Individual Pharmacology Practical Report with the organs Ileum, Aorta and Atrium
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Course
Pharmacology Practical
Institution
Rijksuniversiteit Groningen (RuG)
This is an individual report of the course Pharmacology practical in which the unknown compounds are investigated in the organs: ileum, aorta and atrium. The grade received for this report was a 6.5 and therefor sufficient to pass the course.
,Table of Contents
Introduction and Theory........................................................................................................................3
Materials and Methods.........................................................................................................................9
Small organ bath................................................................................................................................9
Large organ bath................................................................................................................................9
Chemicals.........................................................................................................................................10
Process.............................................................................................................................................11
Ileum............................................................................................................................................11
Aorta............................................................................................................................................11
Atrium..........................................................................................................................................11
Formulae..........................................................................................................................................11
Processing results / Data analysis....................................................................................................12
Results.................................................................................................................................................13
Ileum................................................................................................................................................13
Aorta................................................................................................................................................16
Atrium..............................................................................................................................................21
Discussion............................................................................................................................................24
Ileum................................................................................................................................................24
Aorta................................................................................................................................................26
Atrium..............................................................................................................................................27
Conclusion...........................................................................................................................................28
References...........................................................................................................................................29
Appendices..........................................................................................................................................30
Ileum................................................................................................................................................30
Aorta................................................................................................................................................33
Atrium..............................................................................................................................................35
2
,Introduction and Theory
In this report an experiment will be described in which there are three unknown compounds
A, B and C. These compounds are investigated by using three different types of organs the
ileum, aorta, and atrium. The compounds will be determined by using several reference
compounds to compare with and investigate whether there is a right shift when the unknown
compound is added.
First the types of muscles will be discussed, there are three types of muscles: skeletal
muscle, smooth muscle, and cardiac muscle.
Skeletal muscles are voluntary modulated, the skeletal muscle fibers are large,
multinucleate cells that appear striped or striated. Skeletal muscle can cause
contraction when a somatic motor neuron releases acetylcholine at
neuromuscular junction by calcium influx. Acetylcholine activates nicotinic
receptors that are present on the motor end plate which leads to an entry of
Sodium through acetylcholine receptor-channels which will initiate a muscle
action potential. This action potential in t-tubule alters conformation of
Dihydropyridine L-type calcium channel (DHP) receptor, the DHP receptor opens
Ryanodine receptor-channel (RyR) calcium release channels in the sarcoplasmic
reticulum and calcium enters the cytoplasm. Calcium binds to troponin allowing
actin-myosin binding and displacement of tropomyosin and the myosin heads
execute a power stroke. The actin filaments slide towards the center of
sarcomere and causes the contraction of the skeletal muscle. [2][3]
Smooth muscles have a smooth structure, and cause a slow contraction
compared to skeletal muscle tissue. The smooth muscle does not consist of
sarcomeres. The smooth muscle does have myosin and actin filaments but does
not have troponin and the contraction is differently regulated compared to the
skeletal muscle. Smooth muscle contraction occurs in response to a rise in
calcium concentration and in smooth muscle cells it can occur with and without
an action potential. The action potential in smooth muscle cells is generated by
the L-type calcium channels that allow Calcium entry. Smooth muscle
contraction starts by acetylcholine which activates M3 and activates the Gq
protein which leads to PLC and IP3 which will induce a release of calcium from
the sarcoplasmic reticulum by activation of receptors on the sarcoplasmic
reticulum. There is an increase in intracellular calcium and the calcium binds to
calmodulin. The calcium-calmodulin complex activates myosin light chain kinase
which phosphorylates myosin light chains in myosin heads and increases myosin
ATPase activity. The active myosin crossbridges slide along actin and create
muscle tension which leads to the contraction of smooth muscle cells. IP3 is
generated by activation of many types of G protein coupled receptors and this
pathway does not generate an action potential for the contraction. The RyR
channels are also present in many smooth muscle cells which induce calcium
release.
The contractile machinery of smooth muscle cells is activated when the myosin
light-chain undergoes phosphorylation causing it to become detached from the
actin filaments. This phosphorylation is catalyzed by myosin light-chain kinase,
and this is activated when it binds to calcium. Myosin phosphates reverse this
phosphorylation and causes relaxation of the smooth muscle. This leads to a
balanced effect of promoting contraction and relaxation.
Both enzymes (myosin light chain kinase and myosin phosphate) are regulated
by cAMP and cGMP and many drugs that cause smooth muscle contraction or
relaxation are mediated through the G protein coupled receptors.
3
, Relaxation of smooth muscle cells is induced by inhibition of MLCK or activation
of MLC phosphatase. The relaxation of smooth muscle is associated with a
decrease in calcium and an increase in cAMP or cGMP concentration. [2][3]
Cardiac muscle consists of striated muscle tissues, sarcomeres and one nucleus
per contractile cell. The contraction of cardiac muscles is induced by calcium.
Cardiac muscle contraction starts by an action potential which enters from
adjacent cell. The voltage-gated L-type calcium channels will then open, and
calcium enters the cell. Calcium induces more calcium release through RyR
channels, this local release of calcium causes a calcium spark which is an
increase of calcium in the cytosol of a cardiomyocyte. Summed calcium sparks
create a calcium signal, the calcium ions bind to troponin to initiate contraction
and displaces the tropomyosin. [2][3]
Specifically in the aorta there is also the Nitric Oxide (NO) pathway which can
induce constriction or relaxation. This is induced via endothelial cells instead via
a skeletal muscle, smooth muscle, or cardiac muscle. NO mimic the compound
which is naturally released from the endothelial in the vessels of the body. NO
increases and activates a protein called Guanylyl cyclase and this leads to an
increase in cGMP. cGMP causes a decrease in calcium which will then lead to
relaxation of the vessels (aorta). In the aorta experiment the role of the
endothelium in the action of the unknown compound is negligible. [1][3]
G protein coupled receptors (GPCRs) play an important role in determining the
unknown compounds in this experiment. GPCRs are transmembrane receptors
which sense molecules outside the cell and initiate intracellular transduction
pathways which then leads to a cellular response. GPCRs consist of 7
transmembrane alpha-helices across the membrane. The G protein consist of an
alpha, beta, and gamma subunit. When GDP is bound the G protein coupled
receptor is inactive and when GTP is bound the G protein coupled receptor is
active. There are three types of G protein coupled receptors alpha subtypes
which are Gi, Gs and Gq. [2]
Gs alpha subtype is also known as the stimulating subtype. When a ligand binds
to this Gs protein coupled receptor the GDP present on the alpha subunit will be
exchanged with GTP and this activates the alpha subunit and, in this case the
adenylate cyclase will be activated which leads to an increased conformation of
ATP into cAMP. The increased amount of cAMP will then activate protein kinase A.
The activated protein kinase A cause a decrease of calcium which will lead to
smooth muscle relaxation. cAMP is on itself a relaxing agent, so when there is
more cAMP there will be relaxation. Examples of Gs receptors are the beta1,
beta2 and beta3-adrenergic receptors, histamine H2 receptor and serotonin
5HT4 receptor. [2]
Gi alpha subtype is also known as the inhibiting subtype. The Gi alpha subtype
inhibits the adenylate cyclase which causes less conversion from ATP to cAMP.
There will be a lower amount of cAMP and therefore also less protein kinase A
which lead to constriction due to less calcium release. cAMP is on itself a relaxing
agent, so when there is less cAMP there will be constriction. Examples of Gi
receptors are the alpha2-adrenergic receptor, M2 and M4-cholinergic receptors,
histamine H3, H4 receptors and serotonin 5HT1 receptor.
When a compound binds to the Gq alpha subtype than the Gq signaling pathway
is activated. The Gq subtype stimulates the activation of phospholipase C which
is an enzyme that converts PIP2 into DAG and IP3. DAG will activate Protein
4
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