- Email: [email protected]
Fluid-potentiometer and acetylcholine bioassay with clam entire heart
Pergamon Press
Life Sciences, Vol . 26, pp . 111-115 Printed in the U .S .A,
FLUID-POTENTIOMETER AND ACETYLCHOLINE BIOASSAY WITH CLAM ENTIRE HEART Razuaki Ohsawa Department Faculty of iJniversity Tokyo 113,
of Physiology Medicine of Tokyo Japan
(Received is final form November 20, 1979) Summary Acetylcholine-chloride (Ach) was assayed on isolated entire heart of clam for more than 15 hours with newly designed fluid-potentiometer, a simple balance mechanism with one fulcrum. Beating preparations were accurately depressed by Ach of picogram order and cardiac arrest appeared with a large quantity of Ach . The heart was excited by 5-hydroxytriptamine (5-HT) and the distortion was found in the falling phase . With these facilities the dose-response curve of Ach was measured by frequencies of beat and this bioassay was applied to the measurement of trace amounts of Ach in ultra-purified synaptic vesicles from the brain (1) . The bioassay of Ach is in marked contrast with the chemical analysis (2) in accuracy and principle. In chemical analysis, many samples can not be taken straight on gas chromatography . Therefore, the bioassay has been used for the determination and estimation of small amounts of Ach because it allows the screening of unknown substances and because of the ease of the setting. Isolated heart preparations of certain marine molluscs are highly sensitive to both of Ach and 5-HT and the clam heart generally are extremely selective for Ach (3-5) . Ganglia of Venus contain Ach and 5-HT, which were found by chromatographical analysis and biological tests (5,12) . Intracellular recordings of the auriclewentricular valve in molluscs were found to contain Ach-response in the excitation-contraction coupling phenomena (6) . However the entire heart preparations are not generally described as drawing dose-response curves in such a way that the leech muscle responds to low levels of Ach (7,8) . Materials and Methode Specimens of the clam (Meretrix lusoria)heart, about 10 mud in volume, Were removed and ligatured with thin nylon thread at~each of aorta . This must not be tied too near the heart . The whole heart was then tranafered to the tissue chamber of a microbath (9) with a 1 ml capacity and was perfused with boiled and filtered sea water containing 10 mg/ml eserine sulfate which was run at approximately 1 ml/min into one, end of the bath as shown in Fig.l . The bath was aerated with oxygen . The flow was not stopped and 4 ml of drug solution was added to the well while taking the records continuously . The baseline of the record was carefully adjusted to the resting level of the suspended entire heart using a fluid-potentiometer which applied little inertia to the heart, so that any slight contraction of the heart resulted in 0024-3205/80/020111-05$02 .00/0 Copyright (c) 1980 Pergamoa Press Ltd
112
Fluid Potentiometer and Ach Bioassay
Vol . 26, No . 2, 1980
moving a thin silver electrode . Ach was vacuum-dried over P205 for at least 24hr prior to use . Ach was dissolved in eserinized sea water and dilution was made using 10 and 100 U1 disposal microcap pipettes . The experiments were completed in the spring, early summer and autumn .
fulcrum
fluid potentiometer Fig . 1 Fluid-potentiometer, recording apparatous, and isolated organ bath for clam entire heart . The lever is a straw, 4 cm long from fulcrum to the tip where the thread is tied . The moving of the electrode is dependent on the length of the lever . The other end of the lever is balanced with a very thin shielded silver electrode on one point of fulcrum to diminish stress on the heart . The spiral wire at the top of the balance acts as both electric conductor and spring . When the lever falls or rises, the current through the water resistance between the Ag-electrode and Ag-plate is amplified by the D .C . amplifier and recorded on the pen recorder . The Ag-electrode is held by the fulcrum as close to the Ag-plate as possible . A perfusion pathway, which is oxygenated, is shown by the fine tunnel leading from the reservoir of sea water on the right to the bottom of the micro bath (9) . Results and Discussion The majority (about 70%) of the isolated hearts, prepared as described above, began to beat in a rhythmical manner some 15 hours or more after isolation . The hearts which not beat regularly were discarded . Ach, in amounts as low as 20pg, slowed, graded and reduced the amplitude and frequency of the heart beat (Figs . 2D,2E and 2F) and the effect of Ach can be rapidly disappeared by washing for 5 min . High dose of Ach completely stopped the rhythmical activity of entire heart (Fig .2C) and after a short quiescent state eserinized washing reversed the heart to normal, so that generally the Ach dose was added at 20 min intervals . In control experiments, the effects of several other compounds, including adrenaline, noradrenaline, dopa, dopamine and GABA on the activity and threshold dose of entire heart were tested (Figs . 2G and 2H) .
Fluid Potentiometer and Ach Bioassay
Vol . 26, No, 2, 1980
1
11 3
rrpnnV"nunnqurlnl nnrr qur rugyIIJqurIIlryuunlIHIIl11111111JI1111gIH11111nrrrinuqlr"'ngllllllpr4rrnJlprr'r'rr'rlquulgq ^, ." .' . .. . ...
,!~'l~f
ffFi
.r~ .a .a . ~.
1,
.F~ k'
. . .. . .. ... . . . .. . .. . .. . .. . .. . ... . . . . .. . . . . ... .A . .w ... .. . .. . . . . .
l a e r r r''''I.
..
el rr~hdt;~pMr~~~j~~fd~~~l~ ~ ~~â ~~~~ F~ . ~~~(,!~I p J!!>râhr~~~~~~!f fff . . . .~'"~'Ji"J~J : .~J:J~~i .. . . . . . . . . .a`J: . . . . . . . . . . .1~~J~',l ;' .
. . . . . . . . . . . . . . . . . . . .Y
,, ., .,J . , .,
, :. .. .lfrf!rrrrrrr . . . . .. ..: ::JJ:'.
.
. . .. . . . . . .. . . .. . .. . .. . . .. . . . .. .
~;
~r9~JrfILir~ . . . . . .w . ..,
.~~t! ;
. . .' .iJJ . . .. .pW~~. . .
:" .
. .j
;'~rJF,S~;~1ÎÎ~ ;pfIR~r :f!ü' / : .
. . .~ .v .'4a".eJG~V'YX~w.ble . . .
. . .
.
.... .
. . . . . . . ., .. . . : : . .
.
.. . . .u..
. . . . . . . .. . . . . .u ..
Fig . 2 Responses of the isolated heart of the clam, Meretrix lusoria . The records were made with serial preparations . (A) Normal beat .(B) Continuous beat of 15 hours after record A. (C-R) Responses to Ach . Depressor offects of several compounds on the entire heart . (C) The effect of subsequent Ach dose was shown as the threshold response and the heart was then washed on quiescent state and cardiac arrest . The reversal after washing was steep . (D,E and F) Doses of Ach produce depression and cause irregualritiea in the beat . (G) Action of adrenaline (Ad) resulting in depression and diastolic arrest . (H) Threshold dose with GABA was higher than that with Ach . (I and J) Excitatory effects of 5-HT . A distinctive response recognlzed was an increase in frequency and tone of heart beat . Note the humps between the normal contractions . (R) Combinated doses of Ach and 5-RT did not produce any effect because the effects of the excitatory and inhibitory components offset each other . The heart was treated in all cases . The dowward-pointing arrows indicate drug adding and the upward-pointing ones indicate washing with eserinized sea water . The flow and recording were not stopped . Concentration of Ach : pico grams/ml . Time : 30 seconds for short lines and 10 seconds for long ones .
11 4
Fluid Potentiometer and Ach Bioassay
Vol . 26, No . 2, 1980
Unfortunately, the compounds caused the entire heart to reduce or cease rhythmic activity in a contracted state, but the range of the dose giving minimal responses was very large, such as 10'to l0~times the threshold dose of Ach . In many respects, the effects of the above compounds were also similar to the responses shown by the Mia strip heart reported previously by Cottrell et al . (3) By contrast, low doses of approximately 10 pg . of 5-HT increased both the amplitude and frequency of beat (Figs . 2L and 2J) . An increment oscillation of base line for 5-HT was produced by the frequency increase, and the amplitude was always accompanied by a small secrement hump between the normal contractions .One point of physiological interest ie that while 5-HT excites the entire heart, causing an increase in both amplitude and frequency, the entire heart responds vigorously with active contraction . The mechanism underlying this change is interpreted as the action of cyclic AMP(11) .
ä~ .7V c
~Za = ~o ~ Ach ~ pica g i ml Fig . 3 Ach dose response curve and regression line of Y on x for 16 doses . Dotted lines show dose responses from random sampling . See text the calculation methods of depression . We can detect those differences from the amplitude and tone in the dosereaponaes of Ach and 5-HT, for the reason that 5-HT has the ability to alter completely the response of the entire heart (Figs . 2I and 2J) and this ability ie absent in Ach (Figs . 2A,2B,2C,2D,2E and 2F) . For calculation of the depression of responses due to Ach, each beat was recorded for 4 min . The inhibition of the dose depression (Fig . 3) is taken as follows : Effect of depression in spontaneous beat(1- beat number of dose-response per min . before washing ) g 100 (X) normal beat number per min . before adding dose The linearity of the dose response curve was gained at 2 to 12 picograma per ml for 16 doses of random sampling and the regression line of Y on x was obtained by the method of least squares for intercept and slope as follows : Y = 0 .76x - 0 .05
Vol . 26, No . 2,
1980
Fluid Potentiometer and Ach Bioassay
11 5
Ach content of purified synaptic vesicles in brain cerebral cortex (1) Our methods (1) have madé it possible to isolate homogeneous fractions of synaptic vesicles from the brain tortes of guinea pig in a 0 .1-0 .2 M sucrose solution . The Ach content per fractioned layer of the purified synaptic vesicles has been examined by this method to be ca . 0 .57 n moles Ach/mg protein . The protein was measured by the Lowry method (10) . The value was lower than that obtained by previous workers (13, 14) . It is impossible to determine whether this lower value is due to a leakage of Ach from the homogeneous fraction of synaptic vesicles, or whether it is a reflection of the total loss of Ach from a heterogenity of other transmitter substances in the brain tissue . References 1 . R . Oheawa, Exoerientia 33, 1403 (1977) . 2 . D . J . Jenden and I . Hanin, in Choline and Acetylcholine, I. Hanin Ed . (Raven Press, New York, 1974) . 3 . G . A . Cottrell, B. Powell and M . Stauton, Br . J. Pharmac . 40, 866 (1970) . 4 . M. J . Greenberg, ~. Biochem. Physiol . 14, 513 (1965) . 5 . J . H . Welsh, Nature 1Z3, 955 (1954) . 6 . K. Ruwasawa and D . Hill, in Neurobiology of invertebrate, Savanki, Ed . (Academia Kiado Budabeat, 1973) . 7 . J . C . Szerb, J . Phveiol. London 158 . 8p (1962) . 8 . W. Feldberg and A . Feasard, J . Phvsiol . 101, 200 . (1942) . 9 . E. Heilbronn and V. P . Whittaker, in Preparation and use of aubcellular fractions in neurochemiet, (European molecular biology organization, 1974) . 10 . 0. H. Lowry, N. J . Rosebrouch, A. L. Farr, and R . J . Randall, J . Biol . Chem . 193, 265 (1951) . 11 . G. Marchand -Dumont, Pfluaers Arch . 354, 87 (1975) . 12 . R. W. Wabnits and D. von Wachtendonk, Experientia ~, 707 (1976) . 13 . V. P . Whittaker and M . N . Sheridan, J . Neurochem. 12, 363 (1965) . 14 . W. S . Wilson, R . A . Schulz and J . R . Cooper, J . Neurochem. x,659 (1973) .
Life Sciences, Vol . 26, pp . 111-115 Printed in the U .S .A,
FLUID-POTENTIOMETER AND ACETYLCHOLINE BIOASSAY WITH CLAM ENTIRE HEART Razuaki Ohsawa Department Faculty of iJniversity Tokyo 113,
of Physiology Medicine of Tokyo Japan
(Received is final form November 20, 1979) Summary Acetylcholine-chloride (Ach) was assayed on isolated entire heart of clam for more than 15 hours with newly designed fluid-potentiometer, a simple balance mechanism with one fulcrum. Beating preparations were accurately depressed by Ach of picogram order and cardiac arrest appeared with a large quantity of Ach . The heart was excited by 5-hydroxytriptamine (5-HT) and the distortion was found in the falling phase . With these facilities the dose-response curve of Ach was measured by frequencies of beat and this bioassay was applied to the measurement of trace amounts of Ach in ultra-purified synaptic vesicles from the brain (1) . The bioassay of Ach is in marked contrast with the chemical analysis (2) in accuracy and principle. In chemical analysis, many samples can not be taken straight on gas chromatography . Therefore, the bioassay has been used for the determination and estimation of small amounts of Ach because it allows the screening of unknown substances and because of the ease of the setting. Isolated heart preparations of certain marine molluscs are highly sensitive to both of Ach and 5-HT and the clam heart generally are extremely selective for Ach (3-5) . Ganglia of Venus contain Ach and 5-HT, which were found by chromatographical analysis and biological tests (5,12) . Intracellular recordings of the auriclewentricular valve in molluscs were found to contain Ach-response in the excitation-contraction coupling phenomena (6) . However the entire heart preparations are not generally described as drawing dose-response curves in such a way that the leech muscle responds to low levels of Ach (7,8) . Materials and Methode Specimens of the clam (Meretrix lusoria)heart, about 10 mud in volume, Were removed and ligatured with thin nylon thread at~each of aorta . This must not be tied too near the heart . The whole heart was then tranafered to the tissue chamber of a microbath (9) with a 1 ml capacity and was perfused with boiled and filtered sea water containing 10 mg/ml eserine sulfate which was run at approximately 1 ml/min into one, end of the bath as shown in Fig.l . The bath was aerated with oxygen . The flow was not stopped and 4 ml of drug solution was added to the well while taking the records continuously . The baseline of the record was carefully adjusted to the resting level of the suspended entire heart using a fluid-potentiometer which applied little inertia to the heart, so that any slight contraction of the heart resulted in 0024-3205/80/020111-05$02 .00/0 Copyright (c) 1980 Pergamoa Press Ltd
112
Fluid Potentiometer and Ach Bioassay
Vol . 26, No . 2, 1980
moving a thin silver electrode . Ach was vacuum-dried over P205 for at least 24hr prior to use . Ach was dissolved in eserinized sea water and dilution was made using 10 and 100 U1 disposal microcap pipettes . The experiments were completed in the spring, early summer and autumn .
fulcrum
fluid potentiometer Fig . 1 Fluid-potentiometer, recording apparatous, and isolated organ bath for clam entire heart . The lever is a straw, 4 cm long from fulcrum to the tip where the thread is tied . The moving of the electrode is dependent on the length of the lever . The other end of the lever is balanced with a very thin shielded silver electrode on one point of fulcrum to diminish stress on the heart . The spiral wire at the top of the balance acts as both electric conductor and spring . When the lever falls or rises, the current through the water resistance between the Ag-electrode and Ag-plate is amplified by the D .C . amplifier and recorded on the pen recorder . The Ag-electrode is held by the fulcrum as close to the Ag-plate as possible . A perfusion pathway, which is oxygenated, is shown by the fine tunnel leading from the reservoir of sea water on the right to the bottom of the micro bath (9) . Results and Discussion The majority (about 70%) of the isolated hearts, prepared as described above, began to beat in a rhythmical manner some 15 hours or more after isolation . The hearts which not beat regularly were discarded . Ach, in amounts as low as 20pg, slowed, graded and reduced the amplitude and frequency of the heart beat (Figs . 2D,2E and 2F) and the effect of Ach can be rapidly disappeared by washing for 5 min . High dose of Ach completely stopped the rhythmical activity of entire heart (Fig .2C) and after a short quiescent state eserinized washing reversed the heart to normal, so that generally the Ach dose was added at 20 min intervals . In control experiments, the effects of several other compounds, including adrenaline, noradrenaline, dopa, dopamine and GABA on the activity and threshold dose of entire heart were tested (Figs . 2G and 2H) .
Fluid Potentiometer and Ach Bioassay
Vol . 26, No, 2, 1980
1
11 3
rrpnnV"nunnqurlnl nnrr qur rugyIIJqurIIlryuunlIHIIl11111111JI1111gIH11111nrrrinuqlr"'ngllllllpr4rrnJlprr'r'rr'rlquulgq ^, ." .' . .. . ...
,!~'l~f
ffFi
.r~ .a .a . ~.
1,
.F~ k'
. . .. . .. ... . . . .. . .. . .. . .. . .. . ... . . . . .. . . . . ... .A . .w ... .. . .. . . . . .
l a e r r r''''I.
..
el rr~hdt;~pMr~~~j~~fd~~~l~ ~ ~~â ~~~~ F~ . ~~~(,!~I p J!!>râhr~~~~~~!f fff . . . .~'"~'Ji"J~J : .~J:J~~i .. . . . . . . . . .a`J: . . . . . . . . . . .1~~J~',l ;' .
. . . . . . . . . . . . . . . . . . . .Y
,, ., .,J . , .,
, :. .. .lfrf!rrrrrrr . . . . .. ..: ::JJ:'.
.
. . .. . . . . . .. . . .. . .. . .. . . .. . . . .. .
~;
~r9~JrfILir~ . . . . . .w . ..,
.~~t! ;
. . .' .iJJ . . .. .pW~~. . .
:" .
. .j
;'~rJF,S~;~1ÎÎ~ ;pfIR~r :f!ü' / : .
. . .~ .v .'4a".eJG~V'YX~w.ble . . .
. . .
.
.... .
. . . . . . . ., .. . . : : . .
.
.. . . .u..
. . . . . . . .. . . . . .u ..
Fig . 2 Responses of the isolated heart of the clam, Meretrix lusoria . The records were made with serial preparations . (A) Normal beat .(B) Continuous beat of 15 hours after record A. (C-R) Responses to Ach . Depressor offects of several compounds on the entire heart . (C) The effect of subsequent Ach dose was shown as the threshold response and the heart was then washed on quiescent state and cardiac arrest . The reversal after washing was steep . (D,E and F) Doses of Ach produce depression and cause irregualritiea in the beat . (G) Action of adrenaline (Ad) resulting in depression and diastolic arrest . (H) Threshold dose with GABA was higher than that with Ach . (I and J) Excitatory effects of 5-HT . A distinctive response recognlzed was an increase in frequency and tone of heart beat . Note the humps between the normal contractions . (R) Combinated doses of Ach and 5-RT did not produce any effect because the effects of the excitatory and inhibitory components offset each other . The heart was treated in all cases . The dowward-pointing arrows indicate drug adding and the upward-pointing ones indicate washing with eserinized sea water . The flow and recording were not stopped . Concentration of Ach : pico grams/ml . Time : 30 seconds for short lines and 10 seconds for long ones .
11 4
Fluid Potentiometer and Ach Bioassay
Vol . 26, No . 2, 1980
Unfortunately, the compounds caused the entire heart to reduce or cease rhythmic activity in a contracted state, but the range of the dose giving minimal responses was very large, such as 10'to l0~times the threshold dose of Ach . In many respects, the effects of the above compounds were also similar to the responses shown by the Mia strip heart reported previously by Cottrell et al . (3) By contrast, low doses of approximately 10 pg . of 5-HT increased both the amplitude and frequency of beat (Figs . 2L and 2J) . An increment oscillation of base line for 5-HT was produced by the frequency increase, and the amplitude was always accompanied by a small secrement hump between the normal contractions .One point of physiological interest ie that while 5-HT excites the entire heart, causing an increase in both amplitude and frequency, the entire heart responds vigorously with active contraction . The mechanism underlying this change is interpreted as the action of cyclic AMP(11) .
ä~ .7V c
~Za = ~o ~ Ach ~ pica g i ml Fig . 3 Ach dose response curve and regression line of Y on x for 16 doses . Dotted lines show dose responses from random sampling . See text the calculation methods of depression . We can detect those differences from the amplitude and tone in the dosereaponaes of Ach and 5-HT, for the reason that 5-HT has the ability to alter completely the response of the entire heart (Figs . 2I and 2J) and this ability ie absent in Ach (Figs . 2A,2B,2C,2D,2E and 2F) . For calculation of the depression of responses due to Ach, each beat was recorded for 4 min . The inhibition of the dose depression (Fig . 3) is taken as follows : Effect of depression in spontaneous beat(1- beat number of dose-response per min . before washing ) g 100 (X) normal beat number per min . before adding dose The linearity of the dose response curve was gained at 2 to 12 picograma per ml for 16 doses of random sampling and the regression line of Y on x was obtained by the method of least squares for intercept and slope as follows : Y = 0 .76x - 0 .05
Vol . 26, No . 2,
1980
Fluid Potentiometer and Ach Bioassay
11 5
Ach content of purified synaptic vesicles in brain cerebral cortex (1) Our methods (1) have madé it possible to isolate homogeneous fractions of synaptic vesicles from the brain tortes of guinea pig in a 0 .1-0 .2 M sucrose solution . The Ach content per fractioned layer of the purified synaptic vesicles has been examined by this method to be ca . 0 .57 n moles Ach/mg protein . The protein was measured by the Lowry method (10) . The value was lower than that obtained by previous workers (13, 14) . It is impossible to determine whether this lower value is due to a leakage of Ach from the homogeneous fraction of synaptic vesicles, or whether it is a reflection of the total loss of Ach from a heterogenity of other transmitter substances in the brain tissue . References 1 . R . Oheawa, Exoerientia 33, 1403 (1977) . 2 . D . J . Jenden and I . Hanin, in Choline and Acetylcholine, I. Hanin Ed . (Raven Press, New York, 1974) . 3 . G . A . Cottrell, B. Powell and M . Stauton, Br . J. Pharmac . 40, 866 (1970) . 4 . M. J . Greenberg, ~. Biochem. Physiol . 14, 513 (1965) . 5 . J . H . Welsh, Nature 1Z3, 955 (1954) . 6 . K. Ruwasawa and D . Hill, in Neurobiology of invertebrate, Savanki, Ed . (Academia Kiado Budabeat, 1973) . 7 . J . C . Szerb, J . Phveiol. London 158 . 8p (1962) . 8 . W. Feldberg and A . Feasard, J . Phvsiol . 101, 200 . (1942) . 9 . E. Heilbronn and V. P . Whittaker, in Preparation and use of aubcellular fractions in neurochemiet, (European molecular biology organization, 1974) . 10 . 0. H. Lowry, N. J . Rosebrouch, A. L. Farr, and R . J . Randall, J . Biol . Chem . 193, 265 (1951) . 11 . G. Marchand -Dumont, Pfluaers Arch . 354, 87 (1975) . 12 . R. W. Wabnits and D. von Wachtendonk, Experientia ~, 707 (1976) . 13 . V. P . Whittaker and M . N . Sheridan, J . Neurochem. 12, 363 (1965) . 14 . W. S . Wilson, R . A . Schulz and J . R . Cooper, J . Neurochem. x,659 (1973) .