2. Introduction:
The purpose of this experiment was to test the effects of neuromuscular blocking agents
on striated, skeletal muscle in response to orthodromic electrical stimulation of the phrenic
nerve. An isolated rat diaphragm in a nerve-muscle preparation will be used to measure the
effects of three drugs: tubocurarine, succinylcholine, and edrophonium on nicotinic-
acetylcholine receptors (nAChr).
The phrenic nerve innervates the diaphragm to control breathing and is one of a pair of
nerves. It arises from cervical spinal roots and passes down the thorax to its target destination to
regulate breathing and is myelinated for faster action potential propagation. The diaphragm is a
sheet of muscle that extends across the bottom of the rib cage separating the thoracic cavity from
the abdominal cavity. During inhalation, the diaphragm contracts to enlargen the thoracic cavity
which is necessary for proper respiration. The decrease in pressure created from the enlargened
thoracic cavity is reduced as O2 gets pulled into the lungs. When the diaphragm relaxes, air is
exhaled through the lungs.
Diaphragm muscles are multinucleated and are classified as skeletal. Skeletal muscles are
composed of muscle fibers, which contain many myofibrils with thick filaments of myosin and
thin filaments of actin in sarcomeres. Thin filaments (actin) bound to Z-lines define the I-band.
Thick filaments (myosin) are bound to Z-lines by titin fibers, and the length of the myosin on
both sides of the M-line defines the A-band. The H-zone is defined by the distance between two
adjacent actin filaments connected to different Z-lines.
When the phrenic nerve is stimulated, Ca2+ ions flow in through voltage gated calcium
channels into the nerve cytoplasm to allow storage vesicles to fuse with the presynaptic
membrane and release their contents of acetylcholine into the neuromuscular junction.
Acetylcholine proceeds to bind the nAChRs on the skeletal muscle. Upon the binding of ACh,
ion channels on the postsynaptic membrane open to allow Na+ ions to rush into the muscle fiber,
initiating a graded depolarization. The graded depolarization activates voltage-gated sodium and
potassium channels, and an action potential occurs on the muscle fiber. The action potential (AP)
travels down the sarcolemma and enters T-tubules where L-type calcium channels open in
response to the AP. Ca2+ ions passing through the T-tubule bind to ryanodine receptors in the
adjacent sarcoplasmic reticulum (SR) membrane activates release of additional Ca2+ from the
SR. The extensive meshwork of sarcoplasmic reticulum ensures that it is able to readily diffuse
Ca2+ to all troponin sites. The cross-bridge cycle starts with the increase in calcium ions, which
allows the myosin-binding site to become available. Ca2+ ions bind to troponin to pull
tropomyosin away from the cross-bridge binding site. Once myosin binds to actin, ADP and Pi
which energized the cross bridge, depart to enable the cross-bridge to move. ATP proceeds to
bind to myosin to initiate uncoupling from actin. Partial hydrolysis of the bound ATP re-
energizes the cross-bridge and prepares the myosin for the next cross-bridge cycle. During
contraction, myosin binds to actin and slides it, to pull the Z-lines closer. This arrangement
reduces the length of the I-bands (the distance between the A-band and H- zones). Contractions
occur quickly because each myosin head can bind to ATP and hydrolyze through the cross-
bridge cycle.
An orthodromic current will be used via stimulation by a S44 Grass Stimulator that can
run voltages from 0.5V to 150V. Orthodromic impulses were used to mimic the normal direction
of an action potential running down an axon. The threshold and maximal responses will be found
to keep the tissue at the supramaximal voltage level (one volt above the maximal response
voltage). The frequency used to initiate tetanus will be determined by increasing the stimulation
3. rate until tetanus is induced. Unfused tetanus is defined as partial dissipation of elastic tension
between subsequent stimuli, and fused tetanus is defined as sustained muscle contraction caused
by rapidly repeated stimuli. A sustained contraction will be induced by a high stimulation
frequency. The point of inducing tetanus after the tissue experienced a partial block from either
depolarizing or non-depolarizing blocking drugs was to analyze the different patterns of response
and study the mechanisms of actions of the two drugs.
The three drugs used in this experiment all act upon the somatic nervous system which is
a branch of the peripheral nervous system. The somatic nervous system is further divided into
afferent and efferent nervous systems. Efferent nerves carry impulses from the brain to stimulate
skeletal muscles for contraction via release of acetylcholine (ACh) from upper cell bodies of
upper motor neurons to nicotinic receptors of the alpha motor neurons. That signal is then
relayed to neuromuscular junctions where ACh is released as a neurotransmitter which binds to
postsynaptic nAChRs on the muscle membrane and presynaptic nAChRs. Presynaptic nAChRs
function to facilitate ACh release during sustained synaptic activity (2). The nAChR is a
pentameric complex composed of two -subunits, a single β, γ, and ε subunit. ACh binds
specifically to the subunit to initiate depolarization.
Tubocurarine is the active ingredient in the plant toxin curare, and is rarely used as a
surgical muscle relaxant. This plant alkaloid is a non-depolarizing neuromuscular blocker that is
a competitive antagonist of nicotinic neuromuscular acetylcholine receptors in the somatic
nervous system. It binds to the subunit of the nAChR on the post-synaptic muscle fiber
membrane. Tubocarine should reduce contraction and paralyze the diaphragm when dosed onto
the tissue. It has a slow onset and a slow recovery period (2).
Succinylcholine is a competitive agonist of the nAChr with a quick onset and short
recovery period and has a higher affinity than tubocurarine. It binds to the subunits of
presynaptic and postsynaptic nAChRs in the neuromuscular junction, and elicits two blocking
phases: phase I (with a low concentration of succinycholine) and phase II (with a high
concentration of succinylcholine). The phase I block causes depolarization which opens
receptors’ cation channels, causing Ca2+ to be release from the SR to elicit multiple contractions
which are spastic. However, the prolonged binding prevents the muscle cell from repolarizing by
maintaining the membrane potential above threshold via a constant inflow of cations. In phase II,
the receptor blockade takes on characteristics of a non-depolarizing neuromuscular block,
decreasing muscle contractions to the point where the muscle is flaccid. The strength and number
of contractions is decreased due to desensitization of the receptors and inactivation of voltage-
gated sodium channels at the neuromuscular junction (5). The prolonged block is due to the
metabolism of succinylcholine. Butyrylcholinesterase is the enzyme which hydrolyzes
succinylcholine but is found primarily in plasma. Furthermore, little or no butyrlcholinesterase is
present at the neuromuscular junction. If the experiment were to have taken place in vivo,
succinylcholine would paralyze the tissue for a shorter time period than tubocurarine due to
diffusion away from the neuromuscular junction and hydrolysis by plasma butyrlcholinesterase
(5). Compared to the hydrolysis of acetylcholine by acetylcholinesterase, succinylcholine takes a
longer time because the tissue has been removed from its physiological environment.
Edrophonium is a rapid-onset, short-acting acetylcholinesterase inhibitor that will
increase acetylcholine levels by preventing its hydrolysis. It should enhance the block of skeletal
muscle contractions following succinylcholine, and should reverse the block of skeletal muscle
contractions following tubocurarine (3).
4. Methods:
Vials were initially labeled with the drug names and corresponding bench concentrations to
help keep all material organized. Drug dilutions were then prepared, using Krebs ringer for the
dilutions (Table 1). The PolyVIEW System was then calibrated to 0.00 Volts (balance) and 1.00
Volts (gain), and chart speed was set to 5 mm/sec.
After a euthanized rat was exsanguinated, the dissection process began while making sure the
metal scissors and other metal objects did not come close to the target phrenic nerve or
diaphragm. Care was taken to keep the phrenic nerve and diaphragm moist at all times with
aerated Krebs ringer. The fur, muscle, and skin were removed to uncover the thoracic cavity and
abdominal cavity (Figure 1). The initial cut was made from the rat’s bottom left-side of the
ribcage along the backbone, past the 6th rib towards the head to expose the diaphragm. Time was
taken to stop and look inside to make sure the nerve was not attached to the chest. The ribs were
then cut laterally along the 6th rib from backbone towards the sternum and down the sternum in
between the two diaphragms (Figure 2). The upper portion of rib cage and lungs were removed
using plastic forceps. A cut was made laterally below the rib cage to isolate the rib-diaphragm
tissue instead of below the diaphragm. A cut along the back of the diaphragm freed the ribs-
diaphragm preparation (Figure 3). The phrenic nerve was then isolated by cutting away
connective tissue. A thin suture (3.0) was tied around the top of the nerve, near the top of the
spinal cord (Figure 4). Cuts made from the sternum and backbone towards the vena cava
produced a fan-shaped piece of diaphragm with the strip of ribs on the outside and the tendonous
material around the vena cava as the apex. The phrenic nerve was freed near the thymus using
plastic forceps. A piece of suture (6.0) was threaded through the diaphragm away from the
phrenic nerve, and the other end of the suture was tied to the transducer (Figure 4). While one
person held the diaphragm in place, a second person mounted the tissue underneath the
horseshoe. The phrenic nerve was coiled twice around the electrode wires. The nerve came down
through the plastic loop of the tissue holder and the diaphragm was anchored under the loop with
the back of the bath up against the ribs for support. The excess suture on the phrenic nerve was
wrapped around the plastic tube on the right-side which led down to the electrode wires (Figure
5). The bath was then raised so Krebs ringer (warmed to 32.1°C) covered the prep and nerve
completely (Figure 6). Care was taken to make sure the diaphragm suture did not touch the top of
the mount or the bottom of the horseshoe to prevent any transducer misreading (Figure 7). The
tissue was gently aerated with 95% O2 and 5% CO2.
GRASS S44 stimulator was turned on and stimulation rate was set to 1 pulse per second
(PPS) by setting the large dial to 10, and the small dial to 0.1x. The delay dial was not touched or
used throughout the experiment. The duration was set to 0.3 milliseconds (ms). Pulses were set
by flipping switch into the “up position” to “pulses”, not “DC”. Output switch was flipped up to
the “on” position. The isolator’s coupling switch was set to “direct”. Polarity was set to
“normal”. “multiply input volts by” dial was set at “1”.
The threshold, maximal response, supramaximal response were then established by setting
the simulator to 0.1 Volts and increasing by 0.2 Volts at a time. Normal polarity was used for the
entire experiment. Threshold value was determined to be at 69 Volts. Maximal voltage, where
any further increase did not result in a higher response was determined to be at 130 Volts.
Supramaximal voltage was established by setting the stimulator to one volt higher than maximal
voltage at 131 Volts and was used throughout the entire experiment. The tetanus frequency was
determined by increasing the stimulation rate, using the x1 range initially and then increasing to
5. x10 to run the gamut of the large dial at each frequency/sec until sustained contraction was seen.
The tetanus frequency was determined to be 20 PPS.
Whenever tetanus was required, stimulator was turned off, frequency was set to 20 PPS,
chart speed was increased to 100 mm/min, and then stimulator was turned on. After tetanus was
observed, power was turned off, chart speed and stimulation rate were reset to prior levels, and
power was turned back on.
During drug dosing, stimulator was turned off for washes to prevent destruction of the nerve.
Before each drug administration, at least one-minute of control response was recorded. Drug was
left in contact with tissue until the desired response was elicited. After response, the tissue was
washed and stimulated until the height of the contraction was back to the control level or a new
baseline.1E-6M tubocurarine was administered until 50% paralysis was observed. Following a
wash and establishment of control contractions, 2E-6M tubocurarine was administered until 50%
paralysis was observed, without washing; 3E-5 M edrophonium was administered until an
increase in contractions was observed to return to near control levels. Following a wash and
establishment of control contractions, 2E-6 M tubocurarine was administered until 50%
paralysis, and the nerve was tetanized for 10 seconds. Following a wash and establishment of
control contractions, 3E-6 M tubocurarine was dosed until 10-20% paralysis was observed,
without washing; 3E-6 M succinylcholine was administered. Following a wash and
establishment of control contracations, 6E-6 M succinylcholine was dosed to demonstrate a
phase II block. Following a wash and establishment of control contractions, 6E-6 M
succinylcholine was administered until 50% paralysis then followed by a tetanus stimulation of
10 seconds. Following a wash and establishment of control contractions, 6E-6 M succinylcholine
was administered until 50% paralysis, without washing, 2E-6M tubocurarine was dosed.
Following a wash and establishment of control contractions, 6E-5 M succinylcholine was
administered until 50% paralysis, without washing, 3E-5 M edrophonium was dosed. Voltages,
rates, and duration values were all recorded and included in
Table 1: Drug Dilution Table
Drug [Stock]
in M
[Bench]
in M
Dilution
Factor
Vdrug
(mL)
Vringer
(mL)
Vdose
(mL)
Bath
Vol.
[mL]
[Bath]
in M
Tubocurarine 3E-3 1E-3 3 3 6
1E-3 1E-4 10 2 18 1.0 50 2E-6
0.5 50 1E-6
Edrophonium 5E-2 5E-3 10 2 18
5E-3 2.5E-3 2 5 5 0.6 50 3E-5
Succinylcholine 3E-2 3E-3 10 1 9
3E-3 1E-3 10 1 9 0.5 50 3E-6
1.0 50 6E-6
1E-3
1E-4 10 2 8 1.0 50 2E-6
0.5 50 1E-6
4E-4 2.5 4 6
1.00 50 8E-6
0.75 50 6E-6
0.50 50 4E-6
6. Table 1 Sample Calculations:
Tubocurarine dilution Factor:
[ 𝑆𝑡𝑜𝑐𝑘]
[𝐵𝑒𝑛𝑐ℎ]
=
1𝐸 −3 𝑀
1𝐸 −4 𝑀
= 10
Tubocurarine Vdrug: C1V1 = C2V2
(1E-3 M)(V1) = (1E-4 M)(20 mL)
V1 = 2 mL
Tubocurarine Vringer: 20 mL –Vdrug
20 mL – 2 mL = 18 mL
Tubocurarine VDose: C1V1 = C2V2
(1E-4 M)(V1) = (1E-6 M)(50 mL)
V1 = 0.5 mL
Figure 1: Removal of fur, skin, and muscle covering thoracic and abdominal cavity
Cut fur along line to uncover
thoracic cavity and some of
abdominal cavity
7. Figure 2: Cuts made on rib cage
Figure 3: Isolation of diaphragm and suture of phrenic nerve
Start cutting here
Cut along 6th rib from (b) to (c)
(b)
(c)
Backbone
Sternum
Tie THIN suture (3.0)
around top of nerve
near spinal cord
Cut BEHIND diaphragm to isolate
diaphragm with horseshoe ribs
(for support)
8. Figure 4: Suture of diaphragm and phrenic nerve
Figure 5: Diaphragm mount and electrode wires
Figure 6: Tissue in bath
Thread a piece of THICK suture
(6.0) through diaphragm
Diaphragm and Ribs
Phrenic Nerve
Suture (around
phrenic nerve)
Loop nerve twice
around electrode
wires
Mount
diaphragm here
underneath
horseshoe
Tissue in Krebs
ringer, aerated
with 95% O2
and 5% CO2
9. Figure 7: Suture set-up (Make sure suture does not touch these two features)
Top of Mount
Bottom of Mount
(Horseshoe)
12. Discussion:
The purpose of this experiment was to observe the effects of neuromuscular blocking
agents on the nicotinic receptors in the rat diaphragm in conjunction with orthodromic
stimulation of the phrenic nerve. The utilization of orthodromic currents allows researchers to
recreate the natural biological environment for the tissue preparation. Orthodromic currents run
through the nerve in the normal direction to elicit contractions that would be seen in a living rat.
The phrenic nerve is a pair of nerves arising from the cervical roots that passes down the thorax
to innervate the rat diaphragm and are a part of the efferent division of the somatic nervous
system, which is a branch of the peripheral nervous system.
The tissue used in this experiment differs from the previous experiments in the use of
skeletal muscle tissue instead of cardiac or smooth muscle tissue. Skeletal muscle is a form of
striated muscle tissue that is primarily attached to bundles of collagen fibers known as tendons.
Skeletal muscle is made of muscle fibers that are formed from the fusion of developmental
myoblasts. Myoblasts are a type of embryonic progenitor cell that gives rise to muscle cells. The
skeletal muscle fibers are long, cylindrical, and multinucleated cells made up of myofibrils.
Repeating sacromere sections consist of the myofibrils. Sarcomeres are composed of repeated
fibrous proteins known as myosin and actin. The sarcomere is the functional unit of the cell
responsible for the skeletal muscle’s striated appearance and forms the machinery necessary for
muscle contraction.
Skeletal muscles are the only type of muscle to be stimulated by tetanus to produce
prolonged contractions. These mechanisms take place through the cross bridge mechanism. In a
relaxed muscle, tropomyosin blocks the attachment site for the myosin cross bridge, preventing
contraction. When the muscle cell is stimulated to contract by an action potential, calcium
channels open in the sarcoplasmic reticulum. Ca2+ ions attach to troponin, forming a complex
which pulls tropomyosin away from the cross-bridge binding site. Tetanus induced by repeated
electrical stimulation causes acetylcholine (ACh) to be constantly released from motor end
plates. The excess ACh constantly activates nicotinic acetylcholine receptors (nAChR) on the
skeletal muscle to cause a prolonged depolarization of the cells. Ion channels remain open, and a
rise in Ca2+ ions sustains the cross-bridge cycle. The release of ACh due to tetanus is at such a
high rate that acetylcholinesterase (AChE) is unable to degrade the ACh fast enough to prevent a
prolonged contraction.
STATE CONTROL VALUES FOR EACH DRUG DOSE CYCLE
The initial dose of tubocurarine 1E-6 M elicited a response of 0.28 g which correlates to a
decrease of 53.3% change response and 76.6% paralysis. This was expected because
tubocurarine is a non-depolarizing competitive antagonist of nAChRs at the presynaptic and
postsynaptic neuromuscular junction. Antagonism of the presynaptic receptor downregulated the
modulation of ACh vesicles for release, while antagonism of the postsynaptic receptor inhibited
ACh from initiating the action potential which would have led to an increase of Ca2+ ions for
contraction. The decrease in response after administration of tubocurarine matched the expected
response.
13. Tubocurarine 2E-6 M dosed afterwards elicited a response of 0.4 g which correlates to a
decrease of 61.9% change response and 66.6% paralysis. This was expected because
tubocurarine antagonized both presynaptic and postsynaptic nAChRs of the neuromuscular
junction to inhibit presynaptic release of ACh and postsynaptic depolarization. The following
edrophonium 3E-5 M dose elicited a response of 0.85 g which correlates to an increase of
112.5% change response (compared to prior tubocurarine response) and 29.2% paralysis.
Edrophonium is an acetylcholinesterase (AChE) inhibitor which increased the amount of ACh
available at the neuromuscular junction. The increased levels of ACh outcompeted tubocurarine
for nAChRs presynaptically to upregulate modulation of ACh vesicles for release and
postsynaptically to initiate the action potentials for contraction. The increase in response after
administration of edrophonium matched the expected response of a paralysis reversal.
The next dose of tubocurarine 2E-6 M elicited a response of 0.38 g which correlates to a
decrease of 51.3% change response and 68.3% paralysis. This was expected because
tubocurarine antagonized both presynaptic and postsynaptic nAChRs of the neuromuscular
junction to inhibit presynaptic release of ACh and to inhibit postsynaptic depolarization. At 50%
paralysis, the tissue was tetanized. This elicited a single strong contraction which matched the
control contractions, followed by a flaccid muscle state which registered 0.02 g in response.
Blockade of presynaptic nAChRs by tubocurarine prevents ACh from being made available fast
enough to support sustained tetanus. The presynaptic positive feedback control system, which
serves to maintain the availability of ACh for vesicle release, was downregulated. The ACh
vesicles ready for exocytosis were released at the start of tetanus to elicit the brief contraction.
However, additional ACh vesicles were inhibited from being modulated for vesicle release. The
lack of ACh released is the reason why the tissue became flaccid. A lack of ACh in conjunction
with the antagonized postsynaptic nAChR was the reason why tetanic stimulation only elicited a
brief response before returning to a flaccid state.
A dose of tubocurarine 4E-6 M elicited a response of 0.3 g which correlates to a decrease
of 21.1% change response and 75% paralysis. This was expected because tubocurarine
antagonized both presynaptic and postsynaptic nAChRs of the neuromuscular junction to inhibit
presynaptic release of ACh and postsynaptic depolarization. At 20% inhibition of response due
to tubocurarine, succinylcholine 4E-6 M was dosed to elicit a response of 0.2 g. The
succinylcholine 4E-6 M response correlates to a decrease of 33.3% (compared to prior
tubocurarine response) and 83.3% paralysis. The addition of succinylcholine 4E-6 M should
have reversed the competitive block by tubocurarine because succinylcholine has a higher
affinity for nAChRs. Succinylcholine should have outcompeted tubocurarine to agonize the
presynaptic and postsynaptic nAChRs to upregulate modulation of ACh vesicles for release and
initiate the action potential in the muscle. The lack of block reversal with succinylcholine may be
attributed to the previous tetanus stimulation which may have downregulated presynaptic ACh
release. Furthermore, the concentration of succinylcholine used (4E-6 M) may have been too
high to elicit phase I effects of increasing response. Phase I can only be elicited if a low enough
concentration of the depolarizing blocking agent is used. The experiment could be improved by
running a range of succinylcholine concentrations to find an appropriate dose which will elicit
phase I.
14. A phase II block was exhibited with a dose of 6E-6 M succinylcholine. The dose elicited
a response of 0.05 g which correlates to a decrease of 90.6% change response and 95.8%
paralysis. This was expected because succinylcholine is a competitive depolarizing agonist of
nAChRs. The two phases of a depolarizing block is dependent on the concentration of drug used.
Phase I of the depolarizing block was not seen due to the high concentration of succinycholine
(6E-6 M). Depolarizing neuromuscular blockers such as succinylcholine produces prolonged
depolarization of the end-plate region that results in desensitization of nAChR and inactivation
of voltage-gated sodium channels at the neuromuscular junction. The end result is failure of
action potential generation and a continuous block of contraction (5). The decrease in response
due to succinylcholine 6E-6 M matched with predicted responses of a phase II block.
Succinylcholine 6E-6 M elicited a response of 0.14 g which correlates to a decrease of
50% change response and 81.6% paralysis. The decrease in response was expected because the
use of a depolarizing blocking agent at high concentration induces a phase II block in which
prolonged depolarization of nicotinic receptors leads to receptor desensitization and inactivation
of voltage-gated sodium channels at the neuromuscular junction. At 50% paralysis, the tissue
was tetanized. Tetanus following a phase II block mimics the response seen with tetanus
following a non-depolarizing block (e.g. tubocurarine). While non-depolarizing agents
antagonize nAChRs, the desensitization of receptors due to phase II block essentially produces
the same effect: receptors are no longer responsive to stimulation (6). This elicited a single
strong contraction which surpassed the control contractions, followed by a flaccid muscle state
which registered 0.02 g in response, similar to the tetanus induced response following
tubocurarine. Desensitization of presynaptic nAChRs by succinylcholine prevented ACh from
being modulated for vesicle release fast enough to support sustained tetanus. The presynaptic
positive feedback control system, which serves to maintain the availability of ACh for vesicle
release, was downregulated. The ACh vesicles ready for exocytosis, were released at the start of
tetanus to elicit the brief contraction. However, additional ACh vesicles were inhibited from
being modulated for vesicle release. A lack of ACh in conjunction with the desensitized
postsynaptic nAChRs was the reason why tetanic stimulation only elicited a brief response
before returning to a flaccid state.
Treatment with a low concentration of depolarizing blocking agent, prior to tetanus,
would have elicited a reduced but well sustained contraction. During phase I, nAChRs would be
agonized, but receptor desensitization does not occur due to the low concentration of
depolarizing blocking agent used. An important concept to consider is the economy of ACh
synthesis, storage, and release. Response to stimulation fades over time with repetitive
stimulation (e.g. tetanus) because of a decrease in the amount of ACh released from the
prejunctional nerve terminal with successive stimuli. The quantity of ACh released with each
nerve action potential is inversely proportional to the frequency of stimulation (5). Tetanic
stimulation following phase I would stimulate the presynaptic membrane to release ACh, and the
fade of response due to repetitive stimulation is prevented by succinylcholine’s agonism of
presynaptic nAChRs which up-regulates modulation of ACh vesicles for release.
15. Succinylcholine 6E-6 M elicited a response of 0.1 g which correlates to a decrease of
44.4% change response and 91.7% paralysis. The decrease in response was expected because the
use of a depolarizing blocking agent at high concentration induces a phase II block in which
prolonged depolarization of nicotinic receptors leads to receptor desensitization and inactivation
of voltage-gated sodium channels at the neuromuscular junction. At 50% paralysis relative to
control, a dose of tubocurarine 2E-6 M elicited a response of 0.04 g which correlates to a
decrease of 60% change response and 96.7% paralysis. The decrease in response was expected
because tubocurarine antagonized the remaining functional receptors. The combined effect of
desensitized receptors, inactive ion channels, and antagonism of functional receptors resulted in a
complete neuromuscular block.
On the other hand, a lower concentration of succinylcholine (inducing phase I block)
followed by tubocurarine, may have caused a block reversal. The increase in response due to
agonism of nAChRs by succinylcholine would have been reduced via antagonism by
tubocurarine.
Succinylcholine 6E-6 M elicited a response of 0.05 g which correlates to a decrease of
50% change response and 95.8% paralysis. The decrease in response was expected because the
use of a depolarizing agent at high concentrations induces a phase II block in which prolonged
depolarization of nicotinic receptors leads to receptor desensization and in activation of voltage-
gated sodium channels at the neuromuscular junction. At 50% paralysis a dose of edrophonium
3E-5 M elicited a response of 0.04 g which correlates to a decrease of 20% change response and
96.7% paralysis. Edrophonium is an acetylcholinesterase inhibitor which prevents the hydrolysis
of ACh. While edrophonium reversed the block induced by tubocurarine, the phase II block is
potentiated by the increase of ACh levels. The combined effect of succinylcholine and ACh
repeatedly stimulating nAChRs led to complete receptor desensitization and an enhanced block.
In conclusion, the experiment clearly demonstrated the effects of non-depolarizing and
depolarizing neuromuscular blocking agents on nicotinic receptors in the neuromuscular junction
between the phrenic nerve and diaphragm of the rat. The experiment could be improved by
having students run a range of succinylcholine to find two different doses which will elicit phase
I and phase II block.
16. References:
1. Gottschall, J (March 1981). “The Diaphragm of the Rat and its Innervation.
Muscle Fiber Composition; Perikarya and Axons of Efferent and Afferent
Neurons.” Retrieved November 7, 2012, from: Medicine 4, 405-417.
2. Elsevier (2011). Rang and Dale’s Pharmacology 7e. Retrieved November 7, 2012,
from: www.studentconsult.com
3. Woolsey J et al. (January 2006). “DrugBank: A Comprehensive Resource for in
silico Drug Discovery and Exploration.” Retrieved November 7, 2012, from:
Nucleic Acids Jan, 34 (Database issue):D668-72. http://drugbank.ca/
4. Lab manual
5. Good paper
6. http://bentollenaar.com/_MM_Book/Ch.9.htm
