Experimental Pharmacology-II 23 Experiments

EXPERIMENTNO, 2
HEART
FEFECT OF DRUGS ON ISOLATED FROG

Purpose:
class, the students shall be able to
At the end of practical
cardiovascular system.
1 Know about drugs on the isolated heart of froa.
idt Know the effects of different
experiments on isolated preparation and in situ preparation of frog
2.
3. Know about
liat heart.
the isolated pertused mammalian heart by using
ia
4 Know about experiments on
hal Langendorffs apparatus.
Terminology:
all the physiological events associated with a single
no
Cardiac cycle: It comprises alternately contract in each cardiac cycle. It isdivided
d heartbeat. The atria and ventiricles
ed phases as systole and diastole.
into two
known as systole. It is the part of the cardiac cycle which
Systole: Contraction of heart is
the heart muscle contracts and pumps the blood from
happens right after diastole, when
the ventricle. cycle
the heart is known as diastole. It is the part of the cardiac
Diastole: Dilation of to fill with blood.
when the heart muscle relaxes and allows the chambers
muscle contraction. Positive inotropes increase the force
Inotropic: Affecting the force of
contraction of the heart, whereas negative inotropes weaken it.
of Chronotropic
Chronotropic effects are those that change the heart rate.
Chronotropic: affecting the electrical conduction
and rhythm by
drugs may change the heart rate influence it, such as by changing the rhythm
system of the heart and the nerves that
chronotropes increase heart rate; negative
produced by the sinoatrial node. Positive
chronotropes decrease heart rate.
dromotrope affects atrioventricular node (AV node) conduction. A
Dromotropic: A dromotrope
positive dromotropeincreases AV nodal conduction, and a negative
decreases AV nodal conduction.
Lusitropic: Alusitropeeis an agent that affects diastolic relaxation. of cardiac muscle.
Abathmotrope is an agent that affects excitatbility
Bathmotropic: These are the drugs which increase the force of
contraction of the heart
Cardiotonics:
and increase the cardiac output.

DescrTheiptioauricles
n: and ventricles of the heart usually contract in a synchronized
manner for
Pumping the blood Coordination of
efficiently by a route determined by the valves.
Contraction is achieved by a specialised conducting system. Physiological sinus rhythm is
 Experimental Pharmacology-I 24

characterised by arising in the sinoatrial (SA) node and

conducted
in
Experime
through the atria, impulses sequen
the atrioventricular (AV) node, bundle of His, Purkinje fibres and ventricle
Cardiac cells owe their electrical excitability to voltage-sensitive plasma membrane channe
Heart has three functional parte:
selective for various ions, includina Nat, K* and Ca*.
systenm and blood supply. Cardiovascular disease can involve an.
muscles, conducting
of hearts functional parts.
the isolated frog heart.
Objective: To study the effects of drugs on
Principle:
drugs which influence the heart rate, force of contraction, rhvth
There are several
heart either as therapeutic effect or as side effects. Many drugs used
tone of the
various effects like chronotropic (he
cardiovascular diseases may act on the heart and have (excitabit
(contractility), bathmotropic
rate), dromotropic (conduction velocity), inotropic
andlusitropic (relaxation) action.
increases the rate and forced
Sympathomimeticdrugs like adrenaline and noradrenaline
Chronotropic drugs. There are fel
contraction, hence they grouped as positive inotropic and
rate and force of contraction and hend
drugs like acetylcholine (ACh), which decreases the cholinera
Adrenergic and
they are grouped as negative inotropic and chronotropic drugs.
receptors. Some drugs such
drugs produce opposite effects on heart through respective
calcium chlorise and potassium chloride act directly on the heart.
Requirenments:
Animal/tissue: Frog /frog heart.
Drugs: Calcium chloride, potassium chloride, adrenaline, noradrenaline, isoprenalin
(now known as isoproterenol), propranolol, acetylcholine, atropine.
PSS: Frog ringer solution.
Equipment: Akymograph with drum and smoked paper and starling's heart lever.
Others: Stand, syringe, beaker, stop watch, dissection box, suturing needle, and threau
Procedure:
1. Prepare the stock of frog ringer solution. Also prepare the stock solution or va
drugs to study their effect on isolated frog heart.
needle inth
2. Destroy the nerves in the spinal column of the frog by inserting a sharp forlackd
foramen magnum (pithing) to prevent involuntary reflex contractions and test
needle.
any reflexes by pinching the feet or by touching the cornea of eye withthe
frogboard
3. Lay the frog on its back on the dissecting board. Pin or tap the limbs to the
abdomen. Cutth
4. Make an incision in the loose skin extending from the midline on the Cutth
abdominal muscle up to pectoral girdle without injuring the visceral organs.
pectoral girdle with abone cutter to expose the heart.
 Experimental Pharmacology-I 25 Experiments

heart with the help of a blunt forceps. Trace the

Remove the pericardium layer of the
thread under it.
inferior vena cavaand pass a
V shaped cut in the inferior vena cava with the help of a fine scissor, in
Give asmall
6. insert
order toin the venous cannula (syme's cannula), which is connected to the reservoir
tip of the cannula in the vena cava and tie it in
containingfrog ringer solution. Insert the
the Fig 2.1).
position with the thread firmly (as shown in
for the perfusate to
7.
Allow frog ringer solution to flow slowly. Immediately cut the aorta
Comeout.

Slighty lift the cannula up and carefully isolate the perfused heart by cutting off the
8.
tissues attached to the heart. The
vertical arm of the cannula is fixed with the clamp to a
stand.
the level of fluid in the cannula remains
o Adjust the flow of frog Ringer solution such that
constant.

Attach the apex of the heart with the help of thin thread to the starling's heart lever,
10.
tension such that it gives maximum
which writes on a smoked drum and adjust the
amplitude of contraction (as shown in the fig 2.2).
smoked kymograph drum (as
11. Record the normal response for about 2-3 cm on the
upward and downward
shown in the fig 2.3). The lever records the response by
systole and downward
movement on the drum. The upward movement indicates
movemernt indicates diastole.
record their effect on various
12. Inject different drugs one by one in the cannula and
shown in
parameters like heart rate, force of contraction and tone of the frog heart (as
table 2.1). Also note how antagonist and agonist interact with each other.
the
13. After testing one drug, the next drug is added only after recovery from the effect of
preceding drug.

Venous cannula

Inferior venacava

Sinus venosuS

Auricles

Ventricles

Hg. 2.1: Insertion of the venous cannula in the inferior vena cava
 26
Experimental Pharmacology-1 Reservoir

Syme's Cannula
TR8
Sherrington
recordingdrum

Screw clip
Heart

Rubber tube
Starling heart
lever

Stand

effects of drugs
Fig. 2.2: Isolated frog heart preparation to study the

Normal Potassium chloride Calcium chloride

Adrenaline
Noradrenaline Isoprenaline

Propranolol
Acetylchöline Atropine
Fig. 2.3: The responses of various drugs on the isolated frog heart on theSmoke
:

kymograph drum
 Experimental Pharmacology-II 27
Experiments
Observation:
Table 2.1: Effect of various drugs on isolated heart of froa
Effectson isolated heart of frog
Drugs (Concentration of stock Dose
solution) (ug/ml) Heart.rate Amplitude Tone
(Beats/min)
Normal
1.
2
Potassium chloride (10 mg/ml) 2000

3.
Calcium chloride (10 mg/ml) 2000

Adrenaline (10 ug /ml) 2

4
Noradrenaline (10 g /ml) 2
5.
6.Isoprenaline (10 ug/ml) 2

7.Propranolol (100 ug /ml) 20

Acetylcholine (10 mg/ml) 2

20
9. Atropine (100 ug/ml)
Volume of above solutions to be injected = 0.2 ml
Inference:
Potassium chloride and calcium chloride act directly on the heart. Potassium chloride
(2000 ug/m) stops the heart in diastolic condition and the lever records the response as a
straight line on lower border of the drum. Calcium chloride in lower doses increases the heart
rate and force of contraction. But in the higher doses (2000 ug/ml), it stops the heart in the
systoliccondition and the lever records the response as astraight line on upper border of the
drum.
Direct effects on the heart are determined largely by Bi receptors, although B2 and to a
lesser extent a receptors are also involved. B1-receptors are found mainly in the heart, where
ney are responsible for the positive inotropic and chronotropic effects of catecholamines,
resulting in a markedly increased cardiac output and cardiac oxygen consumption.
Noradrenaline (predominantly aagonist) causes vasoconstriction and increased systolicand
diastolic pressure, with a reflex bradycardia. Isoprenaline (selective Bagonist) strongly
increases cardiac force and rate. Adrenaline combines both aand Bactions.
effect but
B-adrenergic receptor blockers like propranolol as such does not produce any
in higher doses slight decrease in heart rate and force of contraction may be observed. In
presence of propranolol the sympathomimetic drugs fails to produce any effects on heart.
ACh reduces heart rate and force of contraction. In high doses, it stops the heart in
ditheastolic condition and the lever records the response as a straight line on lower border of
drumn same as KCI. This effect is through Mz-receptors, found mainly in the heart.
Atropine does not have any effect, except aslight increase in heart rate and force of
 Experimental Pharmacology-II 28
Experime
contraction sometimes. In presence of atropine, ACh fails to produce any inhibitory action
heart.
Note:
1. The effects of various drugs may also be studied in situpreparation of the perfused fr
heart. In this method pith the frog and place it on the frog board. Cannulate the w
cava with little incision and.tie it. Immediately cut the aorta for the perfusate to
out. Allow frog ringer solution to flow slowly. Attach the apex of the heart to
starling's heart lever with the help of a thread, which. writes on asmoked drum and ad
the tension such that it gives maximum amplitude of contraction. Inject different dn
one by one in the cannula and observe their effect, as mentioned on the abo
procedure.
2. Langendorff's ápparatus is used to study the effect of different drugs on the isolat
perfused mammalian heart. The coronary artery which supplies blO0d into the he
arises from the base of the aorta, just above the aortic valves. If the perfusion is mad
through the aorta, the perfusate will go only to the coronary blood vessels provided th
the pressure of the perfusate is just sufficient to keep the aortic valves closed. Heart
quickly isolated along with the aorta from the body and the aorta is tied to the cannula
the bottom of the perfusion apparatus. Oxygenated Ringer-Locke solution is warmed
37°C in this apparatus by water circulating in the outer jacket. The thread tied to t
hook is passed over pulleys and is.connected to the recording system (heart
tension of the lever is adjusted such that it gives maximum
lever). T
contraction. The norm
records are taken and various drugs are added to study the effect. The
preparation is used to study the autonomic and cardiotonic drugs. This Langendorth
also beenused for the study of the effect of preparation
drugs on coronary flow.
Precautions:
1. Dissect the frog heart
2. Maintain the aseptic carefully.
3. Check the condition of the laboratory.
kymograph drum speed before starting the
4. Properly balance
5. Try to
the starling's heart lever with
smoked
experiment.
minimize the direct handling of tissue. drum.
Questions for the students:
1. Díscuss the
effects of different drugs on the
2. Discuss the
in-Vitro isolated heart of frog.
3. List cardiac experiments carried out by using isolated heart of
4. Why should stimulants
and depressants? frog.
5. Discuss the intravenous potassium and calcium injections be given
effects of slowly
heart. Sympathomimetic and
parasympathomimetic drugs
on thd