Conduit of ATP Release in Astrocytes
B. Corbett
1
Walsh ,
George R.
2
Dubyak
1Dept.
of Physics, Case Western Reserve University, Cleveland, OH 44106
2Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106
Abstract
Cell volume regulation is of particular physiologic
importance in neural tissues due to the adverse effects of
even small changes in brain volume. In the brain,
extracellular ATP acts as an autocrine / paracrine signaling
molecule and participates in volume regulation by activating
a family of P2 receptors. While significant advances have
been made regarding the signaling controlling ATP release,
the actual conduits for the export of ATP have not been
clearly defined. Investigations of ATP release from astrocytes
have identified connexin gap junction hemmichannels as a
candidate conduit for ATP release. ATP Assay and
transfection using 1321N1 Human Astrocytoma and C6 Rat
Glioma are currently underway to determine which conduit
is responsible for releasing ATP within astrocytes.
Methods
Isotonic saline
Background
Adenosine‐5'‐triphosphate (ATP) is not only a universal
energy source constantly produced and utilized by cells, but
it also serves as an ‘extracellular second messenger’. ATP,
acting as a signaling molecule, participates in many biologic
processes. Other functions of ATP include a paracrine role
in coordinating hepatocytes and induce apoptosis in various
cell types. Since ATP is a relatively large and hydrophilic
molecule it thus needs specially designed pathways in order
to exit the cells.
One possible channel being considered are
the connexin gap junction hemmichannels.
These channels exist at the point of cell
contact and serve as aqueous conduits for
ions and hydrophilic molecules smaller than
1kD. Importantly, connexin channels open in
response to hypotonic stress and increased
intracellular Ca2+, two facts used in the Figure 1. A rough
of a
experimental setup. Additionally, numerous depiction
Connexin gap junction
studies involving genetic manipulation of Hemmichannel.
http://en.wikipedia.org/
connexin subunits have produced evidence wiki/
File:Biggapjunct2.png
that connexin channels can function as ATP
release conduits.
Rationale
ATP acts as an autocrine / paracrine signaling molecule that
has numerous downstream physiological effects. Better
understanding of the conduit or pathway of ATP release can
enable pharmaceutical companies to develop inhibitors or
activators, to enable or prohibit certain processes from
occurring.
In relation to ATP release in cellular apoptosis, there may be
major implications by defining new chemotherapy
treatments for cancer patients. It will be crucial to determine
which chemotherapies have the capacity to induce
immunogenic cell death and which type of tumors can
actually generate immunogenic signals after radiotherapy or
chemotherapy.
Both 1321N1 cells and C6 cells contain thrombin
receptors, so when these cells are exposed to
thrombin the thrombin is binding to the cell surface.
Both cell lines also mobilize Ca2+ as a second
messenger in response to being exposed to
thrombin. 1321N1 cells clearly contain whatever
protein(s) are responsible for causing ATP release
when exposed to thrombin because figure 3 and 5
show that they release ATP in response to thrombin.
C6 cells however lack the connexin channels
sequence in their genome and thus have no way to
express it. The C6 cells also do not release ATP when
exposed to thrombin as shown in figure 4. These
results suggest that connexin channels may be
responsible for ATP release when these astrocytes
are exposed to thrombin.
A solution is called isotonic if the concentration of
osmolytes is the same inside the cell as it is
outside. A solution is called hypotonic if more
osmolytes are present inside the cell than are
present outside of the cell. As a result of this
gradient, water will spontaneously diffuse into the
cell to correct this imbalance. Because the plasma
membrane is a fluid organelle, subject to changes
in shape and size, the cell will swell as a result of
the sudden intake of water. A solution is called
hypertonic if there are more osmolytes outside
the cell than inside. Again because of the dynamic
nature of the plasma membrane the cell will
Luciferase added to
shrivel up.
Stimulus,
Rest 30
Cell
monolayer
min
Osmolarity
adjustment
Extracellular sample
10
min
Discussion
Figure 2. Effects of placing red blood cells in an isotonic solution (left), a hypotonic solution (middle) or hypertonic
solution (right). http://biology.unm.edu/ccouncil/Biology_124/Images/tonicity1.jpeg
Experimental 24 well plates were used to simulate conditions under the protocol shown above. Cells were
washed of their cell media and allowed to rest for 30 minutes. Then thrombin was added and the correct
osmolarity adjustments were given to the appropriate wells. Media surrounding the cells was then extracted
and placed in a cuvette with Luciferase (a compound that can be fluorescently detected when bound to ATP).
Results
Future Direction
To begin to verify the hypothesis that connexin
channels may be responsible for ATP release when C6
and 1321N1 cells are exposed to thrombin under
certain osmotic conditions, the C6 cell line will be
transfected with the genomic sequence for connexin
channels.
Plasmid of Connexin Channel to be Introduced to the
cell’s genome for expression
Expression of
Connexin
Channel in Cell
Genome
mRNA
Figure 3. Experimental results from exposing 1321N1 cells to different osmotic conditions in the presence Figure 4. Experimental results from exposing C6 cells to different osmotic conditions in the presence and absence
of thrombin.
and absence of thrombin.
As shown in figure 3, 1321N1 cells release ATP under
hypotonic conditions in the absence of thrombin.
Additionally thrombin causes ATP release under
isotonic conditions but is inhibited under hypertonic
conditions. Hypotonic conditions, when simultaneously
exposed to thrombin, potentiate ATP release.
Looking at figure 4, C6 cells only release ATP in
response to osmotic conditions, specifically hypotonic
conditions. This ATP release was not affected at all by
the presence of thrombin.
Examining the amount of ATP release from both cell
lines in figure 5, it becomes evident that the amount of
ATP release under similar conditions are comparable to
Hypertonicity
Isotonicity
Hypotonicity each cell line (comparing C6 +/- thrombin to 1321 –
Figure 5. Combining figures 3 (1321N1) and 4 (C6) into one graph for easy comparison relative to thrombin under any tonicity).
each other.
Figure 6. A basic depiction of transfection; the introduction of DNA into a recipient eukaryotic cell and its
subsequent integration into the recipient cells chromosomal DNA. This integration will then allow that
DNA
sequent
to
be
translated
into
a
protein.
http://www.labsupplymall.com/images/products/sg/PolyExpress_Degradation.jpg
Once the C6 cells possessed the connexin channels
gene, western blotting will be conducted to verify
that the C6 cells are expressing that protein on their
plasma membrane. Once this has been verified, the
same tonicity experiments in the presence and
absence of thrombin will be conducted. If ATP is
released by the transfected C6 cells, than these
connexin channels may be largely responsible for
releasing ATP when these cells are exposed to
thrombin.
Acknowledgments
This research was supported in part by NIH grant
R01-GM-36387.