role of TRPM is still a matter of debate in the scientific community. Hermosura et al.
(2001) postulated that M is, like the ATP-activated K' channel, an ATP-depletion activated
cation channel (ADAC that plays a key role in maintaining Ca and Mg homeostasis in
response to the cell’s energy state. Previous work has also suggested links between regulation of
the second messenger cyclic adenosine monophosphate (cAMP), an indicator of energy state, and
regulation of calcium levels (Hermosura, personal communication). Several isoforms of the
enzyme adenylyl cyclase, which synthesizes cAMP from adenosine triphosphate (ATP), are
known to be either positively or negatively regulated by Ca“. A spatial association between
membrane-bound adenylyl cyclases and specific ion channels that serve as Ca entry pathways
has also been documented (Mons et al 1998). More recently, the work of Takezawa et al. (2003)
suggested that cAMP up-regulates TRPM activity, while G-proteins have inhibitory effects on
the channel through their regulation of adenylate cyclase activity.
In addition to controlling the entry and exit of calcium from the cell, cells also regulate the
release to the cytosol of calcium stored internally in the endoplasmic reticulum (ER) or, in muscle
cells, the sarcoplasmic reticulum (SR). The two major pathways of intracellular calcium release
are controlled by the inositol 1,4,5 triphosphate receptor (IP,R) and the ryanodine receptor
(RyR). Ca release via the IP,R is regulated by a positive feedback loop in which increased
[Ca increases the open probability of the channel (Wang and Thompson 1995). It has also
been suggested that the affinity of the IP,R for its substrate, and thus the rate of release from
internal stores, is enhanced by the presence of cAMP (Chatton et al 1998). Calcium reuptake into
the internal stores is controlled by the sarcoplasmic-endoplasmic reticulum calcium ATPase
pumps (SERCA pumps).
Calcium signaling pathways and the relationship between plasma membrane channel and the
internal stores have been a focus of study for many years, but these complex pathways are not yet
fully understood. A long-standing model is the capacitative, or store-operated entry model, first
proposed by Putney (1986, 1990). Under this model, Ca* influx occurs subsequent to the
emptying of agonist-sensitive internal calcium stores. A more recent model suggests an
important role for a non-capacitative pathway regulated by arachidonic acid, which is activated at
lower agonist concentrations (Shuttleworth and Thompson 1999).
An important regulatory role for cyclic nucleotides has been suggested in both the influx of
calcium from the extracellular environment and in pathways of release from the internal stores.
In the present experiment, I used HEK-293 cells as a study system in an attempt to better
characterize the roles of cyclic nucleotides in the regulation of intercellular calcium levels, and in
particular to further investigate the role of cAMP in regulation of calcium flow through TRPM.
Materials and Methods:
Materials: 2'-O-dibutryryl-cyclic AMP, 8-bromo-cyclic GMP, ionomycin, gadolinium G, MnQ,
and thapsigargin were purchased from Sigma Chemical Co. Ryanodine, 2-APB and staurosporine
were purchased from CalBioChem.
Tissue Culture: This experiment utilized human embryonic kidney (HEK-293) cells from the
AlO cell line, which were stably transfected with a tetracycline-inducible murine TRPM/
construct to over-express the TRPM ion channel. All cells were grown in 50mL tissue culture
flasks in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and
0.1% penicillin/streptomycin, 0.01% blastocidin, and 0.01% zeocin, and kept in a 37°C incubator
with 5% C. Flasks were passaged every 2-3 days, or when cells were nearly confluent.
For the purpose of imaging, cells were plated on glass chips that had been treated with
high molecular weight poly-lysine to increase adhesion and washed with ultrapure HO prior to
plating. Plated cells were kept in DMEM in mini Petri dishes in the 37°C incubator until cells
were nearly confluent. The usual time between plating and imaging was 48-72 hours, but this
varied as growth rates changed over the course of the experiment. TRPM expression was
induced with tetracycline 24-48 hours prior to an imaging experiment. Cells expressing the
TRPM construct could be distinguished from controls by their rounded morphologies.
Fluorescent Imaging: Cells were loaded with the fluorescent Ca indicator Fluo-3/AM,
5uL/mL, (Molecular Probes) for 40 minutes and washed 3x with a 2 mM Ca saline solution 20
minutes prior to imaging. Images were recorded at 4-second intervals as cell-laden glass cover
slips were perfused with various solutions via a vacuum injection pump apparatus. Each imaging
trial lasted four minutes. All Fluo-3/AM imaging was done on an Olympus Fluoview 300 laser
scanning microscope using a UPLFL 20X objective. Fluoview Physiology FV30O (Olympus
Optical) software was used in data acquisition.
Fura-2/AM (Molecular Probes) dye was used to calibrate AF/Fo values with cellular
calcium concentrations using the ratiometric calibration equation developed by Grynkie wicz
(1985). Freshly prepared 2uM ionomycin was used to release all stored Ca and thus obtain a
maximal fluorescence reading (Fma). 2mM MnC, quenches fura-2 fluorescence and was used to
obtain a minimum reading (Fp). Fura-2 experiments were conducted on an Axiovert S100
microscope (Zeiss); MetaFluor 4.1 (Universal Imaging) was used for data acquisition and analysis.
Dye loading procedures were identical to Fluo-3 experiments, but perfusion was done by hand
with a micropipette.
Data Analysis: At the completion of each experiment, ten representative cells were selected as
regions of interest. The fluorescence intensity of these cells as a function of time over the course
of several trials was determined in Fluoview. Data were then exported to Excel (Microsoft) for
organization and subsequently to IGOR Pro (Wave Metrics) or Imagel (NIH) for further analysis.
Data from the ten individual cells were averaged and corrected for initial fluorescence intensity to
obtain values of AF/Fo. Statistical analysis was carried out using Systat 8.0 (SPSS). Where
applicable, statistics are presented as mean + one standard deviation.
Results:
Dye Fade: The fluorescent intensity of Fluo-3/AM fades with time during continued exposure
to light. To quantify the effects of dye fade, a four-minute dye fade control was run as the first
trial on each chip. Some trials showed a slight increase in fluorescence; others a slight decrease.
The patter of increases and decreases was random, and all trials approximated linearity. The
mean best-fit line slope (N=43) was 9.957E-O5 + 2.756E-4, not significantly different from zero.
Thus dye fade was not considered a significant confounding factor in interpreting the results.
Calibration of intracellular [Ca*: Using the equation developed by Grynkiewicz (1985) and
assuming a fura-2: Ca Kd of 135 nM (Molecular Probes), the concentration of intracellular Cat
of a typical cell in the standard 2 mM Ca solution was determined to be 51.1 nM.
Response to Changes in Extracellular Ca: TRPM expression increased the sensitivity of
intracellular Ca** concentrations to changes in extracellular Ca“. When external [Ca was
decreased from a 2mM solution to OmM EGTA-buffered saline, cells expressing the ion channel
underwent a 26 + 15% drop in fluorescence while control cells experienced a smaller 11 + 2%
decline. When external [Ca was increased from OmM to 2mM, cells expressing the ion channel
underwent a 30 + 28% maximum rise in fluorescence while control cells experienced a decline of
2 + 12%. These results suggest that TRPM is a significant pathway of calcium entry.
Representative plots are shown in Fig 1.
Effects of Exogenous cAMP: The addition of 100uM or ImM exogenous, membrane¬
permeable cAMP to cells incubated in a 2mM Ca solution resulted in a sharp transient rise in
[Cai, followed by dramatic oscillations, or "sparks", with a frequency of approximately
1/minute. A representative plot is shown in Fig 2. Lesser concentrations of cAMP (luM and
1QuM) did not produce either a peak or subsequent oscillations. The addition of ImM CGME
also did not elicit an oscillatory response (Fig 3).
Oscillations continued as long as the cells remained in a medium containing exogenous
cAMP, regardless of changes in extracellular Ca between 2mM and OmM. In the presence of
2mM Ca“, oscillations continued for over an hour. TRPM expression did not have an effect
on the magnitude or frequency of cAMP-induced [Ca oscillations.
In cells expressing TRPM, the previously characterized cellular responses to changes in
external calcium could be detected as a steady rise or fall underlying the oscillations. The steady
change in response to increasing (although not decreasing) external calcium was potentiated by
the presence of cAMP. When external calcium was increased from O to 2mM, the mean increase
in fluorescence without cAMP was 31 + 28% without cAMP and 62 +54% with cAMP (one-
tailed t-test P=.1377).
Pharmacological Manipulation of the Oscillatory Response: A variety of interventions were
made in an attempt to elucidate the mechanism underlying the oscillations. The frequency of
oscillation was quantified by sub-sampling three regions of interest from each trial in which
CAMP was added and counting the number of oscillations per cell in the field of view.
Oscillation frequency under varying conditions was compared with a nested ANOVA and
Student Newman-Keuls post hoc tests (Appendix A). Oscillation frequencies are plotted in Fig.
The cAMP-induced [Ca i peak and subsequent oscillations persisted in the presence of
1OuM ryanodine (RyR inhibitor); indeed, the frequency of oscillation was significantly higher
(SNK). Oscillations also continued in the majority of cells in the presence of 2uM and 1OuM
concentrations of the broad spectrum protein kinase inhibitor staurosporine, but were
significantly less frequent (SNK). The peaks observed in staurosporine-treated cells also had a
different characteristic shape, generally broader and of lesser magnitude (Fig 6a). The ratio of
peak height to width at half height was used as a metric for peak shape. The mean height: width
ratio +SD of twenty randomly selected peaks was 4.92 + 1.63 in the presence of cAMP alone
and 1.98 + 0.78 with staurosporine (two-tailed t-test P.001, Fig 6b). In some trials with
staurosporine, these prolonged oscillations were observed without cAMP addition.
2-aminoethyoxydiphenyl borane (2-APB) inhibits Ca release via the IP, receptor. It does
not affect the release of stored Ca modulated by the ryanodine receptor. Cells pretreated with
100uM 2-APB did not show a peak upon perfusion with a solution of 2mM Ca plus 10OuM
cAMP, regardless of whether additional 2-APB was present in the perfusion solution.
Furthermore, addition of 1O0uM 2-APB to cells oscillating in the presence of 2mM Ca and
100uM cAMP stopped the oscillations. In addition to blocking the oscillatory response, 2-APB
also caused a very gradual rise in [Cai, which could be observed both in dye fade controls and
in trials where cAMP was added.
The Role of Internal Stores: The diterpene thapsigargin specifically blocks SERCA Cat
pumping, which results in the release of calcium from internal stores. The addition of luM
thapsigargin to cells incubated in a 2mM Ca solution caused a gradual increase in free Cat and
subsequent, yet more gradual, decrease. Within 15 minutes, Cai had stabilized below the
initial level. Pretreatment with luM thapsigargin eliminated cAMP-induced [Ca i oscillations in
both control cells and those induced to express the TRPM construct (Fig 5), indicating that the
observed oscillations are dependent on internal Ca stores. Incubation in gadolinium (Gd**),
which creates a fast block to all Ca influx pathways present in the AlO cell line, including
TRPM (Ta 2004), did not affect the results observed with thapsigargin and the subsequent
addition of CAMP.
Discussion:
This experiment addressed two distinct phenomena concerning the regulation of intracellular
calcium levels: (1) the effect of TRPM expression on intracellular calcium concentrations and
the potentiation of the channel by cAMP, and (2) previously uncharacterized [Ca oscillations
induced by the addition of exogenous cAMP. TRPM expression substantially increased the
degree to which [Cali responded to changes in extracellular concentration, suggesting that
RPM does indeed serve as a major pathway of calcium influx under physiologically relevant
conditions. Furthermore, I observed that the steady [Cat i change in response to increasing
(although not decreasing) external calcium was potentiated by the presence of cAMP, lending
support to the suggestion of Takezawa et al (2003) that TRPM activity is up-regulated by
increased concentrations of cAMP.
While the presence of cAMP affected the activity of TRPM, the degree of TRPM
expression had no effect on the cAMP-induced oscillatory response, suggesting that oscillations
are inherent to the HEK cells themselves, and that influx pathways do not play a significant role.
Others (Luo et al 2000, St. Bird and Putney 2004) have reported similar Ca oscillations in
HEK-293 cells, but these have all been dependent on the continued presence of external calcium.
Thus the phenomenon I observed, in which oscillations continued throughout the course of a
four minute imaging trial in OmM Ca EGTA-buffered saline, can be considered novel.
Furthermore, the oscillations persisted at a continuous frequency for over an hour, suggesting
they might continue indefinitely and that the cells had been switched to a different "steady state".
The mechanism underlying these oscillations is of particular interest because it represents a
coupling between the two most common second messengers in the cell: cAMP and Ca (Cooper
and Hausman 2004). Various pharmacological probes were used in an attempt to elucidate this
mechanism. The response was not attenuated when experiments were conducted in the presence
of 1OuM ryanodine, indicating that the ryanodine receptor, a major pathway of calcium release,
does not play a significant or necessary role. In fact, analysis showed that the frequency of
oscillations actually increased in the presence of ryanodine. However, in the presence of 2-APB,
a membrane-permeant inhibitor of the IP, receptor, the addition of cAMP caused no [Ca i peak
or oscillations. This result implicates the IP,R as a key player in the oscillatory response.
While the most significant regulator of IP,-induced calcium release is calcium itself
(Nagakawa et al 1991), there is also a substantial body of evidence which indicates that cyclic
nucleotides also regulate the receptor's activity. There are two primary splice variants of the IP
receptor, the S2+, or neuronal, variant and the S2- non-neuronal type, which lacks 40 amino acid
residues between its two phosphorylation sites (Nagakawa et al 1991). Previous work has shown
that in cells expressing the S2+ variant, Ca release was enhanced when either PKA or PKG
was activated, but that only PKA had an effect in cells expressing the S2- variant (Wagner et al
2003).
The most plausible mechanism based on these results, especially given the lack of response to
cGMP, would be the activation of IP,R via phosphorylation by a protein kinase, possibly protein
kinase A (PKA), also known as cAMP-dependent kinase. Phosphorylation would increase the
open probability of the receptor, enhancing the positive feedback effect due to increased Cat
concentrations. Under this model, the oscillations would continue as long as the receptor
remained phosphorylated and the SERCA pumps continued to replenish the internal stores. This
hypothesis is also consistent with previous work in HEK-293 cells showing a qualitatively
different oscillatory response to be phosphorylation dependent. In cells expressing the Cat
sensing receptor CaR, [Ca i oscillations were shown to be dependent on a negative feedback
mechanism in which protein kinase C(PKQ phosphorylates CaR at an inhibitory threonine site
(Young et al 2002).
However, the results observed when experiments were carried out in the presence of various
concentrations of stauros porine somewhat complicate the picture. Staurosporine, derived from
the bacterium Streptomoes spp., inhibits a broad spectrum of protein kinases and in particular is a
very strong inhibitor of PKA, with an IG, of 7nM (CalBioChem). It was expected that
staurosporine would completely block the oscillatory response, yet mixed results were observed
in the presence of both 2uM and lOuM staurosporine. Oscillations were attenuated in only some
trials, while in the majority of trials, a [Ca peak and oscillations persisted in varying fractions
of the cells. However, the oscillations were significantly less frequent and more prolonged
(indicated by shallower and broader fluorescent peaks).
The differences in peak frequency and characteristic shape suggest that the oscillations
observed in the presence of staurosporine may have been due to a different and possibly
unrelated phenomenon, and that the original cAMP-induced oscillatory phenomenon, with its
characteristic sharp, narrow peaks and 1 per minute oscillation frequency was in fact attenuated
by protein kinase inhibition. The presence of the low-frequency, broad oscillations in
staurosporine trials in which no cAMP was added lends credence to this theory. An alternative
explanation is that the presence of staurosporine somehow altered or tempered the oscillatory
response without completely blocking it. Regardless, such a significant change in the response
suggests some role for protein kinases in the underlying mechanism.
It is clear that much remains to be learned about this unique oscillatory response. In future
work, it would be informative to better characterize the response to staurosporine and see
whether other broad spectrum kinase inhibitors elicit similar oscillations. Testing the effects of
more specific kinase inhibitors, especially those, such as PKI-a, which are highly specific for
protein kinase A, could further explain the underlying mechanism. Investigation into the
underlying kinetics of the oscillations would also be relevant and highly interesting.
Conclusions:
Expression of the ion channel TRPM greatly increases the response of Ca to changes in
external calcium. The presence of 100uM exogenous cyclic AMP potentiates the response of the
ion channel to increasing external Ca* concentrations. In both control cells and those
expressing TRPM, cAMP causes a rapid rise in [Ca followed by oscillations that persist for
up to an hour, an effect not shared by cyclic GMP. The oscillations are dependent on the release
and reuptake of calcium from internal stores and do not depend on the continued presence of
extracellular calcium. The IP, receptor plays a central role in the response, but the ryanodine
receptor is not involved. A plausible mechanism involves phosphorylation of the IP,R by cAMP
dependent kinase, or protein kinase A.
Acknowledgements:
This project could not have been possible without the assistance of many people. Most
importantly, I would like to thank Stuart Thompson for his knowledge, patience, and enthusiasm,
and his assistance with all aspects of the project. Many thanks also to my lab mates, Jennifer
Cribbs, Allison Waters and Christian Reilly, for their never-ending help, camaraderie and good
coffee. Thanks to Amro Hamdoun for his assistance with fura-2 experiments, to the entire Epel
lab for microscope use and contribution of reagents, to Jim Watanabe for statistical advice, and
especially to Chris Patton for assistance with all things technological. As always, love and many
thanks to my parents for supporting me in all I do.
Sources Cted:
Bujag V, Alexeenko V, Zubov A, Glushankova L, Nikolaev A, Wang Z, Kaznacheyeva E,
Bezprozvanny I, Mozhayeva GN (2005) Functional properties of endogenous receptor- and
store-operated calcium influx channels in HEK293 cells. J Biol Chem 280: 16790-16797.
Chatton JY, Cao Y, Liu H, Stucki JW (1998) Permissive role of cAMP in the oscillatory response
to inositol 1,4,5-trisphosphate in rat hepatocytes. Biochem J 330: 1411-1416.
Cooper DMF, Mons N, Karpen JW (1995) Adenylyl cyclases and the interaction between calcium
and cAMP signaling. Nature 374: 421-424.
Cooper GM, Hausman RE, eds. (2004) Cell Signaling. pp 541-589 in The Cll: A Mdeadar
Apprard. ASM Press, Washington DC.
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca indicators with greatly
improved fluorescent properties. J Biol Chem 260: 3440-3450.
Hermosura MC Thompson SH (2005, in press) Manganese influx via TRPM and its regulation
by calcium and magnesium.
Hermosura MC Nadler MS, Inabe K, Perraud A, Zhu Q, Kinet J, Kurosaki T, Penner R
Scharenberg AM, Fleig A (2001) LTRPC encodes an ATP-sensitive calcium channel.
Biophysical J 80: 458.
Luo D, Broad LM, Bird GSJ, Putney JW (2001) Signaling pathways underlying muscarinic
receptor induced [Ca oscillations in HEK293 cells. J Biol Chem 276: 5613-5621.
McHenry R, ed. (1993) "Calcium" in the New Encyclopedia Britannica, 151 ed. Vol. 2. University
of Chicago Press, Chicago IL.
Mons N, Decorte L, Jaffard R, Cooper DMF (1998) Calcium-dependent adenylyl cyclases, key
integrators of cellular signaling. Life Sci 62: 1647-1652.
Nadler MS, Hermosura MC Inabe K, Perraud A, Zhu Q, Stokes AJ, Kurosaki T, Kinet J,
Penner R, Scharenberg AM, Fleig A (2001) LTRPC is a MgeATP regulated divalent cation
channel required for cell viability. Nature 411: 590-595.
Nakagawa T, Okano H, Furuichi T, Aruga J, Mikoshiba K (1991) PNAS 88: 6244-6248.
Putney JW (1986) A model for receptor-regulated calcium entry. Cell Calcium 7:1-12.
Putney JW (1990) Capacitative calcium entry revisited. Cell Calcium 11: 611-624.
Runnels LW, Yue L, Capham DE (2001) TRP-PLIK, a bifunctional protein with kinase and ion
channel activities. Science 291, 1043-1047.
Shuttleworth T, Thompson JL (1999) Discriminating between capacitative and arachidonate-
activated Ca (24) entry pathways in HEK-293 cells. J Biol Chem 274: 31174-31178.
St J Bird G, Putney JW (2004) Capacitative calcium entry supports calcium oscillations in human
embryonic kidney cells. J Physiol 562: 697-706.
TaJ (2004) Ca2+ influx through the mTRPM channel is inhibited by Mg2+ and Gd3 +. Biology
176H final paper.
Takezawa R, Schmitz C Demeuse P, Scharenberg AM, Penner R, Fleig A (2004) Receptor
mediated regulation of the TRPM channel through its endogenous protein kinase domain.
PNAS 101:6009-6014.
Takezawa R, Schmitz C, Cheng H, Bessac BF, Scharenberg AM, Penner R, Fleig A (2003)
TRPM channel activity is inhibited by G proteins and stimulated by cAMP. Biophysical 184.
Part 2: 551a
Wagner LE, Li W, Yule DI (2003) Phosphorylation of type-1 inositol 1,4,5-trisphosphate
receptors by cyclic nucleotide-dependent protein kinases: a mutational analysis of the functionally
important sites in the S2+ and S2- splice variants. J Biol Chem 278: 45811-45817.
Wang SSH, Thompson SH (1995) Local positive feedback by calcium in the propagation of
intracellular calcium waves. Biophysical 69: 1683-1697.
Young SH, Wu SV, Rozengurt E (2002) Ca2+ stimulated Ca2+ oscillations produced by the
Ca2+ sensing receptor require negative feedback by protein kinase C.J Biol Chem 277: 46871-
46876.
Appendix A: ANOVA
A two factor nested design was used with movies nested within pharmaceutical treatment.
Sub-sampled regions of interest from the field of view served as individual replicates (n-3)
Source
F-ratio
Treatment
46.189
0.00
11.547
26.150
Movie
12.806
0.442
00.001
6.090
(Treatment)
Error
4.930
0073
Treatment
Description
LS Mean
cAMP alone
0.052
1.282
Ryanodine
2.086
0.078
2-APB
0.010
0.090
Thapsigargin
0.055
0.022
0798
0.049
Staurosporine
Post Hoc Tests: SNK Results
2-APB
Thapsigargin
Staurosporine
cAMP alone
Ryanodine
0.798
0.010
1.282
2.086
0.022
All treatments are significantly different at the.05 level with the exception of 2-APB and
thapsigargin, both of which effectively attenuated any oscillatory response.
Figure Legends:
Fig 1. Characteristic responses of cells expressing TRPM7 to changes in external
calcium. Perfusion occurred from 20 to 40 seconds.
a. Individual cells, decreasing [Ca from 2mM to OmM
b. Ten-cell average, decreasing Ca from 2mM to OmM
c. Individual cells, increasing [Ca from OmM to 2mM
d. Ten-cell average, increasing [Ca from OmM to 2mM
Fig 2. Characteristic response to addition of exogenous cyclic AMP. ImM CAMP was
added from 20 to 40 seconds.
a. Individual cells
b. Ten-cell average
Fig 3. Frequency of oscillation in the presence of cGMP and cAMP. The number of
oscillating cells in the region of interest was counted during repeated 2-minute sampling
periods over the course of a 1 hour imaging trial. Heavy black bars indicate the periods in
which the reagents were present.
Fig 4. Oscillations per cell for different pharmacological treatments. Three rectangular
regions were sub-sampled from the video of each trial. Data shown are least-squares means
with error bars connoting standard error. See Appendix A: ANOVA for a more detailed
comparison across treatment groups.
Fig 5. Effects of thapsigargin on internal Cat stores.
a. Addition of TuM thapsigargin occurs from 20 to 40 seconds.
b. [Cai gradually decreases in the subsequent four minutes.
c. Fifteen minutes later, perfusion with 100uM cAMP (from 20-40 s) has no discernible
effect.
Fig 6. Oscillations are broader and less frequent in the presence of staurosporine
a. Representative plot of individual cell oscillations in the presence of 1OuM
staurosporine (compare to Fig 3a)
b. The ratio of peak height to peak width at half height was used as a metric for
comparing peak shapes. Graphed values are means (n-20) with error bars denoting
one standard deviation. Peaks were significantly broader with staurosporine (2-tailed
t-test P.001)
Fig la
0
0.9 -
0.8 -
07-
Fig 1b
1.00-
0.95
0.90 -
0.85 -
0.80 -





W


100
150
50
200
Time (seconds)



100
150
50
200
Time (seconds)
V
Fig lc
1.4 -

1.3-
1.1 -
1.0-
Fig 1d
1.20:
1.15-
1.10:
1.05-
1.00 -



50


100
150
Time (seconds)

100
150
Time (seconds)
200
V

50
200
Fig 2a
2.5 —
2.0-
1.5-
1 10
50
Fig 2b
2.0-
1.8-
1.6
1.4 -
1.2:
10
ap;
W

100
150
Time (seconds)

100
150
Time (seconds)
A
200

200
50
Fig 3
50-
2 40-
8
6 30

20-
10:
sa
0+
ktaaataa-
14
30
10
20
50
Time (minutes)
ImM CGMP 1mM CAMP
Fig 4
2.5
1.5
0.5
-0.5
CAMP alone Ryanodine
+

2-APB Thapsigargin Staurosporine
Fig. Sa
2.0 -
1.8 -
1.6 -
1.4 -
1.2 -
1.0 -
Fig 5b
1.0-
09-
0.8 -
2 07-

Fig 5c
1.03 —
1.02:
1.01-
100
0.99-
0.98 -
0.97:
50
50
100
150
Time (seconds)
100
150
Time (seconds)




150
100
Time (seconds)
200
200

200
50
Fig 6a
3.0-
L
2.5
2.0-
1.5 -
1
50
Fig 6b
Without Staurosporine
V







100
150
200
Time (seconds)
PO.001
With Staurosporine (1OuM)