In vitro screening methods ach, ne and 5-HT

1. In vitro screening methods Ach, NE andIn vitro screening methods Ach, NE and
5-HT5-HT
Presented by :-Presented by :-
Prof. Anup A. Patil.Prof. Anup A. Patil.
. (Dept. of Pharmacology). (Dept. of Pharmacology)
GIPER,Limb,Satara.GIPER,Limb,Satara.

2. ContentsContents
IntroductionIntroduction
AcetylcholineAcetylcholine
In vitro screening methods for AcetylcholineIn vitro screening methods for Acetylcholine
NorepinephrineNorepinephrine
In vitro screening methods for NorepinephrineIn vitro screening methods for Norepinephrine
5-HT5-HT
In vitro screening methods for 5-HTIn vitro screening methods for 5-HT
ReferencesReferences

3. AcetylcholineAcetylcholine
Acetylcholine is a major neurohumoral transmitter atAcetylcholine is a major neurohumoral transmitter at
autonomic as well as somatic sitesautonomic as well as somatic sites
Acetylcholine has functions both in the peripheralAcetylcholine has functions both in the peripheral
nervous system (PNS) and in the central nervous systemnervous system (PNS) and in the central nervous system
(CNS) as a neuromodulator.(CNS) as a neuromodulator.
In the PNS, acetylcholine activates muscles, and is aIn the PNS, acetylcholine activates muscles, and is a
major neurotransmitter in the autonomic nervousmajor neurotransmitter in the autonomic nervous
systemsystem..
Since a shortage of acetylcholine in the brain has beenSince a shortage of acetylcholine in the brain has been
associated with Alzheimer's disease some drugs thatassociated with Alzheimer's disease some drugs that
inhibit acetylcholinesterase are used in the treatment ofinhibit acetylcholinesterase are used in the treatment of
that diseasethat disease

4. In vitro screening methods for AcetylcholineIn vitro screening methods for Acetylcholine
In vitro estimation of acetylcholine-esterase.
In vitro estimation of butyrylcholine-esterase.
Release of [3
H]ACh and other transmitters from rat brain
slices.
A novel in vitro model for screening and evaluation of
anti-asthenopia
Guinea pig ileum
Isolated eye of rodents

5. In vitro estimation of acetylcholine-esterase.
Purpose and rationale:-Purpose and rationale:-
To screen drugs for inhibition of acetylcholine-esteraseTo screen drugs for inhibition of acetylcholine-esterase
activity.activity.
Inhibitors of this enzyme may be useful in treatment ofInhibitors of this enzyme may be useful in treatment of
Alzheimer's disease.Alzheimer's disease.
It is also called as true or specific cholinesterase.It is also called as true or specific cholinesterase.
Found in nerve cells, skeletal muscle, smooth muscle,Found in nerve cells, skeletal muscle, smooth muscle,
various glands and red blood cells.various glands and red blood cells.
Physiological role of AChE is rapid hydrolysis andPhysiological role of AChE is rapid hydrolysis and
inactivation of acetylcholine.inactivation of acetylcholine.
inhibitors of AChE shows marked cholinomimetic effect.inhibitors of AChE shows marked cholinomimetic effect.

6. Procedure :-Procedure :-
Preparation of reagent (EIIman’s reagent)Preparation of reagent (EIIman’s reagent)
Substrate – 1) Acetylthiocholine 21.6 mg/ml is made usingSubstrate – 1) Acetylthiocholine 21.6 mg/ml is made using
distilled water.distilled water.
2) 5,5’ Dithiobis-2-nitrobenzoic acid2) 5,5’ Dithiobis-2-nitrobenzoic acid
(0.01M)DTNB. 39.6 mg DTNB dissolved in 10 ml of pH 7(0.01M)DTNB. 39.6 mg DTNB dissolved in 10 ml of pH 7
phosphate buffer (0.01M) and 15 mg of bicarbonatephosphate buffer (0.01M) and 15 mg of bicarbonate
added into it.added into it.
3) This reagents are prepared by using3) This reagents are prepared by using
phosphate buffer (pH 7),because this pH reagents arephosphate buffer (pH 7),because this pH reagents are
more stablemore stable

7. Preparation of enzyme homogenatePreparation of enzyme homogenate
Male wistar rats are decapitated, and whole brain isMale wistar rats are decapitated, and whole brain is
taken out quickly.taken out quickly.
The cerebral cortex, cerebellum, medulla oblongata andThe cerebral cortex, cerebellum, medulla oblongata and
midbrain are removed and suspended in phosphatemidbrain are removed and suspended in phosphate
buffer.buffer.
Different regions brains removed homogenate isDifferent regions brains removed homogenate is
prepared in tissue homogenizer (Approximately 20 mg ofprepared in tissue homogenizer (Approximately 20 mg of
the per ml of phosphate buffe )the per ml of phosphate buffe )
2.6 ml of phosphate buffer added to cuvette followed by2.6 ml of phosphate buffer added to cuvette followed by
0.4 ml of aliquot of homogenate.0.4 ml of aliquot of homogenate.
Substrate (acetylthiocholine)20 microlitres added to itSubstrate (acetylthiocholine)20 microlitres added to it
100 micro liter this mixture used for photocell for100 micro liter this mixture used for photocell for
measuring the absorbancemeasuring the absorbance

8. Change in absorbance per minute is calculated.Change in absorbance per minute is calculated.
The rate is calculated by following equation:-The rate is calculated by following equation:-
R =R =
∆ A
1.36 (104
)
1
(400 3120) Co
∆A = Change in absorbance per minute (mean change
in absorbance from the 1st
to 7th
min.is taken )
Co = Original concentration of the tissue (20 mg/ml)
R = Rate in moles substrate hydrolyzed per minute perR = Rate in moles substrate hydrolyzed per minute per
gram of tissuegram of tissue

9. Statistical analysisStatistical analysis
Rat brain acetyl cholinesterase activity of differentRat brain acetyl cholinesterase activity of different
groups are analyzed using one way variance (ANOVA)groups are analyzed using one way variance (ANOVA)
followed by Dunnet's ‘t’ test.followed by Dunnet's ‘t’ test.

10. Release of [3
H]ACh and other transmitters from rat brain
slices.
Purpose and RationalePurpose and Rationale::
Electrically stimulated release of [Electrically stimulated release of [33
H] Ach, used as aH] Ach, used as a
biochemical screen for agents having possible action ofbiochemical screen for agents having possible action of
enhancing or inhibiting release of [enhancing or inhibiting release of [33
H]Ach through a directH]Ach through a direct
muscarinic interaction or other indirect interactionsmuscarinic interaction or other indirect interactions
..
The compound’s effect on [The compound’s effect on [33
H]Ach release may provideH]Ach release may provide
evidence for a wide variety of release activitiesevidence for a wide variety of release activities
The electrically stimulated release technique helps inThe electrically stimulated release technique helps in
measuring the presynaptic activity of test compounds.measuring the presynaptic activity of test compounds.

11. ProcedureProcedure::
a.a. Reagents:Reagents:
1) Krebs-Henseleit bicarbonate buffer, pH 7.4 (KHBB).1) Krebs-Henseleit bicarbonate buffer, pH 7.4 (KHBB).
[Methyl-[Methyl-33
H]-choline chloride (80-90Ci/mmol).H]-choline chloride (80-90Ci/mmol).
0.25nmol of [0.25nmol of [33
H]choline added to 2.5ml of KHBB toH]choline added to 2.5ml of KHBB to
obtain final concentration of 100nM.obtain final concentration of 100nM.
2) 10mM stock solution of the test drug prepared in2) 10mM stock solution of the test drug prepared in
suitable solvent and diluted to get a concentration ofsuitable solvent and diluted to get a concentration of
1010µM with KHBB.µM with KHBB.
3) Hemicholinium-3 or HC-3: 10mM stock solution in3) Hemicholinium-3 or HC-3: 10mM stock solution in
water. 2ml of this solution diluted to 1ltr with KHBB towater. 2ml of this solution diluted to 1ltr with KHBB to
obtain final concentration of 20obtain final concentration of 20µM.µM.

12. Tissue PreparationTissue Preparation::
Male Wistar rats are decapitated and cortical, striatal orMale Wistar rats are decapitated and cortical, striatal or
hippocampal tissue is removed on ice.hippocampal tissue is removed on ice.
0.4m slices are prepared using tissue slicer.0.4m slices are prepared using tissue slicer.
Placed in cold, oxygenated buffer (10-20ml), incubatedPlaced in cold, oxygenated buffer (10-20ml), incubated
for 30min. at 35for 30min. at 35°C.°C.
The buffer is decanted, leaving the slices behind afterThe buffer is decanted, leaving the slices behind after
incubation.incubation.
Then 2.5ml of buffer is added with enough [Then 2.5ml of buffer is added with enough [33
H]choline toH]choline to
get the final concentration of 100nM, incubated andget the final concentration of 100nM, incubated and
shaken for 60min. at 35shaken for 60min. at 35°C under oxygen.°C under oxygen.
Then buffer is decanted and the slices are placed onThen buffer is decanted and the slices are placed on
nylon mesh in the stimulation chamber.nylon mesh in the stimulation chamber.

13. Assay:Assay:
Buffer is pumped through the chamber for 42min. at aBuffer is pumped through the chamber for 42min. at a
constant flow rate of 0.7ml/min to establish a stableconstant flow rate of 0.7ml/min to establish a stable
baseline.baseline.
The released [3H]Ach under these conditions is due toThe released [3H]Ach under these conditions is due to
the hydrolysis by acetylcholinesterase.the hydrolysis by acetylcholinesterase.
The perfusion buffer is changed to KHBB containingThe perfusion buffer is changed to KHBB containing
2020µM HC-3.µM HC-3.
The potent choline uptake inhibitor is included to preventThe potent choline uptake inhibitor is included to prevent
the reuptake of any [the reuptake of any [33
H]choline formed from theH]choline formed from the
hydrolysis of [hydrolysis of [33
H]Ach.H]Ach.
The evoked release has been shown to be mostlyThe evoked release has been shown to be mostly
[[33
H]Ach rather than [H]Ach rather than [33
H]choline, whereas spontaneousH]choline, whereas spontaneous
release under control, drug-free conditions is mostlyrelease under control, drug-free conditions is mostly
[[33
H]choline.H]choline.

14. EvaluationEvaluation::
Percentage of fractional release is calculated for eachPercentage of fractional release is calculated for each
fraction.fraction.
Percent fractional release is defined as the amount ofPercent fractional release is defined as the amount of
radio-labeled compound released divided by amountradio-labeled compound released divided by amount
present in the tissue.present in the tissue.
Spontaneous release (SP) values are average of twoSpontaneous release (SP) values are average of two
fractions preceding the first fraction in that range afterfractions preceding the first fraction in that range after
the stimulation period.the stimulation period.
Stimulated (S) values are the summed differencesStimulated (S) values are the summed differences
between the percent fractional release during thebetween the percent fractional release during the
stimulation and appropriate SP values.stimulation and appropriate SP values.

15. A novel in vitro model for screening and evaluation of
anti-asthenopia
Purpose and Rationale:Purpose and Rationale:
AsthenopiaAsthenopia or eye strain is an ophthalmologicalor eye strain is an ophthalmological
conditioncondition
fatigue,fatigue,
red eyes, eye strain,red eyes, eye strain,
pain in or around the eyespain in or around the eyes
blurred vision, headache andblurred vision, headache and
occasional double vision.occasional double vision.

16. Symptoms often occur after reading, computer work, orSymptoms often occur after reading, computer work, or
other activities that involve tedious visual tasks.other activities that involve tedious visual tasks.
Author undertook study to develop a simple in vitroAuthor undertook study to develop a simple in vitro
model that can replicate fatiguing of ciliary muscle,model that can replicate fatiguing of ciliary muscle,
A novel technique thus developed uses ciliary muscles
removed from rabbit eyeballs.
The technique can be used to evaluate and screen drug
candidates under development to treat patients suffering
from asthenopia

17. ProcedureProcedure
Reagent :- 1) Cyanocobalamine.Reagent :- 1) Cyanocobalamine.
2) Acetylcholine chloride.2) Acetylcholine chloride.
3) Ketamine hydrochloride.3) Ketamine hydrochloride.
4) Xylazine hydrochloride4) Xylazine hydrochloride
Animal :- Male New Zealand white rabbits weighing aboutAnimal :- Male New Zealand white rabbits weighing about
3 kg (12 week-old)3 kg (12 week-old)
Experimental protocol:-Experimental protocol:-
Rabbits were anesthetized with intramuscular injection of
ketamine hydrochloride and xylazine hydrochloride(20
and 10 mg/kg, respectively)
The eyeball taken from the rabbits was sclerotonized
and amputated into half at the equatorial position.

18. The ciliary muscle was carefully ablated from the sclera
after removal of the crystalline lens and then cut into a
strip of 3 mm in width and 6 mm in length.
The ciliary muscle was suspended at 37C in a Magnus
tube filled with Krebs-Henseleit (KH) solution bubbled
with a mixed gas of 5% carbon dioxide and 95% oxygen
.
Tension was applied at a loading weight of 0.4 g using a
tension transducer
The ciliary muscle can be stimulated electrically,
mechanically, or chemically.
Author use a chemical stimulant, acetylcholine, involved
in ciliary activity

19. After a 30-min equilibration of the suspended muscle in
KH solution, the ciliary muscle was stimulated by the
addition of 10-4
mol/L acetylcholine.
The resultant tension was monitored as a function of
time until it has reached a plateau value.
The ciliary muscle was then washed with KH solution to
bring the tension to baseline.
Upon establishment of the baseline, the 2nd stimulation
was applied by the addition of 10-4
mol/L acetylcholine.

20. This process was repeated multiple times and at each
time.
After the 9th stimulation, the ciliary muscle was washed
with KH solution to establish the baseline.
Then KH solution containing varying concentrations of
cyanocobalamin was added to the ciliary muscle.
After incubation for 30 min at 37C, the 10th stimulation
was applied in the presence of cyanocobalamin.
The tension signal was monitored by the data collection
software.

21. The net tension (contraction rate) was calculated by
subtracting the baseline tension from the tension after
stimulation.
The percent change in contraction rate was calculated
by dividing the net tension of the 2nd or subsequent
acetylcholine stimulation (the i-th stimulation) by the net
tension of the first stimulation, as shown by the following
equation:
Contraction
rate (%)
=
net tension of the 1st stimulation
100
net tension of the i-th stimulation

22. ==
tension of the i-th stimulation -baseline tension
tension of the 1st stimulation - baseline tension
100
All data are presented as the mean S.D. Dunnett’s
multiple comparison test was performed to analyze the
significance between two means

23. Conclusion:-
A novel in vitro model for asthenopia was developed
using ciliary muscles obtained from the eyeballs of
rabbits.
Using acetylcholine as a stimulant, fatiguing of the
muscle was produced.
which can be recovered by cyanocobalamin treatment.
The procedure is simple and consistent and can be used
to screen drug candidates to treat patients suffering
asthenopia.

24. NorepinephrineNorepinephrine
Norepinephrine acts as transmitter at post ganglionicNorepinephrine acts as transmitter at post ganglionic
sympathetic sites.sympathetic sites.
Norepinephrine is synthesized from dopamine byNorepinephrine is synthesized from dopamine by
dopamine β-hydroxylase.dopamine β-hydroxylase.
It is released from the adrenal medulla into the blood asIt is released from the adrenal medulla into the blood as
a hormone.a hormone.
Norepinephrine may be used for the indicationsNorepinephrine may be used for the indications
attention-deficit/hyperactivity disorder, depression andattention-deficit/hyperactivity disorder, depression and
hypotension.hypotension.

25. In vitro screening methods forIn vitro screening methods for
NorepinephrineNorepinephrine
Inhibition of [3H]-norepinephrine uptake in rat brain
synaptosomes.
Inhibition of norepinephrine uptake in patients with
major depression treated with paroxetine

26. Inhibition of [3H]-norepinephrine uptake in rat brain
synaptosomes
PURPOSE AND RATIONALE
The neuronal re-uptake mechanism for norepinephrine is
the most important physiological process for removing
and inactivating norepinephrine in the synaptic cleft.
This uptake is inhibited by cocaine, certain
phenylethylamines and antidepressants.
In the brain, the hypothalamus shows the highest level
and greatest uptake of noradrenaline.
Therefore, this region Is used for testing potential
antidepressant drugs

27. PROCEDURE
Tissue preparation
Male Wistar rats are decapitated and the brains rapidly
removed.
The hypothalamic region is prepared, weighed, and
homogenized in 9 volumes of ice-cold 0.32 M sucrose
solution using a Potter-Elvejhem homogenizer.
The homogenate is centrifuged at 1 000 g at 0–4 °C for
10 min. The supernatant is decanted and used for the
uptake experiments

28. Assay
200 μl of tissue suspension are incubated with 800 μl
62.5 nM 3H-norepinephrine in Krebs-Henseleit
bicarbonate buffer and 20 μl of the appropriate drug
concentration (or the vehicle) at 37 °C under a 95%
O2/5% CO2 atmosphere for 5 min.
For each assay, 3 tubes are incubated with 20 μl of
vehicle at 0 °C in an ice bath.
After incubation all tubes are immediately centrifuged at
4000 g for 10 min

29. The supernatant fluid is aspirated and the pellets
dissolved adding 1 ml of solubilizer (Triton X-100 + 50%
ethanol, 1 : 4).
The tubes are vigorously shaken, decanted into
scintillation vials, and counted in 10 ml of liquid
scintillation cocktail
Active uptake is the difference between cpm at 37 °C
and 0 °C.

30. EVALUATION
• The percent inhibition at each drug concentration is
the mean of 3 determinations.
IC50 values are derived from log-probit analysis.
IC50 values for the standard drugs desipramine and
nortriptyline are around 20 nM.

31. 5-HT5-HT
5-HT is a monoamin neurotransmitter synthesized in5-HT is a monoamin neurotransmitter synthesized in
serotonergic neuron in the central nervous system (CNS)serotonergic neuron in the central nervous system (CNS)
In the central nervous system, serotonin is believed toIn the central nervous system, serotonin is believed to
play an important role as a neurotransmitter, in theplay an important role as a neurotransmitter, in the
inhibition of anger, aggression, body temperature, mood,inhibition of anger, aggression, body temperature, mood,
sleep, vomiting,, and appetitesleep, vomiting,, and appetite..
About 90 % of body content of 5-HT is localized in theAbout 90 % of body content of 5-HT is localized in the
intestine; most of rest in platelets and brainintestine; most of rest in platelets and brain

32. In vitro screening methods for 5-HTIn vitro screening methods for 5-HT
Inhibition of [3H]-serotonin uptake in synaptosomes
[3
H ] 5-HT binding assay
Contraction of isolated blood vessels
Contraction of Human isolated middle meningeal Artery.

33. Inhibition of [3H]-serotonin uptake in synaptosomes
PURPOSE AND RATIONALE
Altered serotoninergic function determines the mood
changes associated with affective disorders.
Antidepressants block the reuptake of 5-HT.
3
H-5-HT transport in brain has been found to be
saturable, sodium and temperature-dependent, to be
inhibited by several agents, such as ouabain, tryptamine
analogs, and tricyclic antidepressants.
Therefore, the test can be used to detect compounds
that inhibit serotonin uptake into rat brain synaptosomes
and may be potential antidepressants.

34. PROCEDURE
Tissue preparation
Male Wistar rats are decapitated and the brains rapidly
removed.
Either the whole brain or cerebellum or the
hypothalamus is weighed and homogenized in 9
volumes of ice-cold 0.32 M sucrose solution using a
Potter-Elvejhem homogenizer.
The homogenate is centrifuged at 1 000 g at 0–4 °C for
10 min. The supernatant is decanted and used for further
uptake experiments.

35. Assay
Two hundred μl of tissue suspension are mixed with 800
μl 62.5 nM 3
H-5-HT solution in Krebs-Henseleit
bicarbonate buffer and 20 μl of drug solution in the
appropriate concentration (or the vehicle as control).
The tubes are incubated at 37 °C under 95% O2/5% CO2
atmosphere for 5 min.
For each assay, 3 tubes are incubated with 20 μl of the
vehicle at 0 °C in an ice bath.
After incubation all tubes are immediately centrifuged at
4 000 g for 10 min.

36. The supernatant is aspirated and the pellets are
dissolved by adding 1 ml of solubilizer (Triton X100 +
50% ethanol, 1 + 4).
The tubes are vigorously shaken, decanted into
scintillation vials, and counted in 10 ml of liquid
scintillation counting cocktail.
Active uptake is the difference between cpm at 37 °C
and 0 °C.

37. EVALUATION
The percent inhibition of each drug concentration is the
mean of 3 determinations.
IC50 values are calculated from log-probit analyses.

38. [3
H ] 5-HT binding assay
PURPOSE AND RATIONALE
[3
H ] 5-HT binding assay is done for screening of
potential antimigraine drugs.
The assay is performed using rat or bovine tissue in the
presence spiroperidol.
Which inhibit the binding 5-HT to 5-HT1A and 5-HT2
receptors.

39. PROCEDURE:-PROCEDURE:-
Male wistar rats are used for assay.Male wistar rats are used for assay.
The rats are sacrificed, decapitated and their straitaThe rats are sacrificed, decapitated and their straita
removed and weight.removed and weight.
These are homogenized in 20 volumes of 0.05M trisThese are homogenized in 20 volumes of 0.05M tris
buffer, pH 7.7 and subsequently centrifused at 48000gbuffer, pH 7.7 and subsequently centrifused at 48000g
foe 10 mins.foe 10 mins.
The supernatant is discarded and pallets suspended inThe supernatant is discarded and pallets suspended in
same volume of 0.05M tris buffer and incubated at 37same volume of 0.05M tris buffer and incubated at 37oo
cc
for 10 minutes.for 10 minutes.
This is again at 48000g for 10 min.This is again at 48000g for 10 min.

40. The final pellet is suspended in 0.05 M Tris bufferThe final pellet is suspended in 0.05 M Tris buffer
containing 10 mM pargyline, 4 mM calcium chloride andcontaining 10 mM pargyline, 4 mM calcium chloride and
0.1% ascorbic acid.0.1% ascorbic acid.
The binding assay consist of 50The binding assay consist of 50 µµll [3
H ] 5-HT , 5050 µµl ofl of
spiroperidol (final concentration 1spiroperidol (final concentration 1 µµM), 800M), 800 µµl of tissuel of tissue
preparation , 80preparation , 80 µµl of 0.05 M tris buffer with calciuml of 0.05 M tris buffer with calcium
chloride , pargyline and ascorbic acid and 20chloride , pargyline and ascorbic acid and 20 µµl ofl of
vehicle/ 5-HT / test drug.vehicle/ 5-HT / test drug.
Following incubation at 25Following incubation at 25 oCoC
for 15 min, the binding isfor 15 min, the binding is
terminated by vacuum filtration through Whatman GF/Bterminated by vacuum filtration through Whatman GF/B
filtersfilters
The filters are then washed twice using 5 ml of ice-coldThe filters are then washed twice using 5 ml of ice-cold
0.05 M Tris buffer0.05 M Tris buffer

41. Radioactivity is determined in 10 ml of liquiscintRadioactivity is determined in 10 ml of liquiscint
scintillation cocktail.scintillation cocktail.
Evaluation :-Evaluation :-
Specific binding is determined in the presence orSpecific binding is determined in the presence or
absence of 10absence of 10-5-5
M 5-HTM 5-HT
ICIC5050 value are determined from percent specific binding atvalue are determined from percent specific binding at
each concentrationeach concentration
The KThe Kii value is determined by cheng prusoff equation.value is determined by cheng prusoff equation.

42. cheng prusoff equationcheng prusoff equation
KKii ==
IC50
1+ [(L)] / KD
[(L)] = The concentration of free radioligand used
for assay.
KD = Dissociation constant of radioligand for
receptor.
K = absolute inhibition constant.K = absolute inhibition constant.

43. ReferencesReferences
H. Gerard Vogel and Wolfgang H. Vogel, “DrugH. Gerard Vogel and Wolfgang H. Vogel, “Drug
Discovery and Evaluation: Pharmacological Assays”,Discovery and Evaluation: Pharmacological Assays”,
22ndnd
Edition, 2002, Springer Publications.Edition, 2002, Springer Publications.
S. K. Gupta, “Drug Screening Methods”, 1S. K. Gupta, “Drug Screening Methods”, 1stst
Edition,Edition,
2004, Jaypee Series,2004, Jaypee Series,
““Evaluation of sandalwood oil treatment during growthEvaluation of sandalwood oil treatment during growth
spurt period on the learning and memory in rats” byspurt period on the learning and memory in rats” by
Mr.Shidharkumar S. Biradar.Mr.Shidharkumar S. Biradar.
Iwao Kastuyama , Tsutomu Arakawa ,A novel in vitro
model for screening and evaluation of anti-asthenopia
J. Pharmacology sci. 93.222-224(2003)