The effect of muscarinic acetylcholine receptor drugs on

porcine urinary bladder contractility in vitro

Honours Biology & Pharmacology Program, McMaster University,
Hamilton
SUMMARY
1. The purpose of this experiment was to assess the effects of various drugs acting
on distinct muscarinic (M2 and M3) and adrenergic (alpha-1) receptors found on
the porcine urinary bladder. In addition, the potency of atropine was determined
through Schild plot analysis.
2. The purpose of this experiment was to examine how various drugs can regulate a
contraction response in a porcine urinary bladder through receptor-ligand binding
of both muscarinic and adrenergic receptor agonists.
3. The addition of eserine, a cholinesterase inhibitor, increased muscle contraction.
The pEC50 of acetylcholine rose from 6.780.04 to 8.420.13, which was
accompanied by an observed increase in steepness on its concentration-effect
curve.
4. A reduction in pEC50 (from 6.630.04 to 6.200.07), as well as a reduction in
maximal response (from 94.15 6.51% to 85.7616.15%), was observed upon the
addition of phenoxybenzamine, an antagonist to carbachol.
5. The presence of isoproterenol led to a decrease in the pEC50 (from ___ to ___) on
the carbachol concentration-effect curves, signifying muscle relaxation. The
addition of propranolol prevented this response from occurring, and therefore
promoted contraction (pEC50 values rose from ____ to ____).
6. The pA2 value for atropine was derived to be 8.92.
7. The findings in this present study suggest that contraction and relaxation of the
porcine urinary bladder can be observed following pharmacological manipulation
of the receptor population.

MATERIALS AND METHODS

Organ bath preparation
Prior to every experiment, strips of porcine urinary bladder smooth muscle were
bound to the Grass FT03 force transducer and submerged into organ baths in order to
measure contraction force. Contraction data was collected using the Grass Model 7D
polygraph and displayed using Windaq software. The tissues were submerged in warmed
(37C), aerated (95% O2, 5% CO2) physiological salt solution (PSS) (4.6 mmol/L of KCl,
1.16 mmol/L of MgSO4 and NaH2PO4 each, 2.5 mmol/L of CaCl2, 115.5 mmol/L of NaCl,
21.9 mmol/L of NaHCO3, and 11.1 mmol/L of dextrose), and at a resting force of 20mN.
Tissues were initially challenged with KCl (3M) as an initial contraction, and this
response was used to normalize tissue response from experiment to experiment. KCl
challenge was repeated at the end of the experiment in order to verify that tissues which
did not contract were viable during the course of the experiment.

Effects of eserine on acetylcholines concentration-effect curve

Eserine, at a final concentration of 1x10-5 M, was added to the organ baths. After a
30-minute equilibration period, acetylcholine was added to the organ baths in increasing
half log increments, starting at 1x10-9M and ending with 4.4x10-5M. Increasing
concentrations were only added after a response had reached a plateau, or if there was no
observed response. Changes in contraction amplitude or frequency were recorded with a
polygraph and displayed using Windaq software. This data was then input into GraphPad
Prism in order to construct concentration-effect curves. The concentration-effect curve
for acetylcholine was constructed by plotting % response
contraction forcebaseline force
(% response = )
KCl challenge conctraction forcebaseline force
against -log[acetylcholine]. Agonist potency was calculated by using a nonlinear
regression within GraphPad Prism and are reported as concentrations that caused a half-
maximal effect (pEC50). Standard deviations were also included in the concentration-
effect curve.

Effects of phenoxybenzamine on carbachols concentration-effect curve

 Phenoxybenzamine, at a final concentration of 3x10-7, was added to the organ
baths. After a 30-minute equilibration period, carbachol was added to the organ baths in
increasing half log increments, starting at 1x10-9M and ending with 4.4x10-5M. These
additions occurred in the same manner as acetylcholine. After obtaining the necessary
data, concentration-effect curves were constructed as outlined above.

Effects of propranolol on isoproterenols concentration-effect curve

Tissues were then divided into two groups; one group was pretreated with
isoproterenol (1x10-6M) and left to equilibrate for 30 minutes, whereas the other group
was treated with propranolol (1x10-6M) and left to equilibrate for 15 minutes.
Isoproterenol was then added to the baths that were pretreated with propranolol and were
left to equilibrate for 10 minutes. Carbachol was then added to all baths for a final
concentration of 1x10-6. Control tissues only received the addition of carbachol. The data
was recorded and concentration-effect curves were constructed as outlined above.

Determination of atropines potency on muscarinic acetylcholine receptors

Tissues were treated with varying log concentrations of atropine (1x10-9 M to
3x10-7 M) and were then left to equilibrate for 30 minutes. Carbachol was then added to
all organ baths in the same manner as previously stated above. Control tissues only
received the addition of carbachol. Concentration-effect curves were also constructed as
described above. In order to generate a Schild plot, and therefore determine the potency
of atropine, the concentration-ratios at the EC50 values for each concentration-effect curve
were calculated. Students t-test was used to compare EC50 values of agonist responses in
the porcine urinary bladder, and p<0.05 was considered significant.

RESULTS
 The addition of eserine, as indicated by the vertical black dotted line seen in
Figure 1, produced a steady increase in contraction force. In the control baths, there was
no observed change in contraction force.

insert tracing

Figure 1. The change in contraction force over time after the addition of eserine.

As illustrated in Figure 2, the addition of eserine produced a leftward shift in the

acetylcholine concentration-effect curve, without affecting the maximal efficacy. In the
absence of eserine (control), the pEC50 value was 6.780.04. Upon the addition of
eserine, the pEC50 value increased to 8.420.13. Two sample data points in the trial above
were excluded from the graph.

Figure 2. The effects of eserine (1x10-5 M) on the concentration-effect curve of

acetylcholine. In both conditions, n=4. All data is displayed as mean standard deviation.
Figure 3. The effects of phenoxybenzamine (3x10-7 M) on the concentration-effect curve
of carbachol. In both conditions, n=4. All data is displayed as mean standard deviation.

The carbachol induced contraction, expressed as % KCl induced contraction, was

altered in the presence of phenoxybenzamine. In the first trial (Figure 3a), the addition of
phenoxybenzamine produced a slight rightward shift in carbachols concentration-effect
curve. The pEC50 value increased from -6.63 to -6.20 in the first trial upon addition of
phenoxybenzamine. The presence of phenoxybenzamine in the second trial was also
found to increase the pEC50 value from -6.22 to -5.96, which was also accompanied by a
rightward shift. In both trials, there was a slight decrease in maximal response. The first
trial shows a larger decrease in maximal response (8.39%) comparative to the second trial
(7.22%).

insert tracing

Figure 4. The change in contraction force over time after the addition of isoproterenol on
tissues that were either pretreated with propranolol or not pretreated with propranolol.
Figure 5. The effects of isoproterenol (1x10-6 M) and propranolol (1x10-6 M) on the
concentration-effect curve of carbachol. In both conditions, n=4. All data is displayed as
mean standard deviation.

The direct effects of propranolol and its interaction with isoproterenol on detrusor
contractility can be observed in Figures 4 and 5. The addition of isoproterenol produced a
gradual decrease in contraction. This effect was then cancelled out upon the addition of
propranolol (Figure 4). The tissues that were pretreated with solely isoproterenol were
found to have a greater pEC50 value (-6.080.03) than those treated with both
isoproterenol and propranolol (-6.260.04). Across all treatments, there were no
significant differences in pEC50 values. The maximal contractile response for tissues
treated with only isoproterenol differed by only 0.19% when compared to tissues that
were treated with both isoproterenol and propranolol.
Figure 6. The effects of increasing concentrations of atropine (n=1, 1x10-9 to 3x10-7 M)
on carbachols concentration-effect curve. For the control, n=2. All data is displayed as
mean standard deviation.

Carbachols concentration-effect curve was shifted rightward in the presence of

atropine. As concentrations of atropine were increased, pEC50 values were also increased.
When the lowest concentration of atropine was added (1x10-9 M), a pEC50 value of
-5.162.246 was obtained. The pEC50 values increased to a maximum of -4.261.015 at
the highest concentration of atropine (3x10-7 M). Of the six concentrations of atropine
and two controls, only two concentrations of atropine (3x10-7 M and 1x10-7 M) did not
reach a maximal contraction response, as shown in Figure 6.
Figure 7. Schild plot for the action of atropine in antagonizing the contractility induced
by carbachol on porcine urinary bladder.

A schild plot based on the results of the experiment on the effect of atropine on
the contractile response of porcine urinary bladder to carbachol is shown in Figure 7. The
slope was determined to be 4.64, with an R2 value of 0.93. This slope is therefore
considered to not be close to unity due to its large deviation from 1. Through
interpretation of the x-axis on the Schild plot, the pA2 value was determined to be 8.92.
DISCUSSION
Smooth muscle activity in the porcine urinary bladder is controlled by different
mechanisms; many of which can be manipulated pharmacologically. Through
interference with the signal transduction of receptors found on the smooth muscle, we
were able to explore the effects that various drugs had on contractility. Atropine was
found to produce an insurmountable antagonistic effect, whereas, phenoxybenzamine
produced a surmountable antagonistic effect. The application of eserine displayed
inhibition of agonist breakdown, allowing for increased action of acetylcholine. Also
studied was functional antagonism through use of isoproterenol, which was then
interfered with through use of propranolol. Through analysis of concentration-effect
curves, these different phenomena were observed.
The urinary bladder exhibits a heterogeneous population of muscarinic receptors;
most notably M2 and M3 subtypes (Yamanishi, 2000). Acetylcholine, an endogenous
neurotransmitter, acts as an agonist to both M2 and M3 subtypes in order to produce an
overall net effect of contraction. Activation of phospholipase C, with the subsequent
formation of inositol triphosphate (IP3) and diacylglycerol (DAG) to release calcium from
intracellular stores and activate a protein kinase C, is the prototypical signalling pathway
of M3 receptors. M3 activation occurs via the alpha-subunits of Gq G-proteins, whereas
M2 activation occurs via the Gi G-proteins. Stimulation of M2 receptors inhibits adenylate
cyclase (AC) and therefore the formation of cyclic-AMP, producing an inhibition of the
relaxation that would normally be caused by beta-adrenergic receptors (Frazier, 2007).
After eserine was administered to the baths, there was a noticeable increase in
contraction (Figure 1). Eserine is an acetylcholineseterase inhibitor, which prevents the
hydrolysis of acetylcholine to acetate and choline. Addition of eserine permits the local
concentration of acetylcholine to rise and overstimulate receptors, leading to a steady
increase in contractility (OBrien, 2013). Consistent with these findings was the work
done by Bell (1966) on the smooth muscle of a toad. From this study, it was concluded
that in the presence of eserine, the contractions that were produced by acetylcholine were
greatly increased. This was attributed to an atropinic action of eserine at muscarinic
receptors on the muscle cell and an enhanced release of acetylcholine from nerve
endings.
There were differences and similarities noted when comparing the effects of
acetylcholine on muscle contraction to the effects of both acetylcholine and eserine on
muscle contraction. One of the major differences was a decrease in pEC50 when
acetylcholine acted alone (Figure 2). This decrease signifies that a certain level of
contraction can be obtained at a lower concentration of acetylcholine when eserine is
present, comparative to when eserine is not present. A striking similarity can be drawn
when looking at the efficacy of both trials. It was noted that the addition of eserine did
not alter the maximal response obtained, which seemingly coincides with previous
published literature on cholinesterase inhibitor drugs, specifically eserine. Nizobe and
Livett (1982) concluded that there was no observed decrease or increase in the efficacy of
nicotine in the absence or presence of eserine.
When a receptor has been activated, there are many mechanisms that limit
overstimulation of a receptors signalling pathway. These mechanisms serve to regulate
the density of functional receptors on the cell surface. Upon consistent activation,
phosphorylation of receptors by second-messenger dependent protein kinases occurs.
This receptor phosphorylation leads to receptor desensitization through inhibition of G-
protein coupling and promotion of receptor internalization. Receptor desensitization can
be defined as the tendency of a response to wane, despite the presence of a stimulus of
constant intensity (Foreman et al., 2011). This phenomenon was observed after repeating
the experiment once more and can be signified by the slight decrease seen in both
maximal response and pEC50 in Figure 3.
Since carbachol is not easily metabolized by cholinesterase, it was used in this
experiment to examine different types of antagonism. Phenoxybenzamine is classified as
an irreversible non-surmountable antagonist. This irreversible antagonist effectively
reduces the number of receptors that are available for agonist binding. With this
knowledge, it was expected that phenoxybenzamine would reduce the maximal efficacy
of carbachol, which is signified as a downward shift in its concentration-effect curve. In
the both the first and second trial, the addition of phenoxybenzamine exhibited effects on
carbachols potency as well as maximal efficacy (Figure 3a and b). The decrease in
potency observed in the these trials could be attributed to the concept of spare receptors.
As described by Konno and Takayanagi (1985), receptors may be considered spare when
the maximal response is elicited by an agonist at a concentration that does not produce
full occupancy of the available receptors. When testing on guinea-pig ileal strips,
McPherson et al. (1985) also observed rightward parallel shifts in their concentration-
effect curves for carbachol upon the addition of phenoxybenzamine. They suspected that
the actions of phenoxybenzamine may have reflected an interaction of the antagonist with
receptor-operated calcium channels.
The addition of isoproterenol, a nonselective adrenergic agonist, induced muscle
relaxation. Based on the second messenger theory of Sutherland and Rall (1960), the
beta-adrenergic agonist binds to a receptor, forming a complex, which in turn activates
the enzyme adenylate cyclase on the inner surface of the cell membrane. This leads to
accelerated conversion of adenosine triphosphate (ATP) to cyclic AMP (cAMP). The
increase in cAMP is postulated in turn to lead to an uptake of calcium into intracellular
storage sites, resulting in the relaxation of smooth muscle. Isoproterenol and
acetylcholine have opposite pharmacological effects, where acetylcholine induces
contraction and isoproterenol induces relaxation. This phenomenon of opposing effects
has been termed as functional antagonism and was dually noted upon the addition of
propranolol. The presence of propranolol effectively diminished the effects of
isoproterenol. This functional antagonism can be recognized in Figure 4. An increasing
amount of isoproterenol led to a downward shift of carbachols concentration-effect
curve, which was then disrupted upon the addition of propranolol. Across all eight
treatments, there was no significant observed change in pEC50 values. In a study done by
Fernandes et al. (1992), the investigators further illustrate that stimulation of M2
muscarinic receptors in smooth muscle plays an important role in functional antagonism
by reducing the relaxation caused by agents such as isoproterenol.
 In a study done by Konno and Takayanagi (1985), the addition of atropine, a
competitive antagonist of muscarinic receptors, produced a parallel shift to the right in
the concentration-response curves for carbachol. These results were consistent with the
results that were produced in this experiment (Figure 6). Atropines competitive
antagonistic properties were also reflected in the linearity of the obtained Schild plot
(Figure 7). The results of this experiment described the pA2 value of atropine to be 8.92,
which is consistent with the findings of Konno and Takayanagi (1985), who concluded
that the pA2 value of atropine, derived from experiments performed on rabbit smooth
muscle, was 8.97 +/- 0.25. insert concluding statements about atropine
The results of this experiment successfully demonstrated how various drugs
influence different receptor subtypes on the porcine urinary bladder and ultimately, alter
the final response of the tissue. This experiment was a fairly quick and easy approach to
assessing the physiology and pharmacology of bladder tissue in a well controlled
environment. Finally, ______.
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