Bioassay of epinephrine and

nor-epinephrine
1- Blood pressure of cats or dogs
2- The rat uterus method
3- The rabbit intestine method
4- Other isolated organs
A- Organs containing α receptors
B- Organs containing β receptors

1- Blood pressure method

Principle:
The assay depends on that the increase in BP is
proportional to the dose
Procedure:
• Cat is anaesthetized with chloralose
• The drug is injected in the femoral vein
• The blood pressure is recorded from the femoral
artery
• The potency of the T preparation can be
determined by matching its response with a
submaximal dose of the S preparation

Disadvantage:
• The blood pressure method is sensitive
but is affected by reflex mechanisms
regulating the BP
• An increase in BP stimulates receptors
in the carotid sinus and cardio-
inhibitory center leading to return of
BP to normal
How to abolish the reflex mechanisms?
1- blood pressure of spinal cat
The brain is destroyed to the fourth cervical
segment to avoid vasomotor reflexes
2- Blood pressure of pithed rat
The brain and the spinal cord are destroyed
3- Blood pressure of hexamethomium treated
rat
Ganglion blockers block both sympathetic
and parasympathetic ganglia. This lowers
and stabilizes the BP

2- The rat uterus method

Most sensitive method to differentiate
between E & NE
Principle:
• In non-pregnant rat uterus only β-2
receptors are found, they cause
relaxation of the uterus
• The method is sensitive to E, NOT
sensitive to NE

• The activity of the uterus is different in

the different stages of oestrus cycle
(Proestrus, Oestrus, Metoestrus &
Dioestrus)
• The rat uterus in dioestrus has no
spontaneous contraction
• The rat uterus in oestrus stage has
spontaneous contraction
 Oestrus stages can be determined by
microscopic examination of a vaginal smear
A- Proestrus (Preovulatory, 12 hrs)
Nucleated cells
B- Oestrus (Ovulatory, 14 hrs)
Squamous or cornified cells
C- Metoestrus (Postovulatory, 21 hrs)
Denucleated cells
D- Dioestrus (Resting, 57 hrs)
Leukocytes

Assay of E on rat uterus in dioestrus (Indirect assay)

1- The uterus of non-pregnant rat is isolated and
suspended in De Jalon solution (modified tyrode's
solution containing 1/2 the usual amount of
glucose and 1/4 the quantity of calcium)
2- This modification is important to avoid
spontaneous contractions of the uterus
3- Acetylcholine is used to induce contraction of the
uterus and a suitable submaximal dose is chosen
4- Epinephrine is added to the bath 30 seconds
before the addition of Ach

5- The doses of the S & T which cause 50%

reduction in response to Ach are determined
6- The matching technique is performed and the
relative potency is calculated

Assay of E on the rat uterus in oestrus (Direct assay)

1- Oestrus stage can be induced by injection of
diethylstilbsterol 24 hours before the assay
2- Uterine muscle is suspended in Lock's solution at
37°C
4- The assay depends on that E causes reduction in
uterine contraction
3- The rabbit intestine method
Principle:
The rabbit intestine contains α & β
receptors. Stimulation of both types of
receptors causes relaxation. The degree
of inhibition is proportional to the
dose. The assay may be direct or
indirect

A- Indirect assay
• Used when myogenic contraction of the
muscle is very low
• Acetylcholine is used to stimulate the
intestine and a suitable submaximal dose is
chosen
• The doses of the S & T which cause 50%
reduction in response to Ach are
determined
• The sequence of the matching technique is
applied

B- Direct assay
• Used only when the myogenic
contraction of the muscle is very high
• After determination of the submaximal
dose of the S, the dose of the T
producing the same response is
determined and matching technique is
performed
4- Other isolated organs
A- Organs containing α receptors and so respond by
contraction
i- Guinea pig vas deferens
ii- Rat seminal vesicle
iii- Rabbit aortic strip
iv- Cat spleen
B- Organs containing β receptors and so respond by
relaxation
i- Guinea pig tracheal chain
ii- Isolated guinea pig auricles

Sympatholytics
A- Adrenergic receptor blockers
1- α-blockers e.g.,
• Phentolamine (non-selective)
• Prazosin (selective α1 blocker)
• Yohimbine (selective α2 blocker)
2- β- blockers e.g.,
• Propranolol (non-selective)
• Atenolol (selective β1 blocker)
• Butoxamine (selective β2 blocker)

3- α & β- blockers e.g.,

Labetalol & Carvedilol
B- Adrenergic neuron blockers
1- NE release e.g., Guanethidine
2- NE storage e.g., Reserpine
3- NE synthesis e.g., α-Methyldopa
C- Ganglion blockers e.g.,
1- Depolarizing e.g., Nicotine in large dose
2- Competitive e.g., Hexamethonium
 Screening methods
1- Un-anesthetized cat nictitating membrane
• Used to differentiate ganglion blockers from
adrenergic neuron blockers & α-blockers
• Ganglion blockers cause relaxation of the
membrane & passive mydriasis
• Adrenergic neuron blockers & α-blockers
cause relaxation of the membrane but do
not cause mydriasis

2- Cardiovascular test
• α- blockers reverse the pressor action of E
& abolish the action of NE
• β- blockers reduce or abolish the
hypotensive effect of isoprenaline
• Ganglion blockers inhibit the response to
both carotid occlusion & vagal stimulation
• Reserpine inhibits the hypertensive effect of
indirectally acting drugs (e.g.,
amphetamine) due to depletion of NE stores

3- Finkleman preparation
• This is a nerve muscle preparation
consisting of a piece of rabbit intestine with
attached mesentery & sympathetic nerve
• The muscle is suspended in Tyrode solution
at 37°C & the adrenergic nerve is
stimulated electrically
• The response to both electrical stimulation
and exogenous E will be relaxation
• This preparation is used to differentiate
between adrenoceptor blockers &
adrenergic neuron blockers
• Adrenergic neuron blockers inhibit the
response to electrical stimulation only
• α & β-blockers prevent the action of both
nerve stimulation & exogenous E
• α & β-blockers can be differentiated by the
use of selective agonists e.g., NE or
isoprenaline
• α-blockers abolish the relaxant effect of NE
& β-blockers abolish the relaxant effect of
isoprenaline

Finkleman preparation

Special test for α blockers

Adrenaline reversal action
• Adrenaline stimulates both α & β-receptors,
but its effect on α receptors is the
predominant effect
• α- blockers reverse the action of adrenaline
& giving hypotension
 Special test for β-blockers
Langendorff preparation
Principle:
• Catecholamines cause positive inotropic and
chronotropic effects on the isolated perfused
mammalin heart known as Langendorff
preparation
• These actions are inhibited by β-blockers
Procedure:
1- Rabbit is injected with heparin to prevent clot
formation

2- The rabbit is killed & the heart is isolated and

washed several times in Locke solution to remove
the blood
3- The ventricles are connected to a lever for
recording the size of contraction
4- A funnel is used to collect the fluid, the collected
volume is proportional to the force of contraction
5- The dose response curve of isoprenaline is
performed and a submaximal dose is chosen
6- The doses of S & T (β-blockers) which cause 50%
reduction in the response to isoprenaline are
determined & matching technique is performed

Langendorff preparation
Special test for ganglion blockers
Antagonism of nicotine induced convulsions
• A suitable convulsive dose of nicotine is
determined in groups of mice
• The drug under test is ingected to a group
of mice before the administration of
convulsive dose of nicotine
• Protection against nicotine-induced
convulsions indicates a ganglion blocker