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A sensitive method for the bioassay of acetylcholine
EUROPEAN JOURNAL OF PHARMACOLOGY25 (1974) 287-290. NORTH-HOLLANDPUBLISHINGCOMPANY
A SENSITIVE METHOD FOR THE BIOASSAY OF ACETYLCHOLINE M.T. KHAYYAL*, H.M. TOLBA**, M.B. EL-HAWARY** and S. ABD E1-WAHED** Departments of Pharmacology, *Faculty of Pharmacy, and **Faculty of Medicine, Cairo University, Cairo, Egypt
Received 27 July 1972
Accepted 24 September 1973
M.T. KHAYYAL, H.M. TOLBA, M.B. EL-HAWARY and S. ABD EL-WAHED,A sensitive method for the bioassay ofacetylcholine, European J. Pharmacol. 25 (1974) 287-290. A new sensitive method has been developed for the biological assay of acetylcheline using a serosal strip of the rabbit fundus. The preparation responded to the muscarinic effect of acetylcholine. It has the advantage of being highly sensitive to acetylchoime, in a dose of 0.5 ng/ml, particularly when eserinized. This sensitivity is maintained throughout the experiment and is thus at least equal to, if not more than that of other methods described. The preparation lacks rhythmic activity and shows good discrimination between different small quantities of acetylcholine. Acetylchofine assay
Rabbit fundus
1. Introduction Several methods have been described for the biological assay of acetylcholine depending on the drug's muscarinic or nicotinic effects. The most sensitive of these methods is the eserinized dorsal muscle of the leech (Mann et al., 1939; and Quastel et al., 1936), which is sensitive to mnogram (ng) quantities of acetylcholine. The fact that leeches are not always available to all laboratories and the method is too time consuming, led us to search for a new simple alternative method for the bioassay of acetylcholine. Various parts of the gastrointestinal tract of the rabbit have been tested for their sensitivity to the drug. It was observed that the strip obtained from the fundus of the stomach was sensitive to relatively large doses of acetylcholine. However, stripping off its mucosal layer greatly enhanced its sensitivity. The present work describes this preparation and its application as a new method for assaying acetylcholine.
Serosal strip
obtained as follows. Either male or non-pregnant female rabbits were sacrificed and the stomach dissected out and transferred to a Petri dish of oxygenated Tyrode solution, which contained (g/1 of distilled water): NaC1 8.0, KCI 0.2, CaC12 "6H20 0.396, MgC12 "6H20 0.214, NaH2PO4 0.05, Glucose 1.0, and NaHCO3 1.0. A transverse strip, about 4 cm in length and 3 - 4 mm in width, was taken from the fundus as shown in fig. I. About 4 - 6 strips could be obtained from one rabbit and if kept in a refrigerator, the strips lasted for several hr without loss of sensitivity. The strip, as removed, consisted of both serosal and mucosal layers. By means of blunt dissection, the
M•
tayer
2. Materials and methods The preparation, described as the serosal strip, was
Fig. 1. Preparation of the serosal layer of the fundus strip from the rabbit stomach.
M.T. Khayyal et al., Bioassay of acetylcholine
288
mucosa of the strip was carefully separated from the serosa (fig. 1), care being taken to avoid undue stretching of the serosal layer. The preparation was then suspended in a 10 ml organ bath containing Tyrode solution bubbled with pure oxygen and kept at a temperature of 37°C. The strip was connected to an isotonic frontal recording lever and left to equilibrate for about 1 hr before starting the experiment. The strip could be used for 6 - 8 hr without any appreciable change in sensitivity. The sensitivity to acetylcholine was greatly potentiated by adding eserine salicylate to the bathing fluid in a concentration of 5/ag/rnl. The effects of the following drugs were studied: acetylcholine chloride, (-)-adrenaline bitartrate, (-)-noradrenaline bitartrate, 5-hydroxytryptamine creatinine sulphate, histamine diphosphate, and angiotensin. Dose-response relationships were studied and 4-point assays for acetylcholine were performed. A contact time of 1 min was used and a cycle of 5 - 1 0 min was allowed between 2 successive drug additions to permit return of the responses to the base line. All doses were calculated in terms of the base.
110
10C
9{
O0
70
60
50
40 i
30
20
i
I
i
z i J O,S
i
[ 1
I
I
I
I
I
I
[ 10
I
I
i
i
i I II
l 100
Fig. 3. Log dose-response relationship of acetylcholine. The points on the graph represent the responses shown in fig. 2. Log doses (ng base) are shown on the abscissa and responses (Lq ram) are shown on the ordinate scale.
3. Results r2
3.1. Sensitivity The response of the serosal strip to various doses of acetylcholine is shown in fig. 2 and the results
/ li o.;
• 1
T.N. 1 mls.
t
4
!
2
• t
Acetytchetlne
It
s
& ts
/
•
!=
s2
6& nglmt
Base
Fig. 2. Responses of the rabbit fundus serosal strip to graded doses of acetyleholine.
"~
I
[
0.25
I
i
|
O,S
I
J|i
O.TS
i
|
1
Fig. 4. Graphical assessment of the 4-point assay for aceWlcholine. Log doses (ml) are shown on abscissa and responses (in ram) are shown on the ordinate scale. $I, $2 and T I , T 2 represent the doses of standard and test respectively, selected such that SI/S 2 = TI/T2. i m l o f standard contained I00 ng. The capacity of the bath was I0 ml.
M.T. Khayyal et aL, Bioassay of acetylcholine
T
20 nglmt MCh b a t e
100 n~/ml Adr.
100 ~,g/mt Noradr.
O.S 100 100 ~g/mt ng/m( nglmt O|~e~z~ Adr. Noradr.
289
A A 20 1 20 nglmt pg/m! nglmt ACh Atrop. ,A~h,
Fig. 5. A tracing Showing the effect of phenoxybenzamine (dibenzyline) on the response of the rabbit fundus serosal strip to acetylcholine, adrenaline and noradrenaline. 0.5 t~g/rrd phenoxybenzamine completely abolished the effect of the catecholamines without affecting the acetylcholine response.
interpreted graphically in fig. 3. The strip responded to 0.5 ng/ml acetylcholine and permitted precise assay of 1 ng of the drug. This order of sensitivity was obtained in all 15 preparations. The log doseresponse relationship showed a good linearity (fig. 3). A graphical representation of the 4-point assay is shown in fig. 4, where the two regression lines were found to be parallel, the limits of error of the relative potency being nearly +7.5% of the mean (p = 0.05).
3.2. Specificity A comparison of acetylcholine with some of the other possible interfering substances in biological extracts is given in table 1. Comparison was carried out using non-eserinized Tyrode solution. However, when eserine was added to the perfusion fluid, it selectively enhanced the sensitivity to acetylcholine, whilst not
affecting the sensitivity to the other substances. All the substances listed were found to be excitatory but to a far less extent than acetylcholine. The response to adrenaline and noradrenaline was completely abolished by phenoxybenzamine, in a bath concentration of 0.5 ktg/ml. The presence of phenoxybenzamine did not affect the response to acetylcholine (fig. 5). However, the response of the latter was completely antagonized by atropine (1/~g/ml) but greatly potentiated by eserine salicylate (5/~g/ml) (fig. 6). Lack of sensitivity to histamine, 5-HT, and angiotensin render their presence in tissue extracts of no significance when assaying for acetylcholine. The antagonists to
0.2 .ug I mt
Compound
Equiactive dose
Acetylcholine , (-)-Adrenaline (-)-Noradrenaline 5-HT Histamine Angiotensin
1 8 10 M 1000 > 1000 > 1000
]--
,.
/ 1 Juglmt
Table 1 Approximate equiactive doses (ng of base) of the compounds contracting the serosal strip of the rabbit fundus in a 10 ml organ bath.
L
P
[ atropine
w,
f
).2 .ufllmt A.ChP 0.2 ,IJgImt A.Ch.
z,. D,
S Pg Imt
P
0.2 ,PgI m(
P
A.Ch.
eserine ~
/ - -
Fig. 6. Record showing the effect of atropine and eserine on the response of the rabbit fundus serosal strip to acetylcholine. The latter effect was completely abolished by atropine (1 ~g/ml) and potentiated by eserine (5 ug/ml).
290
M.T. Khayyal et al., Bioassay of acetylcholine
histamine and 5-HT, mepyramine and methysergide respectively, did not affect the acetylcholine response. Effects of bradykinin, substance P or prostaglandins were not investigated.
4. Discussion
The serosal strip of the rabbit fundus has proved to be a highly sensitive and reliable method for the assay of acetylcholine and is comparable in its sensitivity to that of the leech preparation. However, it has the advantage of being less time consuming and less elaborate in its preparation. It also lacks the rhythmic activity characteristic of the rabbit intestine. In contrast to the dorsal muscle of the leech which responds to the nicotinic action of acetylcholine, the serosal strip being a smooth muscle responds to the muscarinic effect of the drug. Thus the response to acetylcholine was completely blocked by atropine. The serosal preparation is far more sensitive than other methods based on the muscarinic action of acetylcholine. The method may also be used to assay
atropine-like drugs using acetylcholine as an agonist. The presence of interfering substances e.g. adrenaline, noradrenaline, histamine, 5-HT, may be completely eliminated by adding appropriate antagonists to the perfusion fluid without affecting the tissue response to acetylcholine. It is concluded that the serosal strip is a stable, sufficiently sensitive method for the assay of acetylcholine particularly when present in tissue extracts in ng quantities. The preparation may be also of value in the biological assay and/or screening of other pharmacologically active compounds. This matter is currently under investigation.
References Mann, PJ.G., M. Tennenbaum and J.H. Quastel, 1939, Acetylcholine metabolism in central nervous system. The effects of potassium and other cations on aeetylcholine liberation, Biochem. J. 33,822. Quastel, J.H., M. Tennenbaum and A.H.M. Wh~tley, 1936, Choline ester formation in cholinesterase activities in tissues in vitro, Biochem. J. 30, 1668.
A SENSITIVE METHOD FOR THE BIOASSAY OF ACETYLCHOLINE M.T. KHAYYAL*, H.M. TOLBA**, M.B. EL-HAWARY** and S. ABD E1-WAHED** Departments of Pharmacology, *Faculty of Pharmacy, and **Faculty of Medicine, Cairo University, Cairo, Egypt
Received 27 July 1972
Accepted 24 September 1973
M.T. KHAYYAL, H.M. TOLBA, M.B. EL-HAWARY and S. ABD EL-WAHED,A sensitive method for the bioassay ofacetylcholine, European J. Pharmacol. 25 (1974) 287-290. A new sensitive method has been developed for the biological assay of acetylcheline using a serosal strip of the rabbit fundus. The preparation responded to the muscarinic effect of acetylcholine. It has the advantage of being highly sensitive to acetylchoime, in a dose of 0.5 ng/ml, particularly when eserinized. This sensitivity is maintained throughout the experiment and is thus at least equal to, if not more than that of other methods described. The preparation lacks rhythmic activity and shows good discrimination between different small quantities of acetylcholine. Acetylchofine assay
Rabbit fundus
1. Introduction Several methods have been described for the biological assay of acetylcholine depending on the drug's muscarinic or nicotinic effects. The most sensitive of these methods is the eserinized dorsal muscle of the leech (Mann et al., 1939; and Quastel et al., 1936), which is sensitive to mnogram (ng) quantities of acetylcholine. The fact that leeches are not always available to all laboratories and the method is too time consuming, led us to search for a new simple alternative method for the bioassay of acetylcholine. Various parts of the gastrointestinal tract of the rabbit have been tested for their sensitivity to the drug. It was observed that the strip obtained from the fundus of the stomach was sensitive to relatively large doses of acetylcholine. However, stripping off its mucosal layer greatly enhanced its sensitivity. The present work describes this preparation and its application as a new method for assaying acetylcholine.
Serosal strip
obtained as follows. Either male or non-pregnant female rabbits were sacrificed and the stomach dissected out and transferred to a Petri dish of oxygenated Tyrode solution, which contained (g/1 of distilled water): NaC1 8.0, KCI 0.2, CaC12 "6H20 0.396, MgC12 "6H20 0.214, NaH2PO4 0.05, Glucose 1.0, and NaHCO3 1.0. A transverse strip, about 4 cm in length and 3 - 4 mm in width, was taken from the fundus as shown in fig. I. About 4 - 6 strips could be obtained from one rabbit and if kept in a refrigerator, the strips lasted for several hr without loss of sensitivity. The strip, as removed, consisted of both serosal and mucosal layers. By means of blunt dissection, the
M•
tayer
2. Materials and methods The preparation, described as the serosal strip, was
Fig. 1. Preparation of the serosal layer of the fundus strip from the rabbit stomach.
M.T. Khayyal et al., Bioassay of acetylcholine
288
mucosa of the strip was carefully separated from the serosa (fig. 1), care being taken to avoid undue stretching of the serosal layer. The preparation was then suspended in a 10 ml organ bath containing Tyrode solution bubbled with pure oxygen and kept at a temperature of 37°C. The strip was connected to an isotonic frontal recording lever and left to equilibrate for about 1 hr before starting the experiment. The strip could be used for 6 - 8 hr without any appreciable change in sensitivity. The sensitivity to acetylcholine was greatly potentiated by adding eserine salicylate to the bathing fluid in a concentration of 5/ag/rnl. The effects of the following drugs were studied: acetylcholine chloride, (-)-adrenaline bitartrate, (-)-noradrenaline bitartrate, 5-hydroxytryptamine creatinine sulphate, histamine diphosphate, and angiotensin. Dose-response relationships were studied and 4-point assays for acetylcholine were performed. A contact time of 1 min was used and a cycle of 5 - 1 0 min was allowed between 2 successive drug additions to permit return of the responses to the base line. All doses were calculated in terms of the base.
110
10C
9{
O0
70
60
50
40 i
30
20
i
I
i
z i J O,S
i
[ 1
I
I
I
I
I
I
[ 10
I
I
i
i
i I II
l 100
Fig. 3. Log dose-response relationship of acetylcholine. The points on the graph represent the responses shown in fig. 2. Log doses (ng base) are shown on the abscissa and responses (Lq ram) are shown on the ordinate scale.
3. Results r2
3.1. Sensitivity The response of the serosal strip to various doses of acetylcholine is shown in fig. 2 and the results
/ li o.;
• 1
T.N. 1 mls.
t
4
!
2
• t
Acetytchetlne
It
s
& ts
/
•
!=
s2
6& nglmt
Base
Fig. 2. Responses of the rabbit fundus serosal strip to graded doses of acetyleholine.
"~
I
[
0.25
I
i
|
O,S
I
J|i
O.TS
i
|
1
Fig. 4. Graphical assessment of the 4-point assay for aceWlcholine. Log doses (ml) are shown on abscissa and responses (in ram) are shown on the ordinate scale. $I, $2 and T I , T 2 represent the doses of standard and test respectively, selected such that SI/S 2 = TI/T2. i m l o f standard contained I00 ng. The capacity of the bath was I0 ml.
M.T. Khayyal et aL, Bioassay of acetylcholine
T
20 nglmt MCh b a t e
100 n~/ml Adr.
100 ~,g/mt Noradr.
O.S 100 100 ~g/mt ng/m( nglmt O|~e~z~ Adr. Noradr.
289
A A 20 1 20 nglmt pg/m! nglmt ACh Atrop. ,A~h,
Fig. 5. A tracing Showing the effect of phenoxybenzamine (dibenzyline) on the response of the rabbit fundus serosal strip to acetylcholine, adrenaline and noradrenaline. 0.5 t~g/rrd phenoxybenzamine completely abolished the effect of the catecholamines without affecting the acetylcholine response.
interpreted graphically in fig. 3. The strip responded to 0.5 ng/ml acetylcholine and permitted precise assay of 1 ng of the drug. This order of sensitivity was obtained in all 15 preparations. The log doseresponse relationship showed a good linearity (fig. 3). A graphical representation of the 4-point assay is shown in fig. 4, where the two regression lines were found to be parallel, the limits of error of the relative potency being nearly +7.5% of the mean (p = 0.05).
3.2. Specificity A comparison of acetylcholine with some of the other possible interfering substances in biological extracts is given in table 1. Comparison was carried out using non-eserinized Tyrode solution. However, when eserine was added to the perfusion fluid, it selectively enhanced the sensitivity to acetylcholine, whilst not
affecting the sensitivity to the other substances. All the substances listed were found to be excitatory but to a far less extent than acetylcholine. The response to adrenaline and noradrenaline was completely abolished by phenoxybenzamine, in a bath concentration of 0.5 ktg/ml. The presence of phenoxybenzamine did not affect the response to acetylcholine (fig. 5). However, the response of the latter was completely antagonized by atropine (1/~g/ml) but greatly potentiated by eserine salicylate (5/~g/ml) (fig. 6). Lack of sensitivity to histamine, 5-HT, and angiotensin render their presence in tissue extracts of no significance when assaying for acetylcholine. The antagonists to
0.2 .ug I mt
Compound
Equiactive dose
Acetylcholine , (-)-Adrenaline (-)-Noradrenaline 5-HT Histamine Angiotensin
1 8 10 M 1000 > 1000 > 1000
]--
,.
/ 1 Juglmt
Table 1 Approximate equiactive doses (ng of base) of the compounds contracting the serosal strip of the rabbit fundus in a 10 ml organ bath.
L
P
[ atropine
w,
f
).2 .ufllmt A.ChP 0.2 ,IJgImt A.Ch.
z,. D,
S Pg Imt
P
0.2 ,PgI m(
P
A.Ch.
eserine ~
/ - -
Fig. 6. Record showing the effect of atropine and eserine on the response of the rabbit fundus serosal strip to acetylcholine. The latter effect was completely abolished by atropine (1 ~g/ml) and potentiated by eserine (5 ug/ml).
290
M.T. Khayyal et al., Bioassay of acetylcholine
histamine and 5-HT, mepyramine and methysergide respectively, did not affect the acetylcholine response. Effects of bradykinin, substance P or prostaglandins were not investigated.
4. Discussion
The serosal strip of the rabbit fundus has proved to be a highly sensitive and reliable method for the assay of acetylcholine and is comparable in its sensitivity to that of the leech preparation. However, it has the advantage of being less time consuming and less elaborate in its preparation. It also lacks the rhythmic activity characteristic of the rabbit intestine. In contrast to the dorsal muscle of the leech which responds to the nicotinic action of acetylcholine, the serosal strip being a smooth muscle responds to the muscarinic effect of the drug. Thus the response to acetylcholine was completely blocked by atropine. The serosal preparation is far more sensitive than other methods based on the muscarinic action of acetylcholine. The method may also be used to assay
atropine-like drugs using acetylcholine as an agonist. The presence of interfering substances e.g. adrenaline, noradrenaline, histamine, 5-HT, may be completely eliminated by adding appropriate antagonists to the perfusion fluid without affecting the tissue response to acetylcholine. It is concluded that the serosal strip is a stable, sufficiently sensitive method for the assay of acetylcholine particularly when present in tissue extracts in ng quantities. The preparation may be also of value in the biological assay and/or screening of other pharmacologically active compounds. This matter is currently under investigation.
References Mann, PJ.G., M. Tennenbaum and J.H. Quastel, 1939, Acetylcholine metabolism in central nervous system. The effects of potassium and other cations on aeetylcholine liberation, Biochem. J. 33,822. Quastel, J.H., M. Tennenbaum and A.H.M. Wh~tley, 1936, Choline ester formation in cholinesterase activities in tissues in vitro, Biochem. J. 30, 1668.