Estimating the Near-Membrane Concentration
of Phosphodiesterase in HEK-293 Cells
Jonathan McLachlan1, Tom Rich2, Silas Leavesley3, and Audi Byrne4.
of Chemistry, Spring Hill College, Mobile, AL; 2Department of Pharmacology, University of South Alabama, Mobile, AL; 3Department of Chemical Engineering,
University of South Alabama, Mobile, AL; 4Department of Mathematics, University of South Alabama;
Abstract
Cyclic adenosine monophosphate (cAMP) is an important, ubiquitous
second messenger in cells. Phosphodiesterase (PDE) is the critical
family of enzymes that hydrolyze cAMP in order to modulate cAMP
signaling. cAMP concentrations are thought to be compartmentalized,
that is, unique at different locations throughout a cell. Because PDE
reacts with specific local concentrations of cAMP within each
compartment, it may be possible to estimate the concentration of a
known type of PDE in a specific area of the cell. This project is focused
on determining the concentration of PDE in the near plasma
membrane via cAMP measurements using cyclic nucleotide-gated
CNG ion channels and computer modeling techniques. The activity of
CNG channels was used to estimate cAMP levels near the plasma
membrane. Modeling techniques were implemented in order to
estimate subcellular PDE concentrations based upon the time course
and amplitude of measured cAMP signals. Results indicate that that
PDE levels near the plasma membrane are substantially lower than in
the rest of the cell. These results will help us to better understand
how signaling specificity is achieved within the cAMP pathway.
Introduction
cAMP signals are known to regulate several cellular processes
including gene transcription, proliferation, and regulation of
endothelial barrier permeability[4]. PDE is one of the many enzymes
which reacts with cAMP. Because PDE concentrations and types vary
in a human cell, the rate of cAMP changes at a different rate based
upon time and location in a cell; suggesting compartmentalization of
cAMP. We will model this rate and determine the concentration PDE
in the near plasma membrane with varying initial conditions.
•Simulation Method:
•A 3-D cell is modeled containing two
separate compartments (bulk and near
plasma membrane).
•cAMP is introduced by pipette and
allowed to diffuse;
•Both diffusion and reaction occur at
each time step
•By matching the model’s outcome to
the experimental data, [PDE] can be
determined within a margin of error.
Results
-4
The Effect of DcAMP on Flux
Conclusions
• Mathematical models are useful when attempting to discern useful
4
information, confirm experimental results, and plan future experimentation.
3.5
•PDE concentrations in the near plasma membrane can be estimated with
both experimental methods and mathematical models
3
•Near-membrane PDE values are very low, with a concentration of
around.03µM from simulations
2.5
2
1.5
1
Future Directions
0.5
0
0
100
200
300
• Study effects of PDE inhibitors (rolipram/IBMX) and fit the model to data
• Utilize optical biosensors to detect near-membrane cAMP levels
• Full validation of mathematical model for different values of cAMP initially
distributed
400
DcAMP(M2/s)
Figure 1: The relationship between the cAMP
diffusion coefficient (D) in the cytosol and the
flux between the near plasma membrane and the
bulk cytosol. PDE concentration was held
constant at 0.03 µM.
Materials & Methods
The Effect of Varying DcAMP
On Estimated Value of [PDE,PM]
0.045
[PDE] in Near Plasma Membrane (µM)
•Experimental Method:
•CNG channels were used to monitor cAMP levels in
HEK-293 cells.
•Spread of cAMP was shown by changes in voltage on
the surface of the cells.
•Known relations to diffusion rates and PDE/cAMP
interactions result in an experimental PDE value in the
near membrane.
x 10
4.5
Flux Between Bulk and
Near Plasma Membrane
1Department
.
0.04
0.035
0.03
0.025
Literature Cited
Lehninger, Albert L, David L. Nelson, and Michael M. Cox. Lehninger Principles of
Biochemistry. New York: W.H. Freeman, 2005.
Murray, Fiona. " The interplay of multiple molecular and cellular components is necessary
for compartmentalization of cAMP. Focus on “Assessment of cellular mechanisms contributing
to cAMP compartmentalization in pulmonarymicrovascular endothelial cells.” Am J Physiol Cell
Physiol 302:C837-C838, 2012. Web. 30 May 2013.
Rich, Thomas C., Fagan, Kent A., Tse, Tonia E., Sckaack, Jerome., Cooper, Dermot M.F., and
Karpen, Jeffery W. "A Uniform Extracellular Stimulus Triggers Distinct cAMP Signals in Different
Compartments of a Simple Cell." PNAS vol. 98 no. 23 13049-13054, 2011. Web. 30 May 2013
Schwartz, James H. "The Many Dimensions of cAMP Signaling." Commentary.
www.pnas.org/cgi/doi/10.1073/pnas.251533998. Web. 30 May 2013..
Xin, Wenkuan., Feinstein ,Wei P., Ochoa, Christian D., Zhu, Bing., M. Byrne, Audi., and Rich,
Thomas C. "Estimating the magnitude of near-membrane PDE4 activity in living cells, 2012."
Submitted.
Zhou, H.X.; Rivas, G.; Minton, A.P. (2008). "Macromolecular crowding and confinement:
biochemical, biophysical, and potential physiological consequences". Annu Rev Biophys 37:
375–97. doi:10.1146/annurev.biophys.37.032807.125817. Web. 28 May 2013.
0.02
0.015
0
100
200
300
400
DcAMP(M2/s)
Figure 2: Estimates of near-membrane PDE
levels based upon measurements of CNG
channel activity and mathematical modeling.
Simulations
indicate
that
the
PDE
concentration is approximately 0.03 µM.
ACKNOWLEDGEMENTS
We would like to thank the
National Science Foundation f
or funding this work and the
University of South Alabama
REU program.