Transcript EPINEPHRINE’S EFFECT ON AVIAN EMBRYONIC IN VITRO …

EPINEPHRINE’S
EFFECT ON AVIAN
EMBRYONIC IN VITRO
HEART RATE
Aresh Ramin & Kelly Fetter
Purpose
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To determine the effects of epinephrine
on the 4-day chicken embryonic heart
rate in beats per minute (bpm).
To investigate whether β-adrenergic
receptors are developed in the 4-day
chicken embryonic heart and their
sensitivity to epinephrine.
Previous research has shown that
younger embryos, 2-2.5 days, contain
less β-adrenergic receptor binding
sites, where 5-day old embryos clearly
have the β-adrenergic receptor
binding sites, suggesting sensitivity to
epinephrine (Lipshultz, 1981).
Figure 1: Micrograph of 4day old chicken embryo
Hypothesis
The heart rate of the 4-day chicken embryo
will increase linearly with the exogenous
application of epinephrine and at toxic levels
arrhythmias will be observed.
Basic Information
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The heart of the chicken embryo
develops from the fusion of paired
precardiac mesodermal tubes.
Four distinct regions can be observed
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Conotruncus aorta
Ventricle
Atrium
Sinus venosus pacemaker
Blood flows anteriorly from sinus
venosus to conotruncus.
Atrium begins to expand in
preparation for dividing into left and
right portions (72 hours).
Figure 2: 72-hour chicken embryo showing the
development of the 4 chambers of the heart.
http://www2.lv.psu.edu/jxm57/chicklab/outline.html#introduction
Adrenal Glands
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Triangle-shaped endocrine
glands that sit on top of the
kidneys
Two distinct parts:
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Adrenal Cortex (Outer Core)
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Synthesis of corticosteroid
hormones from cholesterol
Adrenal Medulla (Inner Core)
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Synthesis of catecholamine
hormones
• Norepinephrine
• Epinephrine
Figure 3: Diagram of a adrenal
gland located on top of a kidney
http://www.merck.com/mrkshared/mmanual_home2/fg/fg164
_1.gif
Epinephrine
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Produced primarily from
norepinephrine
Administered by injection,
inhalation, or topically to the
eye
A cardiac stimulant to
increase heart rate and
breathing
Causes vasodilatation of
blood vessels for muscles
Increases psychological
Figure 4: Epinephrine being synthesized
alertness
from norepinephrine in a synthetic pathway
http://en.wikipedia.org/wiki/Image:Catecholamines_biosynthesis.svg
“Fight or Flight”
• The body's primitive,
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automatic, inborn response
that prepares the body to
"fight" or "flee" from
perceived attack, harm or
threat to survival.
Initiated by the
sympathetic nervous
system.
Allows the body systems
to respond to stress.
Figure 5: Molecular structure
of epinephrine
http://www.calstatela.edu/faculty/dfrankl/sbx2000/epin
ephrine.gif
How does epinephrine work?
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The hormone epinephrine binds to the β-adrenergic
receptor binding sites found in cardiac muscle cells.
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The receptors are linked to G -proteins, which are linked to
adenylyl cyclase .
Adenylyl cyclase converts ATP to cAMP (second messenger).
The cAMP molecules are used to activate Protein Kinase A.
Protein Kinase A phosphorylates the target proteins in the heart,
which promotes Ca++ output from the calcium channels.
Muscle cells begin to contract once Ca++ becomes available in
the muscle cell cytosol and bind to the troponin.
Epinephrine strengthens the heartbeat by mobilizing calcium.
Methods
• Five serial dilutions
of a 1 mg/1 mL stock
solution of
epinephrine were
made using CMRL
media:
• 1 x 10-5 mg/mL
• 1 x 10-4 mg/mL
• 1 x 10-3 mg/mL
• 1 x 10-2 mg/mL
• 1 x 10-1 mg/mL
Methods (Continued)
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Set up work station with
necessary equipment.
• Kept goose neck lamps
positioned in a way to
keep the work area
warm.
Chose 4-day egg from
incubator maintaining
horizontal position.
Carried egg to work station
and placed in glass dish
lined with cotton.
Methods (Continued)
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“Windowed” an egg according to the methods
of Cruz, 1993:
a. Placed Scotch Magic tape along the
long axis of the egg and two more pieces
of tape on either side of the center piece
of the egg.
b. Punctured the rounded end of egg with
the tip of a scissor. Used a syringe
needle to withdraw about 4 mL of
albumen and emptied into the large
beaker.
c. Cut an oval opening using the
punctured hole to expose embryo.
d. Recorded the in vivo heart rate, the
number of beats per minute (bpm), 3x’s
at 15 second intervals each, into a table.
Figure 6: Diagram showing a
“windowed” chicken egg
http://www.flickr.com/photos/egosumdaniel/305094250/
Methods (Continued)
• “Explanation” of the chicken embryo according to the
methods of Cruz, 1993:
a.
b.
c.
d.
e.
f.
Placed filter paper doughnut on blastoderm framing the
embryo.
Filled Syracuse dishes with CMRL and placed the dish on the
stage of the dissecting microscope keeping it warm with
gooseneck lamp.
Cut vitelline envelope along edge of the doughnut with scissors.
Lifted doughnut with forceps and embryo spoon to transfer
embryo to the Syracuse dish.
Removed all excess tissue from the chicken embryo.
Recorded the in vitro heart rate (bpm), 3x’s at 15 second
intervals each, into a table.
Methods (Continued)
• Application of the drug
a.
b.
c.
d.
e.
f.
g.
Filled a warm Syracuse dish with the lowest concentration of
drug.
Transferred the embryo from the CMRL into the first drug
concentration.
Recorded the heart rate (bpm) after 1 minute, 3 x’s at 15
second intervals each, into a table.
Used a pipette to remove the drug solution and transferred it
back into the test tube.
Transferred the second lowest concentration into the Syracuse
dish.
Recorded the heart rate (bpm) after 1 minute, 3 x’s at 15
second intervals each, into a table
Repeated steps d-f for the remaining concentrations.
Controls
• In this experiment, two controls were used:
• In vivo heart rate of the embryos in their
shells.
• In vitro “explanted” heart rate of the embryos
in CMRL.
Chicken Embryo Heart Video
Results
Chicken Embryonic Heart Rate Before
Exposure to Epinephrine
Beats per minute
200
150
144
95
100
50
0
Embryo 1
Embryo 2
Embryo 3
Embryo 4
Embryo 5
Average
In Vivo
In Vitro
Condition of Embryo
Figure 7: Histogram showing the average heart rates, in bpm, of all five
experimental embryos prior to exposure to epinephrine (control data).
Results (Continued)
Beats per minute
Chicken Embryonic Heart Rate with Varying
Concentrations of Epinephrine
200
180
160
140
120
100
80
60
40
20
0
152
127
108
154
109
Embryo 1
Embryo 2
Embryo 3
Embryo 4
Embryo 5
Average
1 x 10-5 mg/mL 1 x 10-4 mg/mL 1 x 10-3 mg/mL 1 x 10-2 mg/mL 1 x 10-1 mg/mL
Epinephrine Concentration
Figure 8: Histogram showing the average heart rates, in bpm, for of all five in
vitro experimental embryos exposed to various epinephrine concentrations.
Results (Continued)
Beats per minute
Comparison of the Chicken Embryonic Hearts Rates
200
180
160
140
120
100
80
60
40
20
0
y = 6.7829x + 106.36
Embryo 1
Embryo 2
Embryo 3
Embryo 4
Embryo 5
Average
Linear (Average)
In Vivo
In Vitro
1 x 10-5
1 x 10-4
1 x 10-3
1 x 10-2
1 x 10-1
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
Figure 9: Histogram comparing the effect of various epinephrine
concentrations on the heart rate, in bpm, of all five experimental embryos.
Conclusion
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The results of the experiment supported our hypothesis.
As the concentration of epinephrine increased, the heart rate of
the 4-day chicken embryo increased.
The data shows that at the second highest concentration of
epinephrine, 0.01 mg/mL, the heart rate was the fastest.
Cardiac arrest resulted four out of five times at the highest
concentration of epinephrine, 0.1 mg/mL.
Arrhythmias such as atrial fibrillation, atrial flutter, and
tachycardia were observed at the concentrations of 0.01 mg/mL
and 0.001 mg/mL.
The data suggests that the chicken embryo heart has the βadrenergic receptors at 4-days or 96-hours, allowing sensitivity
to epinephrine.
Future Research
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Use a larger number of experimental embryos.
Conduct research in a controlled environment for the explanted
embryos for stable conditions.
Count the number of beats using time-lapse video microscopy and
with this technology look closer at heart arrhythmias for proper
diagnoses .
Use older embryos in order to apply the drug directly onto the embryo
while it is still in the shell.
Embryos between 2 to 5-days will be stained with antibodies against
the β-adrenergic receptors to validate their expression via
immunofluorescence microscopy.
Other cardiovascular drugs besides epinephrine will be tested on the
chicken embryonic heart to compare/contrast effects on heart rate.
References
 Barry, A. (1950). The effect of epinephrine on the myocardium of
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the embryonic chick. Circulation. 1: 1362-1368.
Cruz, Y.P. 1993. Laboratory exercises in developmental biology.
Academic Press, San Diego, California, 241 pages
“Epinephrine.” Gold Standard. 2007.
http://www.clinciqalpharmacology.com (6 Mar. 2007)
Lipshultz, S., Shanfeld, J.,& Chacko, S. (1981). Emergence of Badrenergic Sensitivity in the Developing Chicken Heart.
Cell Biology. 78, 288-292.
McLaughlin, J.S. and McCain, E.R. (1997). In vivo and in vitro
development of the chicken heart. Tested Studies for Laboratory
Teaching, Volume 19 (C.A. Goldman, Editor). Proceedings of the
19th Workshop/Conference of the Association for Biology
Laboratory Education (ABLE) 19: 331-332.
SPECIAL THANKS TO:
Dr. Jacqueline McLaughlin
Dr. David Dressler
Nadia Abidi
Dan Devine
Steve Wentzel
Soumya Immella
Mrs. Eileen Grodziak
Mrs. Stephanie Derstine
Any Questions?