CARDIOLAB

Cardiolab is a computer simulation of in vivo blood pressure and heart rate responses to
intravenously injected drugs.
This experiment uses both normal anaesthetised and pithed anaesthetised preparations. In
the latter, the central nervous system has been destroyed including the autonomic
cardiovascular reflex pathway (baroreceptor reflex).

Resting Blood Pressure and Heart Rate

Blood Pressure / mmHg

320
200 Normal preparation

Heart rate / bpm

Baroreceptor present
150 240
to sense changes in
blood pressure and
100 160 regulate these
changes.
50 80

0 0
Time (minutes)

Resting Blood Pressure and Heart Rate 320

Blood Pressure / mmHg

200
Pithed preparation
240
150 Baroreceptors have
Heart rate / bpm

been destroyed, hence

160 fall in arterial pressure
100
is not affected by a
negative feedback
50 80
system.

0 0
Time (minutes)

Calculating the Responses

For the changes in heart rate, the response is measured from baseline to peak and using a
ruler to measure the length, and then the scale is applied.
The blood pressures line is thick as it is a combination of both diastolic and systolic pressures.
Changes in BP are measured by taking the mean of the lines and by using a ruler to measure
the length, and then the scale is applied.
Changes can be negative (fall in BP and HR).
 RECEPTORS INVOLVED IN BLOOD PRESSURE & HEART RATE
REGULATION

ALPHA ADRENOCEPTORS

Alphaadrenoceptor agonists bind to adrenoceptors on vascular smooth muscle and induce

smooth muscle contraction and vasoconstriction, thus mimicking the effects of sympathetic
adrenergic nerve activation to the blood vessels.
There are two types of adrenoceptors on vascular smooth muscle; 1 and 2.

1Adrenoceptors

These are the predominant receptor located on vascular smooth muscle.

These receptors are linked to Gqproteins that activate smooth muscle contraction through the
IP3 signalling pathway.
Activation of phospholipase C with generation of IP3 increases intracellular Ca2+ and thus
force of contraction.

2-Adrenoceptors

Depending on the tissue and type of vessel, there are also 2adrenoceptors found on the
smooth muscle.
These receptors are linked to Giproteins, and binding of an agonist to these receptors
decreases intracellular cAMP, which causes smooth muscle contraction.
There are also 2adrenoceptors located on the sympathetic nerve terminals that inhibit the
release of noradrenaline and therefore act as a feedback mechanism for modulating the
release of noradrenaline.
agonists constrict both arteries and veins, however, the vasoconstrictor effect is more
pronounced in the arterial resistance vessels. Constriction of the resistance vessels (small
arteries and arterioles) increases systemic vascular resistance, whereas constriction of the
venous capacitance vessels increases venous pressure.

BETA ADRENOCEPTORS

1Adrenoceptors

1adrenoceptors are located in cardiac nodal tissue, the conducting system and
contracting myocytes.
These receptors primarily bind noradrenaline that is increased from sympathetic adrenergic
nerves. Additionally, they bind noradrenaline and adrenaline that circulate in the blood.
1adrenoceptors are coupled to GSproteins, which activate adenylyl cyclase to form cAMP
from ATP. Increased cAMP activates cAMPdependent protein kinase (PKA) that
phosphorylates Ltype calcium channels, which causes increased calcium entry into the cells.
Increased Ca2+ entry during action potentials leads to enhanced release of calcium by the
sarcoplasmic reticulum; these actions increase inotropy (contractility).
 Gsprotein activation also increases heart rate by opening ion channels responsible for
pacemaker currents in the SA node. PKA phosphorylates sites on the sarcoplasmic reticulum,
which enhances the release of Ca2+ through the ryanodine receptors associated with the
sarcoplasmic reticulum. This provides more Ca2+ for binding the troponinC, which enhances
inotropy.
PKA can phosphorylate myosin light chains, which may also contribute to the positive
inotropic effect of 1adrenoceptor stimulation.

2Adrenoceptors

Vascular smooth muscle (e.g. smooth muscle on blood vessels supplying blood to skeletal
muscle) has 2adrenoceptors that have a high binding affinity for circulating adrenaline and a
relatively lower affinity to noradrenaline released by sympathetic nerves.
These receptors are coupled to a Gsprotein (like those in the heart), which stimulates the
formation of cAMP. Although increased cAMP enhances cardiac myocytes contraction, in
vascular smooth muscle an increase in cAMP leads to smooth muscle relaxation.
This is because cAMP inhibits myosin light chain kinase which is responsible for
phosphorylating smooth muscle myosin.
Thus increases in intracellular cAMP caused by 2 agonists inhibits myosin light chain kinase
thereby producing less contractile force, i.e. promoting relaxation.
2 in other tissues. Activation of 2adrenoceptors in the lungs causes bronchodilation.
Activation of 2adrenoceptors also leads to hepatic glycogenolysis and pancreatic release of
glucagon, which increases plasma glucose concentration. The heart also has 2adrenoceptors
but the predominant receptor type in number and function is the 1.

CHOLINERGIC MUSCARINIC RECEPTORS

M2 Muscarinic Receptors

The vagus (parasympathetic) nerves that innervate the heart release acetylcholine as their
primary neurotransmitter.
ACh binds to M2 muscarinic receptors that are found principally on cells comprising the SA and
NA nodes.
Muscarinic receptors are coupled to the Giprotein; therefore vagal activation decreases
cAMP. Giprotein activation also leads to the activation of KACh channels that increase
potassium efflux and hyperpolarises the cells.
Increases in vagal activity to the SA node decreases the firing rate of the pacemaker cells by
decreasing the slope of the pacemaker potential (phase 4 of the action potential). This
decreases heart rate (negative chronotropy).

M3 Muscarinic Receptors

M3 receptors are coupled to the Gqprotein family of receptors. Activation of M3 receptors

mediate relaxation of smooth muscle (mainly vascular), which results from the release of nitric
oxide from neighbouring endothelial cells.
Activation of Gq results in the activation of phospholipase C with generation of IP3 increases
intracellular Ca2+.
This intracellular Ca2+ binds to calmodulin to form the Ca2+-calmodulin complex.
 Ca2+-calmodulin activates the nitric oxide synthase (NOS) enzyme which synthesises nitric
oxide from arginine.
Nitric oxide activates guanylyl cyclase in the smooth muscle which stimulates the production of
cGMP from GTP.

Actions of Nitric oxide

1. Nitric oxide acting through cGMP can stimulate a cGMPdependent protein kinase that
activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light
chains, leading to relaxation.
2. Increased cGMP inhibits calcium entry into the cell, thereby increasing intracellular calcium
concentrations and causing smooth muscle relaxation.
3. Nitric oxide also activates K+ channels, which leads to hyperpolarisation and relaxation.

HISTAMINE RECEPTORS

The histamine receptor subtypes in blood pressure and heart rate control are the H1 and H2
receptors.
In the cardiovascular system, histamine dilates small blood vessels, resulting in flushing,
decreased peripheral vascular resistance (vasodilation) and fall in systemic blood pressure.
Vasodilation involves both H1 and H2 receptors. Though both H1 and H2 receptors participate in
the vascular effects, it is the H1mediated action that predominates.

H1Histamine Receptor & Vasodilation

This receptor is found throughout the body especially in smooth muscle, on vascular
endothelial cells, in the heart and CNS.
This receptor is Gqprotein linked which activates phospholipase C. This leads to the fall in
systemic blood pressure just as M3 receptors do.

H2Histamine Receptor & Increased Heart Rate

H2histamine receptors are found on gastric parietal cells, vascular smooth muscle,
neutrophils, CNS, heart and uterus.
Direct cardiac effects of histamine include both contractility and increased pacemaker rate.
The H2receptor is Gproteincoupled, linked to intercellular Gs which stimulates adenylyl
cyclase and increases cAMP.
 DRUG EFFECTS ON BLOOD PRESSURE & HEART RATE

Receptor Action on HR and BP Antagonists

stimulation
1 Stimulation leads to vasoconstriction which in turn Prazosin
leads to increase in BP
1 Increase HR and force of contraction. Atenolol;
propranolol
2 Stimulation leads to vascular and non-vascular smooth Propranolol
muscle relaxation (vasodilation) and thus decrease in
BP.
M2 Decrease in HR. Atropine
M3 Decrease in systemic BP. Atropine
H1 Decrease in systemic BP Mepyramine
H2 Slight increase in HR. Ranitidine

PRELIMINARY SKETCHES

Drugs Acting on the Noradrenergic System

Drug Receptor specificity

Adrenaline 1 = 2 > 1* = 2. At high plasma levels = .
Noradrenaline 1 = 1 > 2 = 2
Phenylephrine 1 selective
Isoprenaline 1 = 2

Adrenaline

Adrenaline acts on 1, 2, 1 and 2 adrenoceptors.

(a) Activation of 1 receptors present on peripheral arterioles leads to an increase in blood
pressure due to vasoconstriction.
(b) Activation of 1 receptors present on the heart leads to an increase in heart rate.
(c) Activation of 2 receptors on arterioles in skeletal muscle leads to a decrease in blood
pressure due to vasodilation.
The increase in blood pressure (1) and the decrease in blood pressure (2) lead to a biphasic
blood pressure response.
Adrenaline is more selective and this can be seen when low doses of adrenaline are injected
into the specimen. Also, adrenaline has a higher affinity for 2 than 1.
 Normal doses of adrenaline
Blood Pressure / mmHg

320
200 1 activation increases HR.
1

Heart rate / bpm

1 activation on peripheral
150 240 arterioles causes
vasoconstriction
1 2 activation on peripheral
100 160
arterioles (skeletal muscle)
cases vasodilatation
50 80
2
0 0
Time (minutes)
Blood Pressure / mmHg

1 320

Heart rate / bpm

240
1 Higher doses of adrenaline
160 Activation of 1 masks the 2
response.
80
2
0
Time (minutes)
Blood Pressure / mmHg

320
1
Heart rate / bpm

240
Low doses of adrenaline
2 > 1 > 1
160
1
80
2
0
Time (minutes)

Noradrenaline

Noradrenaline acts as an agonist on 1, 2 and 1 adrenoceptors.

(a) Activation of 1 receptors present on peripheral arterioles leads to an increase in blood
pressure due to vasoconstriction.
(b) Activation of 1 receptors present on the heart leads to an increase in heart rate.
The blood pressure element of the sketch does not show a biphasic shaped due to
noradrenalines inability to activate 2 adrenoceptors.
 320
Blood Pressure / mmHg

Heart rate / bpm

240

160
1
80

0
Time (minutes)

Phenylephrine

Phenylephrine is an 1selective adrenoceptor agonist.

Activation of 1 receptors present on peripheral arterioles leads to an increase in blood
pressure due to vasoconstriction.
Due to phenylephrines 1selective nature, it does not stimulate 1 adrenoceptors on the
heart, and thus there is a minimal response change in heart rate due to the increase in blood
pressure.
Blood Pressure / mmHg

1
Heart rate / bpm

Phenylephrine 30 nmol/kg
There is no 1 effect. The small
change in heart rate is a result
of the increase in blood
No 1 effect pressure.

Time (minutes)
Blood Pressure / mmHg

1
Heart rate / bpm

Phenylephrine 50 nmol/kg
At a higher concentration, the
effect of phenylephrine on 1 is
No 1 effect shown, as there is still a very
small change in HR but a large
increase in BP.
Time (minutes)

Isoprenaline

Isoprenaline acts on 1 and 2 adrenoceptors.

(a) Activation of 1 receptors present on the heart leads to an increase in heart rate.
(b) Activation of 2 receptors on arterioles in skeletal muscle leads to a decrease in blood
pressure due to vasodilation.
 Isoprenaline has a strong aftereffect on blood pressure (by activating 1adrenoceptors) at
high concentrations.
Blood Pressure / mmHg

Heart rate / bpm

Isoprenaline 0.5nmole/kg

2
eff
Time (minutes)
Blood Pressure / mmHg

1
Isoprenaline 10nmole/kg
Heart rate / bpm At a higher concentration, there
is a strong after-effect due to
the high HR (via 1 stimulation)
giving a somewhat biphasic
Strong after-effect blood pressure curve.
due to increased HR
2
eff
Time (minutes)

Drugs Acting on the Cholinergic System

Acetylcholine

Acetylcholine stimulates muscarinic cholinoceptors causing a decrease in heart rate (M2) and
blood pressure (M3).
Blood Pressure / mmHg

M2
Heart rate / bpm

Acetylcholine 4nmole/kg

M3 (Nitric oxide mediated)

Time (minutes)
Blood Pressure / mmHg

Heart rate / bpm

M2

Acetylcholine 50nmole/kg

M3 (Nitric oxide mediated)

Time (minutes)
Blood Pressure / mmHg

Heart rate / bpm

M2
Acetylcholine 100nmole/kg

M3 (Nitric oxide mediated)

Time (minutes)

Vagus Nerve Stimulation

Stimulation of the vagus nerve results in a decrease in heart rate but no significant change in
blood pressure despite the presence of M3 receptors on blood vessels.
This is due to the fact that the blood vessels are not innervated by the parasympathetic system
via the vagus nerve and thus no acetylcholine reaches the muscarinic receptors on these
vessels and no significant change of blood pressure occurs.
There is a much larger decrease in heart rate due to innervations of the heart by the vagus
nerve (on the SA and AV nodes) hence the release of acetylcholine results in a much greater
decrease in the force of contraction and heart rate.
Blood Pressure / mmHg

Heart rate / bpm

M2

Small indirect effect on BP due to

decrease in HR (no parasympathetic
innervation of blood vessels)
Time (minutes)
Histamine

At low concentrations, histamine produces a decrease in blood pressure by acting on the H1

receptor. There is no effect on heart rate at this point.
Increasing the concentration of histamine leads to a greater decrease in blood pressure via the
H1 receptor and the effect on heart rate (which is increased) is now seen by acting on the H2
receptor.
Blood Pressure / mmHg

No effect

Heart rate / bpm

Histamine 2nmole/kg

H1 (nitric oxide mediated)

Time (minutes)
Blood Pressure / mmHg

H2
Heart rate / bpm

Histamine 50nmole/kg

H1 (nitric oxide mediated)

Time (minutes)

Summary of Receptors, Mechanisms & Effects

G Signal Transduction Receptors Effects

protein
GS Activation of adenylyl cyclase All adrenoceptors Increase in HR.
Increase in cAMP Histamine H2 Smooth muscle
Activation of cAMP-dependent PKA dilation (decrease in
blood pressure)
Gq Activation of PLC 1adrenoceptors Smooth muscle
Hydrolysis of PIP2 to IP3 and DAG M3 and histamine contraction (increase
DAG activates PKC and IP3 (nitric oxide in blood pressure)
phosphorylates proteins mediated)
Gi Inhibition of adenylyl cyclase M2 Decrease in HR.
Decrease in cAMP 2adrenoceptors
No activation of cAMP-dependent
PKA.
 LOG DOSE RESPONSE CURVES

Noradrenaline

Blood Pressure Heart Rate

0.1 1 10 100 1000 1

0.1 10 100 1000
0 0
Log noradrenaline concentration nmol/kg Log noradrenaline concentration nmol/kg

The pithed curve plateaus much higher and is steeper compared to the nonpithed because:
(a) In the pithed preparation (without a spinal cord or baroreceptor reflex), blood pressure and
heart rate cannot be controlled due to the lack of activation of baroreceptors.
(b) There is therefore no negative feedback.
(c) Sympathetic tone is abolished.

Phenylephrine & Isoprenaline

Blood Pressure Heart Rate

0.1 1 10 100 1000 10000

Log 0drug concentration nmol/kg 0.1 1 10 100 1000
0
Log drug concentration nmol/kg
 Adrenaline

Blood Pressure Heart Rate

80

0.1 1 10 100 1000 10000 0.1 1 10 100 1000

0 0
Log adrenaline concentration nmol/kg Log drug concentration nmol/kg

Estimated ED50 Values

Drug ED50 BP (nmol/kg) ED50 BP (nmol/kg) ED50 HR (nmol/kg)

Noradrenaline 6 18
Phenylephrine 40 - -
Isoprenaline - 0.3 0.9
Adrenaline 9 0.4 1.5
Order of potency NOR>ADR>PHE ISO>ADR ISO>ADR>NOR

PROVING AGONISM & ANTAGONISM

Showing that Adrenaline has both and Effects

1000nmol/kg

500 nmol/kg
Propranolol
200nmol/kg

Adrenaline
Adrenaline

Adrenaline
Adrenaline

9nmol/kg
9nmol/kg

9nmol/kg
9nmol/kg

Atenolol
Prazosin

Blood Pressure / mmHg

1 unaffected
1 blocked
Heart rate / bpm

1 blocked
2 unaffected
s 2 blocked
Time (minutes)
 PAST EXAMINATION QUESTIONS

1. In a CARDIOLAB computer simulation experiment, explain with the aid of diagrams how the
intravenous injection of the following drugs would affect the blood pressure and heart rate:

(a) adrenaline
(b) phenylephrine
(c) adrenaline, soon after a high dose of propranolol
(d) phenylephrine, soon after a high dose of prazosin.

Indicate the receptor subtypes involved.

(100%)

2. A CARDIOLAB computer simulation experiment was used to illustrate cardiovascular blood

pressure (BP) and heart rate (HR) responses expected following intravenous injection of
various drugs in vivo. Sequential doses of adrenaline (ADR) from 0.5 to 16 nmol/kg were
initially injected into the preparation, in order to construct a dose response relationship
(doses were doubled at each step).

(a) Sketch the responses to ADR you would expect to observe, explain how the responses
are generated and the receptor types involved
(25%)

A dose of 500 nmol/kg prazosin was then applied and two doses of ADR (0.5 nmol/kg and
16 nmol/kg) repeated in its presence.

(b) What effect would prazosin alone be expected to have on the baseline level of HR and BP
and why? Sketch the responses. Sketch also the expected responses to 0.5 nmol/kg and
16 nmol/kg ADR in presence of prazosin. Explain your answer.
(25%)

In a different experiment, sequential responses to the following drugs were obtained:

noradrenaline (10 nmol/kg), isoprenaline (5 nmol/kg), histamine (5 nmol/kg) and
acetylcholine (10 nmol/kg). Finally, a vagus nerve stimulation was selected from the drug
menu and applied.

(c) Sketch the expected HR and BP responses to each drug and the vagus nerve stimulation.
Explain how they are generated and the receptor types involved. Explain also any
differences that might be expected in the acetylcholine and vagus nerve responses.
(50%)

3. In a CARDIOLAB computer stimulation experiment, sequential single responses to the

following were obtained: noradrenaline (10 nmol/ kg), isoprenaline (5 nmol/kg), histamine (5
nmol/kg) and acetylcholine (10 nmol/kg). A vagus nerve stimulation was then applied.
Sketched the expected HR and BP responses to each drug and the vagus nerve stimulation.
Explain how they are generated and the receptor types involved. Explain also any differences
that might be expected in the responses to acetylcholine and vagus nerve stimulation.
(100%)
4. A CARDIOLAB computer simulation was set up to measure normal cardiovascular blood
pressure (BP) and heart rate (HR) responses expected after intravenous injection of various
drugs in vivo. Doses of 30 nmol/kg phenylephrine (PHE) and 0.5 nmol/kg isoprenaline (ISO)
were initially injected sequentially into the preparation.

(a) Sketch the BP and HR response to PHE and ISO you would expect to observe, explain
how each response is generalised and the receptor types involved. How would you expect
the response to PHE to alter in a pithed preparation in which cardiovascular reflex
pathways have been destroyed? Sketch the responses and explain your answer
(25%)

A dose of 1000 nmol/kg atenolol was then applied and the doses of PHE and ISO
repeated in its presence.

(b) What effect would atenolol alone be expected to have on the baseline level of HR and BP
and why? Sketch the response. Sketch also the expected response to 30 nmol/kg PHE
and 0.5 nmol/kg ISO in the presence of atenolol. Explain your answer.
(25%)

In a different experiment, sequential responses to the following drugs were obtained:

noradrenaline (10 nmol/kg), adrenaline (10 nmol/kg) and acetylcholine (10 nmol/kg).

(c) Sketch the expected HR and BP responses to each drug. Explain how they are generated
and the receptor types involved. Sketch also the expected response to 0.5 nmol/kg
adrenaline. Explain your answer.
(50%)

5. Describe, with the aid of diagrams, how intravenous administration of the following drugs
would affect systemic blood pressure and heart rate in a CARDIOLAB computer stimulation
of a normal anaesthetised animal:

(a) Phenylephrine
(b) Isoprenaline
(c) Adrenaline

Indicate what receptor subtypes would be stimulated and how the effects could be
pharmacologically antagonised.
(100%)

6. In a CARDIOLAB computer stimulation experiment, explain with the aid of diagrams how the
intravenous injection of the following drugs would affect the blood pressure and heart rate:

(a) adrenaline
(b) phenylephrine
(c) adrenaline, soon after a high dose of propranolol
(d) phenylephrine, soon after a high dose of prazosin.

Indicate the receptor subtypes involved.

(100%)