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Effect of ouabain on the catecholamine content of heart and adrenal gland of rabbits
Life Sciences Vol. 9 Part I, Printed is Great Hrita~n
pp . 395-401, 1970 .
Pergamon Presa
EFFECT OF OUABAIN ON THE CATECHOLAMINE C~ITENT OF HEART AND ADRENAL GLAND OF RABBITS A.R . Royce and M .L . Chatterjee Department of Pharmacology, School of Tropical Medicine, Calcutta (Received 13 February 1969 ; in final form 19 January 1970) Many drugs and chemical agents are being discovered to have the property of releasing catecholamines from tissues . An absence of activity in the reserpinised animals or tissues, which is present normally, has been used as an indication of such release . This is an indirect evidence and more over in reserpinised animals, specially the hearts, which is in a state of failure by such treatmentl , there are marked morphological and pathological changes which resemble myocardial degeneration . Therefore there is every possibility that such hearts may not respond to drugs having cardiac activity and consequently such drugs may be branded as catecholamine liberator . Recently Tanz 2 observed that the positive inotropic action of ouabain on hearts might be due to catecholamine release because in reserpine pretreated animals, the action is either lost or very much attenuated . That such a mechanism for ouabain is possible has been strengthened by two facts . (A) There are many points of similarities of actions between ouabain and catecholamine on heart which may be explained on the basis of catecholamine release . (B) Ouabain induced augmentation of heart action has been significantly reduced or abolished by a previous treatment with beta * Present address : Department of Pharmacology, B .S . Medical College, Bankurs, West Bengal, India . 395
398
CATECHOLANIINE RELEASE
Vol. 9, No. 7
blockers like D .C .I . or by reserpinisation as seen by Tanz 3 . Again these are all indirect evidences and till now little attempt has been made to estimate the tissue catecholamines content before and after ouabain treatment to demonstrate directly that there is release of amines which may partly explain the mechanism of ouabain action so far as its positive inotropic action on the heart is concerned . With this idea in the back ground, it has been proposed to investigate the catecholamine content of heart and adrenal gland of rabbits before and after ouabain treatment . MATERIALS AND METHODS Over night fasting adult male rabbits weighing between 1 .0 and 1 .5 kg were taken and divided at random into five groups having nine animals in each group . Daily five rabbits were taken . One served as control . The other four were injected with ouabain 0 .2 mg/kg intravenously and sacrificed at an intervals of 15, 30, 60 and 120 minutes by a sudden blow on the nape of the nake followed by section of the carotids . Heart and adrenal glands were quickly taken out, dried by blotter and weighed . Estimation of catecholamines were done according to the method of Shore and Olin 4 . Estimation of adrenaline was done for adrenal glands because of its higher adrenaline content and for heart, noradrenaline only was estimated for the similar reasons . The extraction procedure was run with 3 ml of tissue homogenate using proportionately smaller volume of all the reagents . 'Blank' and 'added recovery' were also ran side by side and the readings were taken in Farrand spectrofluorometer . For final values, necessary corrections were made by taking into consideration the values showed by 'blank' and 'added recovery' .
Vol . 9, No . 7
CATECHOLANIINE RELEASE
397
RESULTS The catecholamine content of heart and adrenal gland before and after ouabain treatment has been presented in the Tables .
The data clearly indicate that practically there is no
significant change in the noradrenaline content of rabbit heart after ouabain treatment (Table 1) . TABLE
1
Control catecholamine (CA) content of rabbit heart and its modification by ouabain at various time intervals . (Noradrenaline content = microgram per gm fresh tissue)
Expt .
Control
15 mins .
30 mins .
60 mins .
1.
2.5
2.3
4 .1
1 .2
4 .8
2.
2 .8
4 .1
2 .7
4 .2
2 .3
3.
4 .7
2 .1
3.2
2.0
5 .0
4.
1 .9
1 .2
2.3
2.5
1 .0
5.
2 .6
2 .6
2 .6
1 .3
1 .3
6.
3 .2
1 .9
2 .4
4 .0
2 .4
7.
3 .0
2 .0
3 .0
3.3
1 .9
8.
2 .7
4 .2
2 .2
2 .6
5 .2
9.
1 .8
3 .0
1 .8
3 .6
3 .0
No .
Av .t S .E . 2.8+0 .3
2 .6+0 .31
2 .6+0 .22
2 .7+0 .35
120 mins .
2 .9+0 .55
'P' value in respect to the CA content of the control and 30 minutes lies between 0 .3 and 0 .2, t = 1 .2 (not significant)
The maximum lowering of catecholamine by ouabain is found in 15 minutes and 30 minutes which is only seven percent less than
398
CATECHOLAbIINE RELEASE
Vol. 9, No. 7
the control value . But in adrenal gland ouabain is seen to lower the amine content .
The maximum lowering of the amine content
occured in first fifteen minutes of the drug (Table 2) when the level went down by 24 .5 percent of the control level . This difference of adrenal catecholamine content between control and 15 minutes following ouabain has been found to be, on calculations, TABLE
2
Control catecholamine (CA) content of rabbit adrenal gland and its modification by ouabain at various time intervals,
(Adrenaline content = milligram per gm of
fresh gland) Expt . No .
Control
15 rains .
30 rains .
60 rains .
120 mies .
1.
0 .52
0 .36
0 .48
0 .32
0 .28
2.
0 .67
0.40
0 .19
0.62
0 .86
3.
0 .32
0.56
0 .27
0.72
0 .88
4.
0 .84
0 .49
0 .58
0 .56
0 .65
5.
0 .57
0.51
0 .48
0 .55
0,64
6.
0 .48
0 .29
0 .51
0 .63
0 .51
7.
0.37
0 .33
0.52
0 .46
0,32
8.
0.68
0 .41
0.79
0 .65
0 .59
9.
0.73
0 .52
0.63
0 .49
0 .48
Av . +S .E . 0 .57+0,06
0 .43+ 0.04
0 .49+0.06
0 .55+0.04
0 .57+ 0.07
'P' value in respect to the CA content of the control and 15 minutes lies between 0 .05 and 0 .02,
t = 2.2 (Significant)
statistically significant .
Subsequent estimations at 30, 60 and
Vol . 9, No . 7
CATECHOLAMINE RELEASE
S99
120 minutes showed that complete recovery took place within the period understudy . DISCUSSION The catecholamine content of heart of normal rabbits has been found to be 2 .8 + 0 .3 (S .E .) microgram per gm of fresh cardiac tissue which is slightly higher than the values for the same
. obtained by different workers like Von Euler S and others 4 ~ 6,7 Thè catecholamine content of adrenal gland of normal rabbits have been found to be 0 .57 + 0 .06 (S .E .) milligram per gm of fresh adrenal gland,
Values for the same obtained by workers like West B
was 0 .48 mg/gm and that by Muscholl and Vogt 9 was 501 microgram per gm .
In both these tissues, our values have been found to be
on the slightly higher side . It is clear from the data presented that ouabain shows no modification in the catecholamine content of rabbits heart, the adrenal gland catecholamine significantly lowered .
Assuming that
the mechanism of storage and release of amine is the same for both heart and adrenal gland, it seems rather contradictory that in one tissue the drug causes release and fails to do so in some other tissue .
The following arguments have been put forward for
explanation . 1 . That inspite of release of cardiac catecholamine,
the
rate of its synthesis surpassed the rate of release . 2. Catecholamines from intracellular storage particle in the myocardium being released by ouabain react with the target cells or receptors,
Some unused portion may be taken up by intra
cardiac sympathetic nerve endings and ganglion cells .
This bound
form of inactive amines might have been reflected in the estimation of cardiac catecholamine following ouabain.
CATECHOLAMINE RELEASE
400
Vol . 9, No. 7
3. In our unpublished observations, we have evidences of monoamine oxidase inhibitory activity of ouabain .
Therefore even
when ouabain releases amines from heart it may not lower the amine content by virtue of MAO inhibition .
This property is also shown
by ephedrine which is a MAO inhibitor and a catecholamine releaser . 4. Drugs like guanethidine l0 have been found to have tissue specific catecholamine releasing property because it liberates catecholamine from heart but fails to do so in adrenal gland . Similar mechanism may also play here . SUMMARY Investigations have been done to see if ouabain induces release of catecholamines from heart and adrenal gland of rabbits . Results suggest that there is little change of cardiac catechol amine content even up to 120 minutes of observation after administration of ouabain.
However ouabain is found to lower catechol-
amine from adrenal gland within fifteen minutes of administration which is replenished within 120 minutes .
The possible reasons
for such action have been discussed . ACKNOWLEDGEMENT The authors wish to thank the authorities of Indian Institute of Experimental Medicine, Calcutta for kindly allowing them to use spectrofluorometer .
Thanks are also due to M/s . Glaxo
Laboratories (India.) Private Ltd . for financial help of the work . REFERENCES 1.
P. Withrington and E . Zaimis . 380, (1961) .
2.
R . Tanz .
Fed . Proc .
~.1 .
126,
Brit . J . Pharmacol . (1962 b) .
17
Yol . 9, No. 7
CATECHOLAMINE RELEASE J . Pharm . Exa . Theras .
401
3.
R. Tanz .
~,
209,
(1964) .
4.
P .A . Shore and J .S . Olin . 299, (1958) .
9.
U .S . Von Euler .
6.
A. Bertler ., A. Carlsson and E . Rosengren . Naturearissenshaf ~gn . 4~, 521, (1996) .
7.
E .T . Angelakos, K . Fuxe and M .L . Torthu ns . (1963) . Sca . 5Q, 184,
8.
G.B . liest .
9.
E . Muscholl and M . Vogt .
10 .
H. Sheppard and J . Zimmern~an . (1999) .
J . Pharmacol . Exo . Therao .
~,~, 1
The Harvey Lecture Series '59 . p .43, (1961) .
Quart . Rev . Biol .
30,
116,
J . Physiol .
Acta . Physiol .
(1955) .
14~,
132,
Pharraacoloaist .
~,
(1958) . 69,
pp . 395-401, 1970 .
Pergamon Presa
EFFECT OF OUABAIN ON THE CATECHOLAMINE C~ITENT OF HEART AND ADRENAL GLAND OF RABBITS A.R . Royce and M .L . Chatterjee Department of Pharmacology, School of Tropical Medicine, Calcutta (Received 13 February 1969 ; in final form 19 January 1970) Many drugs and chemical agents are being discovered to have the property of releasing catecholamines from tissues . An absence of activity in the reserpinised animals or tissues, which is present normally, has been used as an indication of such release . This is an indirect evidence and more over in reserpinised animals, specially the hearts, which is in a state of failure by such treatmentl , there are marked morphological and pathological changes which resemble myocardial degeneration . Therefore there is every possibility that such hearts may not respond to drugs having cardiac activity and consequently such drugs may be branded as catecholamine liberator . Recently Tanz 2 observed that the positive inotropic action of ouabain on hearts might be due to catecholamine release because in reserpine pretreated animals, the action is either lost or very much attenuated . That such a mechanism for ouabain is possible has been strengthened by two facts . (A) There are many points of similarities of actions between ouabain and catecholamine on heart which may be explained on the basis of catecholamine release . (B) Ouabain induced augmentation of heart action has been significantly reduced or abolished by a previous treatment with beta * Present address : Department of Pharmacology, B .S . Medical College, Bankurs, West Bengal, India . 395
398
CATECHOLANIINE RELEASE
Vol. 9, No. 7
blockers like D .C .I . or by reserpinisation as seen by Tanz 3 . Again these are all indirect evidences and till now little attempt has been made to estimate the tissue catecholamines content before and after ouabain treatment to demonstrate directly that there is release of amines which may partly explain the mechanism of ouabain action so far as its positive inotropic action on the heart is concerned . With this idea in the back ground, it has been proposed to investigate the catecholamine content of heart and adrenal gland of rabbits before and after ouabain treatment . MATERIALS AND METHODS Over night fasting adult male rabbits weighing between 1 .0 and 1 .5 kg were taken and divided at random into five groups having nine animals in each group . Daily five rabbits were taken . One served as control . The other four were injected with ouabain 0 .2 mg/kg intravenously and sacrificed at an intervals of 15, 30, 60 and 120 minutes by a sudden blow on the nape of the nake followed by section of the carotids . Heart and adrenal glands were quickly taken out, dried by blotter and weighed . Estimation of catecholamines were done according to the method of Shore and Olin 4 . Estimation of adrenaline was done for adrenal glands because of its higher adrenaline content and for heart, noradrenaline only was estimated for the similar reasons . The extraction procedure was run with 3 ml of tissue homogenate using proportionately smaller volume of all the reagents . 'Blank' and 'added recovery' were also ran side by side and the readings were taken in Farrand spectrofluorometer . For final values, necessary corrections were made by taking into consideration the values showed by 'blank' and 'added recovery' .
Vol . 9, No . 7
CATECHOLANIINE RELEASE
397
RESULTS The catecholamine content of heart and adrenal gland before and after ouabain treatment has been presented in the Tables .
The data clearly indicate that practically there is no
significant change in the noradrenaline content of rabbit heart after ouabain treatment (Table 1) . TABLE
1
Control catecholamine (CA) content of rabbit heart and its modification by ouabain at various time intervals . (Noradrenaline content = microgram per gm fresh tissue)
Expt .
Control
15 mins .
30 mins .
60 mins .
1.
2.5
2.3
4 .1
1 .2
4 .8
2.
2 .8
4 .1
2 .7
4 .2
2 .3
3.
4 .7
2 .1
3.2
2.0
5 .0
4.
1 .9
1 .2
2.3
2.5
1 .0
5.
2 .6
2 .6
2 .6
1 .3
1 .3
6.
3 .2
1 .9
2 .4
4 .0
2 .4
7.
3 .0
2 .0
3 .0
3.3
1 .9
8.
2 .7
4 .2
2 .2
2 .6
5 .2
9.
1 .8
3 .0
1 .8
3 .6
3 .0
No .
Av .t S .E . 2.8+0 .3
2 .6+0 .31
2 .6+0 .22
2 .7+0 .35
120 mins .
2 .9+0 .55
'P' value in respect to the CA content of the control and 30 minutes lies between 0 .3 and 0 .2, t = 1 .2 (not significant)
The maximum lowering of catecholamine by ouabain is found in 15 minutes and 30 minutes which is only seven percent less than
398
CATECHOLAbIINE RELEASE
Vol. 9, No. 7
the control value . But in adrenal gland ouabain is seen to lower the amine content .
The maximum lowering of the amine content
occured in first fifteen minutes of the drug (Table 2) when the level went down by 24 .5 percent of the control level . This difference of adrenal catecholamine content between control and 15 minutes following ouabain has been found to be, on calculations, TABLE
2
Control catecholamine (CA) content of rabbit adrenal gland and its modification by ouabain at various time intervals,
(Adrenaline content = milligram per gm of
fresh gland) Expt . No .
Control
15 rains .
30 rains .
60 rains .
120 mies .
1.
0 .52
0 .36
0 .48
0 .32
0 .28
2.
0 .67
0.40
0 .19
0.62
0 .86
3.
0 .32
0.56
0 .27
0.72
0 .88
4.
0 .84
0 .49
0 .58
0 .56
0 .65
5.
0 .57
0.51
0 .48
0 .55
0,64
6.
0 .48
0 .29
0 .51
0 .63
0 .51
7.
0.37
0 .33
0.52
0 .46
0,32
8.
0.68
0 .41
0.79
0 .65
0 .59
9.
0.73
0 .52
0.63
0 .49
0 .48
Av . +S .E . 0 .57+0,06
0 .43+ 0.04
0 .49+0.06
0 .55+0.04
0 .57+ 0.07
'P' value in respect to the CA content of the control and 15 minutes lies between 0 .05 and 0 .02,
t = 2.2 (Significant)
statistically significant .
Subsequent estimations at 30, 60 and
Vol . 9, No . 7
CATECHOLAMINE RELEASE
S99
120 minutes showed that complete recovery took place within the period understudy . DISCUSSION The catecholamine content of heart of normal rabbits has been found to be 2 .8 + 0 .3 (S .E .) microgram per gm of fresh cardiac tissue which is slightly higher than the values for the same
. obtained by different workers like Von Euler S and others 4 ~ 6,7 Thè catecholamine content of adrenal gland of normal rabbits have been found to be 0 .57 + 0 .06 (S .E .) milligram per gm of fresh adrenal gland,
Values for the same obtained by workers like West B
was 0 .48 mg/gm and that by Muscholl and Vogt 9 was 501 microgram per gm .
In both these tissues, our values have been found to be
on the slightly higher side . It is clear from the data presented that ouabain shows no modification in the catecholamine content of rabbits heart, the adrenal gland catecholamine significantly lowered .
Assuming that
the mechanism of storage and release of amine is the same for both heart and adrenal gland, it seems rather contradictory that in one tissue the drug causes release and fails to do so in some other tissue .
The following arguments have been put forward for
explanation . 1 . That inspite of release of cardiac catecholamine,
the
rate of its synthesis surpassed the rate of release . 2. Catecholamines from intracellular storage particle in the myocardium being released by ouabain react with the target cells or receptors,
Some unused portion may be taken up by intra
cardiac sympathetic nerve endings and ganglion cells .
This bound
form of inactive amines might have been reflected in the estimation of cardiac catecholamine following ouabain.
CATECHOLAMINE RELEASE
400
Vol . 9, No. 7
3. In our unpublished observations, we have evidences of monoamine oxidase inhibitory activity of ouabain .
Therefore even
when ouabain releases amines from heart it may not lower the amine content by virtue of MAO inhibition .
This property is also shown
by ephedrine which is a MAO inhibitor and a catecholamine releaser . 4. Drugs like guanethidine l0 have been found to have tissue specific catecholamine releasing property because it liberates catecholamine from heart but fails to do so in adrenal gland . Similar mechanism may also play here . SUMMARY Investigations have been done to see if ouabain induces release of catecholamines from heart and adrenal gland of rabbits . Results suggest that there is little change of cardiac catechol amine content even up to 120 minutes of observation after administration of ouabain.
However ouabain is found to lower catechol-
amine from adrenal gland within fifteen minutes of administration which is replenished within 120 minutes .
The possible reasons
for such action have been discussed . ACKNOWLEDGEMENT The authors wish to thank the authorities of Indian Institute of Experimental Medicine, Calcutta for kindly allowing them to use spectrofluorometer .
Thanks are also due to M/s . Glaxo
Laboratories (India.) Private Ltd . for financial help of the work . REFERENCES 1.
P. Withrington and E . Zaimis . 380, (1961) .
2.
R . Tanz .
Fed . Proc .
~.1 .
126,
Brit . J . Pharmacol . (1962 b) .
17
Yol . 9, No. 7
CATECHOLAMINE RELEASE J . Pharm . Exa . Theras .
401
3.
R. Tanz .
~,
209,
(1964) .
4.
P .A . Shore and J .S . Olin . 299, (1958) .
9.
U .S . Von Euler .
6.
A. Bertler ., A. Carlsson and E . Rosengren . Naturearissenshaf ~gn . 4~, 521, (1996) .
7.
E .T . Angelakos, K . Fuxe and M .L . Torthu ns . (1963) . Sca . 5Q, 184,
8.
G.B . liest .
9.
E . Muscholl and M . Vogt .
10 .
H. Sheppard and J . Zimmern~an . (1999) .
J . Pharmacol . Exo . Therao .
~,~, 1
The Harvey Lecture Series '59 . p .43, (1961) .
Quart . Rev . Biol .
30,
116,
J . Physiol .
Acta . Physiol .
(1955) .
14~,
132,
Pharraacoloaist .
~,
(1958) . 69,