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The effect of insulin on the subcellular distribution and the inotropic effect of 3H-digoxin in the guinea pig heart
Life Sciences Vol . 9, Part I, pp. 1135-1139, 1970. Printed in Great Britain
Pergamon Press
THE EFFECT OF INSULIN ON THE ffUBCZIJULAR DISTRIBUTION AND THE INOTROPIC EFFECT OF 3H-DIOO%IN IN THE GUINEA PIG HEART Nak Doo Kim,
Leslie E. Bailey and Peter E. Dresel
Department of Pharmacology and Therapeutics Faculty of Medicine, University of Manitoba Winnipeg 3, Manitoba, Canada
(Received 20 July 1970; in final form 31 August 1970) Dutta and Marks
1,2
found that digitalis accumulates selectively in the
microsomal fraction isolated from perfused guinea pig heart, this fraction may contain a digitalis receptor .
and suggested that
They 3 also reported that high
[K +] and low [Na+ ] solutions decrease the uptake of ouabain by the microsomal traction, which correlates with the effect of these ions on the inotropic effect of digitalis.
Bailey and Dresel 4 have shown that highly purified insulin in-
creases the rate of development of the positive inotropic effect of ouabain in rabbit atria. We have now used insulin to test for the correlation between the subcellular distribution of tritiated digoxin and its effect on contractility.
Although
the rate of onset of the inotropic responses was not increased in the guinea pig heart by prior treatment with insulin,
the inotropic effect of digoxin was poten-
tiated and the uptake of 3H-digoxin by microsomes isolated from guinea pig ventricle was increased by insulin. Materials and Methods Hearts isolated from 300 g guinea pigs of either sex were perfused through the aorta at a constant pressure of 50 mm Hg with Krebs-Henseleit solution equilibrated with 95% 02- 5% 002 at 30°C .
Isometric contractile force at the apex
was measured by a Grass FT-03 force-displacement transducer and recorded on a Grass 5D polygraph.
The hearts were stimulated with twice threshold voltage at
a rate of 216/min with platinum electrodes placed on the apex and on the right atrium.
Resting tension was adjusted to produce a developed tension which was
1135
1136
INSULIN AND 3 H-DIGUMN
50% of maximum.
After 60 min of equilibration, the hearts were exposed to
digoxin (2 x 10 -7 g/ml) for either 15 or 30 min.
One-half of the digoxin added
to the bath was 3H-Digoxin (New England Nuclear Corp .) . (2 mU/ml,
Vol. 9, No. 19
Recrystallized insulin
Connaught Laboratories) was added to the perfusion medium 30 min prior
to digoxin treatment .
The perfusate from each heart was collected throughout
the perfusion with 3H-digoxin or 3H-digoxin and insulin.
The total volume col
lected was not affected by the presence of insulin in the perfusion fluid.
Each
heart was perfused with drug-free medium for 4 min at the end of the experiment to wash out digoxin from the vascular and interstitial spaces . After perfusion,
the ventricles were minced with scissors in an ice bath
and homogenized with a motor driven Teflon pestle in 10 volumes of 0.33 M sucrose solution containing EDTA (0 .00111), followed by 1 hr of centrifugation at 166,000 g at 4°C.
The pellet was resuspended in 10 volumes of cold sucrose-EDTA solution
and was centrifuged differentially for the isolation of the nuclear (700 g for 10 min), mitochondrial (12,000 g for 15 min) and microsomel (166,000 g for 60 min) fractions .
Aliquots of the homogenate and of each fraction were counted
in a liquid scintillation counter .
Protein was determined by the Lowry5 method,
modified by the addition of 5% deoxycholate to each sample . Results The amounts of 3H digoxin in guinea pig heart after 30 min of perfusion followed by 4 min washout is shown in the Table 1.
Mean contractile force in-
creased by 39 .0 f 2 .6% (Mean t û.E .) after 30 min exposure to digoxin (2 x g/ml) .
10-7
Contractile force in the insulin-treated hearts was increased by
50 .0 t 4 .7%, which was significantly greater than in the untreated hearts (P < 0.05) .
Insulin potentiated the inotropic effects of digoxin although
total tissue uptake appeared to be decreased .
This decrease was not statistic-
ally significant ; however the decrease in the tissue/medium ratio reached statistical significance .
Uptake by the mitochondrial and nuclear fractions was
not changed in the insulin treated hearts .
Uptake of digoxin by the microsomes
of insulin treated hearts was significantly greater than that of controls .
DOULIN AND 3H-DIGOXIN
Vol. 9, No. 19
1137
TABLE 1 The effect of insulin on the uptake and pharmacological effect of 3H-digoxin
Drug (N)
Uptake Nuclei Mitochondria ngi mg Protein
Force Increase
Tissue/ Medium Ratio
39 .0
2.07
414.50 f 12 .19
1 .80 f 0.10
2 .09 f 0.10
f 0.26
50 .0 f 4 .7
1 .85 f 0.05
386.75 f 25 .71
2 .04 f 0.10
2 .29 f 0.14
5.40
< .05
< .05
Digoxin7 2 x 10 g/ml (4) Digoxin PLUS Insulin 2 mU/ml (4) P
f 2.6
Total Tissue ng, g
f 0.06
Microsomes
4.63
f 0.30 < .05
The correlation between the microsomal uptake of 3H-digoxin and its inotropic effect
in
guinea pig hearts is shown in Fig. 1 .
Insulin treated and un-
treated hearts perfused for 15 and 30 min are included .
The coefficient of
correlation is highly significant (r = 0.68, P < 0.01) .
These results suggest
that the positive isotropic effect is in fact related to the uptake of
-
digoxin by the microsomas . Discussion Insulin is known to increase the uptake and pharmacological effects of a wide variety of drugs in vivo 6'7 .
It seemed possible that an increase in the
response to ouabain in vitro4 might be related to an increase in the tissue up take of the drug .
Previous studies on the relationship of the uptake of digi-
talis glycosides to their pharmacological effect have suffered from the fact that tissue uptake was not measured in the same experiments in which the pharisscological effects were established.
We feel that it is important,
especially
1138
INSULINAND 3H-DIGOXIN
Vol . 9, No. 19
50W W
40-
V
Z
V
30~ 20
2
3
4
5
6
7
MKROSOMAL DIGOXIN (nR/mg PROTEIN) FIG. 1 The relationship between the microsomal uptake of 3H-digoxin and the inotropic effect . Each dot represents a heart perfused for either 15 or 30 min and either with or without insulin (2
Wml) .
when interactions between drugs are . studied, that a quantitative relationship be based on simultaneous study of the two variables.
We have confirmed the rela-
tionship of uptake of digoxin by the microsomal fraction to its positive inotropic effect as suggested by Dutta and Marksl' 3, but our experiments indicate that the study of total tissue uptake
is
probably not sufficient to establish
interactions between drugs or comparisons between glycosides 3' 8. The effect of insulin on the inotropic response to the 3H-digoxin in the guinea pig heart was qualitatively different from that seen in the rabbit atria . Insulin increased the magnitude of the change in contractile force. after treatment with 3H-digoxin, but did not affect the rate of onset of the response . Perhaps this discrepancy between guinea pig ventricle and rabbit atria is the
Vol. 9, No. 19
INSULIN AND 3H-DIGOXIN
1139
result of a difference in the experimental preparation or in the species, or more likely the fact that the uptake of the drug by ventricular muscle is greater than that by atrial muscle2 (unpublished observations) .
In any event,
the increased positive inotropic response to 3H-digoxin in the presence of purified insulin was directly related to the uptake of 3H-digoxin by the microsomal fraction, but was not related to the uptake by any other subcellular structure. According to Dutta and Marks
3,
total tissue uptake of 3H-ouabain was always
directly related to microsomal uptake and thus total tissue uptake could be taken as an estimate of microsomal binding.
However,
insulin did not increase
the total tissue uptake of 3H-digoxin in our experiments, but decreased the 3Hdigoxin tissue to medium ratio.
Thus, our results indicate that insulin did not
facilitate the transport of digoxin into the cell, but specifically increased the binding of the drug to a site in a structure sedimenting as a microsome. Acknowledgement - This work was supported by grants from the Medical Research Council of Canada and the Manitoba Heart Foundation . References 1.
S . DUTTA, 8. GOSWAMI, J.O . LINDOVER and B.K . MARKS, Therap . 159, 324 (1968) .
J. Pharmacol. Exp.
2.
8. DUTTA, 8. GOSWAMI, D.K . DATTA, J.O . LINDOWER and B.H . MARKS, Exp. Therap . 164, 10 (1968) .
3.
8 . DUTTA and B.H. MARKS,
4.
L.E . BAILEY and P.E . DRESEL, Life Sciences 8,
5.
O.H . LOWRY, N.J . ROSEBROUGH, A .L . FARR and R.J . RANDALL, 193, 265 (1951) .
6.
J.W. TRAVIS, A.C . KEYL and C.A . DRAGSTEDT, 148 (l956) .
7.
K. WISNIEWSKI and A. DANYZ, Biochem. Pharmacol . 15, 669 (l966) .
8.
K. KUBCHINSKY, H. IAHRTZ, H. L$LLWANN and P.A . VAN ZWIEm, Br . J. Chemother. 30, 317 (1967) .
J. Pharmacol. Exp. Therap . 170, part I,
J. Pharmacol.
318 (1969) .
347 (1969) . J. Bi ol . Che
J. Pharmacol . Exp . Th _ erap .
117, --
aarmac .
Pergamon Press
THE EFFECT OF INSULIN ON THE ffUBCZIJULAR DISTRIBUTION AND THE INOTROPIC EFFECT OF 3H-DIOO%IN IN THE GUINEA PIG HEART Nak Doo Kim,
Leslie E. Bailey and Peter E. Dresel
Department of Pharmacology and Therapeutics Faculty of Medicine, University of Manitoba Winnipeg 3, Manitoba, Canada
(Received 20 July 1970; in final form 31 August 1970) Dutta and Marks
1,2
found that digitalis accumulates selectively in the
microsomal fraction isolated from perfused guinea pig heart, this fraction may contain a digitalis receptor .
and suggested that
They 3 also reported that high
[K +] and low [Na+ ] solutions decrease the uptake of ouabain by the microsomal traction, which correlates with the effect of these ions on the inotropic effect of digitalis.
Bailey and Dresel 4 have shown that highly purified insulin in-
creases the rate of development of the positive inotropic effect of ouabain in rabbit atria. We have now used insulin to test for the correlation between the subcellular distribution of tritiated digoxin and its effect on contractility.
Although
the rate of onset of the inotropic responses was not increased in the guinea pig heart by prior treatment with insulin,
the inotropic effect of digoxin was poten-
tiated and the uptake of 3H-digoxin by microsomes isolated from guinea pig ventricle was increased by insulin. Materials and Methods Hearts isolated from 300 g guinea pigs of either sex were perfused through the aorta at a constant pressure of 50 mm Hg with Krebs-Henseleit solution equilibrated with 95% 02- 5% 002 at 30°C .
Isometric contractile force at the apex
was measured by a Grass FT-03 force-displacement transducer and recorded on a Grass 5D polygraph.
The hearts were stimulated with twice threshold voltage at
a rate of 216/min with platinum electrodes placed on the apex and on the right atrium.
Resting tension was adjusted to produce a developed tension which was
1135
1136
INSULIN AND 3 H-DIGUMN
50% of maximum.
After 60 min of equilibration, the hearts were exposed to
digoxin (2 x 10 -7 g/ml) for either 15 or 30 min.
One-half of the digoxin added
to the bath was 3H-Digoxin (New England Nuclear Corp .) . (2 mU/ml,
Vol. 9, No. 19
Recrystallized insulin
Connaught Laboratories) was added to the perfusion medium 30 min prior
to digoxin treatment .
The perfusate from each heart was collected throughout
the perfusion with 3H-digoxin or 3H-digoxin and insulin.
The total volume col
lected was not affected by the presence of insulin in the perfusion fluid.
Each
heart was perfused with drug-free medium for 4 min at the end of the experiment to wash out digoxin from the vascular and interstitial spaces . After perfusion,
the ventricles were minced with scissors in an ice bath
and homogenized with a motor driven Teflon pestle in 10 volumes of 0.33 M sucrose solution containing EDTA (0 .00111), followed by 1 hr of centrifugation at 166,000 g at 4°C.
The pellet was resuspended in 10 volumes of cold sucrose-EDTA solution
and was centrifuged differentially for the isolation of the nuclear (700 g for 10 min), mitochondrial (12,000 g for 15 min) and microsomel (166,000 g for 60 min) fractions .
Aliquots of the homogenate and of each fraction were counted
in a liquid scintillation counter .
Protein was determined by the Lowry5 method,
modified by the addition of 5% deoxycholate to each sample . Results The amounts of 3H digoxin in guinea pig heart after 30 min of perfusion followed by 4 min washout is shown in the Table 1.
Mean contractile force in-
creased by 39 .0 f 2 .6% (Mean t û.E .) after 30 min exposure to digoxin (2 x g/ml) .
10-7
Contractile force in the insulin-treated hearts was increased by
50 .0 t 4 .7%, which was significantly greater than in the untreated hearts (P < 0.05) .
Insulin potentiated the inotropic effects of digoxin although
total tissue uptake appeared to be decreased .
This decrease was not statistic-
ally significant ; however the decrease in the tissue/medium ratio reached statistical significance .
Uptake by the mitochondrial and nuclear fractions was
not changed in the insulin treated hearts .
Uptake of digoxin by the microsomes
of insulin treated hearts was significantly greater than that of controls .
DOULIN AND 3H-DIGOXIN
Vol. 9, No. 19
1137
TABLE 1 The effect of insulin on the uptake and pharmacological effect of 3H-digoxin
Drug (N)
Uptake Nuclei Mitochondria ngi mg Protein
Force Increase
Tissue/ Medium Ratio
39 .0
2.07
414.50 f 12 .19
1 .80 f 0.10
2 .09 f 0.10
f 0.26
50 .0 f 4 .7
1 .85 f 0.05
386.75 f 25 .71
2 .04 f 0.10
2 .29 f 0.14
5.40
< .05
< .05
Digoxin7 2 x 10 g/ml (4) Digoxin PLUS Insulin 2 mU/ml (4) P
f 2.6
Total Tissue ng, g
f 0.06
Microsomes
4.63
f 0.30 < .05
The correlation between the microsomal uptake of 3H-digoxin and its inotropic effect
in
guinea pig hearts is shown in Fig. 1 .
Insulin treated and un-
treated hearts perfused for 15 and 30 min are included .
The coefficient of
correlation is highly significant (r = 0.68, P < 0.01) .
These results suggest
that the positive isotropic effect is in fact related to the uptake of
-
digoxin by the microsomas . Discussion Insulin is known to increase the uptake and pharmacological effects of a wide variety of drugs in vivo 6'7 .
It seemed possible that an increase in the
response to ouabain in vitro4 might be related to an increase in the tissue up take of the drug .
Previous studies on the relationship of the uptake of digi-
talis glycosides to their pharmacological effect have suffered from the fact that tissue uptake was not measured in the same experiments in which the pharisscological effects were established.
We feel that it is important,
especially
1138
INSULINAND 3H-DIGOXIN
Vol . 9, No. 19
50W W
40-
V
Z
V
30~ 20
2
3
4
5
6
7
MKROSOMAL DIGOXIN (nR/mg PROTEIN) FIG. 1 The relationship between the microsomal uptake of 3H-digoxin and the inotropic effect . Each dot represents a heart perfused for either 15 or 30 min and either with or without insulin (2
Wml) .
when interactions between drugs are . studied, that a quantitative relationship be based on simultaneous study of the two variables.
We have confirmed the rela-
tionship of uptake of digoxin by the microsomal fraction to its positive inotropic effect as suggested by Dutta and Marksl' 3, but our experiments indicate that the study of total tissue uptake
is
probably not sufficient to establish
interactions between drugs or comparisons between glycosides 3' 8. The effect of insulin on the inotropic response to the 3H-digoxin in the guinea pig heart was qualitatively different from that seen in the rabbit atria . Insulin increased the magnitude of the change in contractile force. after treatment with 3H-digoxin, but did not affect the rate of onset of the response . Perhaps this discrepancy between guinea pig ventricle and rabbit atria is the
Vol. 9, No. 19
INSULIN AND 3H-DIGOXIN
1139
result of a difference in the experimental preparation or in the species, or more likely the fact that the uptake of the drug by ventricular muscle is greater than that by atrial muscle2 (unpublished observations) .
In any event,
the increased positive inotropic response to 3H-digoxin in the presence of purified insulin was directly related to the uptake of 3H-digoxin by the microsomal fraction, but was not related to the uptake by any other subcellular structure. According to Dutta and Marks
3,
total tissue uptake of 3H-ouabain was always
directly related to microsomal uptake and thus total tissue uptake could be taken as an estimate of microsomal binding.
However,
insulin did not increase
the total tissue uptake of 3H-digoxin in our experiments, but decreased the 3Hdigoxin tissue to medium ratio.
Thus, our results indicate that insulin did not
facilitate the transport of digoxin into the cell, but specifically increased the binding of the drug to a site in a structure sedimenting as a microsome. Acknowledgement - This work was supported by grants from the Medical Research Council of Canada and the Manitoba Heart Foundation . References 1.
S . DUTTA, 8. GOSWAMI, J.O . LINDOVER and B.K . MARKS, Therap . 159, 324 (1968) .
J. Pharmacol. Exp.
2.
8. DUTTA, 8. GOSWAMI, D.K . DATTA, J.O . LINDOWER and B.H . MARKS, Exp. Therap . 164, 10 (1968) .
3.
8 . DUTTA and B.H. MARKS,
4.
L.E . BAILEY and P.E . DRESEL, Life Sciences 8,
5.
O.H . LOWRY, N.J . ROSEBROUGH, A .L . FARR and R.J . RANDALL, 193, 265 (1951) .
6.
J.W. TRAVIS, A.C . KEYL and C.A . DRAGSTEDT, 148 (l956) .
7.
K. WISNIEWSKI and A. DANYZ, Biochem. Pharmacol . 15, 669 (l966) .
8.
K. KUBCHINSKY, H. IAHRTZ, H. L$LLWANN and P.A . VAN ZWIEm, Br . J. Chemother. 30, 317 (1967) .
J. Pharmacol. Exp. Therap . 170, part I,
J. Pharmacol.
318 (1969) .
347 (1969) . J. Bi ol . Che
J. Pharmacol . Exp . Th _ erap .
117, --
aarmac .