1. Screening of sympathomimetic
And
Sympatholytic drugs
By
Srota dawn
M.Pharm(pharmacology)
1st year, 2nd semester

2. Introduction:
These are the drugs which mimic the responses of
epinephrine or stimulate the sympathetic nervous system.
Secreted from :
Adrenal medulla in mammals
Sympathetic nerve activation occurs in response to the
stimuli including physical activity, physiological stress, blood
loss etc.

3. Sympathomimetic drugs may results in any of
the following events like
i. mydriasis in the eye
ii. enhanced stroke volume
iii. acceleration of the heart rate
iv. dialation of the coronary arteries
v. constriction of the pulmonery vessels
vi. relaxation of bronchial muscles
vii. inhibition of gastric secretion
viii. constriction of gastrointestinal sphincters
ix. stimulation of uterus and
x. constriction of spleen capsule

4. Receptors in the ANS
• Cholinergic receptors:
Inhibitory or excitatory
- Nicotinic: fast
- Muscarinic: slow
 Adrenergic receptors:
slow, excitatory or inhibitory
- α 1 and 2
- β 1 and 2 (and 3)

5. ANS - Adrenergic Drugs
-Adrenergic Receptors
 Divided into 1 and 2 receptors
 Differentiated by their location on nerves
1-Adrenergic Receptors
 Located on postsynaptic effector cells
(the cell, muscle, or organ that the nerve stimulates)
2-Adrenergic Receptors
 Located on presynaptic nerve terminals
(the nerve that stimulates the effector cells)
 Control the release of neurotransmitters
Predominant -Adrenergic Agonist Responses
 Vasoconstriction
 CNS stimulation

6. ANS - Adrenergic Drugs
-Adrenergic Receptors
• All are located on postsynaptic effector cells
– 1-adrenergic receptors—located primarily in the heart
– 2-adrenergic receptors—located in smooth muscle of the
bronchioles, arterioles, and visceral organs
• -Adrenergic Agonist Response:
– Results in:
• Bronchial, GI, and uterine smooth muscle relaxation
• Glycogenolysis
• Cardiac stimulation

7. Screening of
sympathomimetics

8. Screening of sympathomimetics can be done by the
following methods:
in vivo method:
Cat spleen method
 In vitro method:
1. Cat spleen method
2. Rabbit pulmonary method

9. in vivo method:
Cat spleen method
Purpose:
This is one of the useful method for evaluation of substances
affecting the release and subsequent fate of the adrenergic
transmitter from the sympathetic nervous system.
After injecting sympathetic amines electrical stimulation of
the pre- and post- ganglionic nerves spleen contracts. Different
doses of the test compound that alter the release of transmitter
output of spleen are compared with the known standards.
Released amount of NMT output can be measured by
collecting spleen venous effluent and analyzing its NMT content.
This is achieved by the labeling of neuronal stores with
radioactive compound, which is taken up from the splenic arterial
circulation and released after nerve stimulation.

10. Procedure (cat spleen method):
6.A ligature is placed around the portal vein
5.Adrenal glands are also removed(to avoid the effect due to
release of pressor amines)
4.Spleenic nerves are also cut to avoid liberation of
catecholamines from other sites
3.Vascular connection between the spleen & omentum are
being cut
2.Abdomen is cut open (midline incision)& intestine is
removed(from mid duodenum to terminal colon)
1.Healthy cats are selected and anaesthetized using chloroform

11. 7.Heparin is injected to avoid clotting of blood
9.Venous blood samples are collected by diverting effluent
through a cannula by applying positive pressure
10.Blood is collected through chilled, silicon coated, calibrated
centrifuge tubes
11.This method is enough for the collection of 80% of NA
released.
12.The amount of NA present in the blood is estimated by
`SCINTILLATION COUNTER for radioactivity measurement.
8.Abdominal cavity is filled with warm paraffin and aerated with 95% O2
& 5% CO2

12. In vitro method:
1.Cat spleen method:
1.This is the method for the estimation of NA content in IN VITRO PROCEDURE
The method for removal of spleen is same as before
The removed spleen is placed on a plethysmograph filled with liquid paraffin
The organ is perfused at a rate of 7-16 ml/min with modified Krebs solution_(temperature-
37○c ,gassed with carbogen,dextran 3% for maintaining osmotic pressure)
Ascorbic acid at 25 uGu/ml is added to prevent oxidation of NA
The perfusion is started and the samples are collected and estimated for NA

13. 2. Rabbit pulmonary artery method:
Purpose:
The pulmonary artery is very sensitive to sympathomimetic
agents . The artery is mainly consists of vascular smooth muscles innervated by
postganglionic adrenergic sympathetic fibers . The NA is released in response
to the nerve stimulation . The released adrenergic transmitter is measured by
labeling the neuronal stores with radioactive NA & with the use of super fusion
technique to reduce the dilution of amines.

14. Procedure (rabbit pulmonary artery method)
1.Rabbits(1-2kg) and sacrificed by exsanguinations followed by
the removal of main pulmonary artery
2.The artery is cut transversely and spirally into vertical strips
3.The strip is suspended vertically in the organ bath (maintained
at 37°c )
4.The last end of the tissue is tied to glass support, whereas
the other end is connected to the strain gauge transducer
5.Resting tension- 2gm
6.Krebs bicarbonate solution(physiological solution)

15. 7.The tissue is loaded with 3[H] NA by submerging it in 20 ml of Krebs
bicarbonate solution at 37*c and gassed with 95% O2 & 5% CO2
8.Incubate for 60 min,the tissue is washed with fresh Krebs solution
9.During exp. Superfusates are collected in vials in every 3 mins.aliquots of
collected samples are then assayed
10.The electric stimulations are given by the use of potassium electrodes
11.Responses to successive 2 min period of electrical stimulation are
reproducible when applied at 16 min intervals

16. Screening of sympatholytics

17. Methods:
In vivo method:
1.nictitating membrane prolapse in cats
2.alpha () & beta( β ) adrenergic antagonism
in mouse eye
In vitro method:
1.vas deferences of the rat
2.splenic strip of the cat
3.pithed rats for evaluation of sympathomimetic and
sympatholytic activity
4.to assess β 1 and β 2 adrenoreceptors agonism &
antagonism

18. 1.Nictitating membrane prolapse in cats
1.Anaesthsised cats are used, drugs are administered orally
2.Sympatholytics exert relaxant effect on the nictitating membrane of cats
3.For each test drug 5 – 10 animals are used
4.The relative activity of different compounds is calculated by dividing the
mean duration of the membrane prolapse of a group in hours by the dose
in mg/kg.

19. 2.Alpha () & beta( β ) adrenergic antagonism in
mouse eye
Nor epinephrine, epinephrine and isoproterenol have the
property of inducing mydriasis,this effect is blocked by
alpha () & beta( β ) adrenergic blockers.  blockers block
the mydriatic effect of nor- epinephrine, β blockers block
the effect of isoproterenol and  β blockers block the
effect of epinephrine.

20. Procedure :
Mice(15-20 gm) are used
Animals are divided into groups as per requirement
Vehicle is administered in sc route (for control)
Test compounds are added to the test group
Std Nor-epinephrine given in i.v.
Pupil diameters are measured(before and after drug administration)
The mean value is compared between groups

21. In vitro method:
1.vas deferences of the rat
Male wister cats(275-300 gm)are used
Animals are killed by stunning
A midline abdominal incision is performed to disect out vas deferens
Tissue is suspended in an organ bath(tyrode, aerated,35*c)
Contractions are recorded(NA is administered repeatedly in concentration
of 0.5,1,2,4 ug/ml
Test drug is added are the reduction in response is recorded
Phentolamine is used as standard)

22. 2.splenic strip of the cat
Cat of either ser weighing around(1-2.8 kg) are used
The spleen is removed and a25 to 30 mm long strips of spleen are prepared
Strip is then suspended in an organ bath(krebs,38*c, aerated)
Tension used – 0.5 gm,magnification- 5-6 times
To induce contraction,NA/A is added
Test drug is then added(followed by agonist)
Phentolamine is used as standard(%reduction of activity of epinephrine or
nor epinephrine is determined)