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Thyroxine and intestinal mitochondria
Comp. Biochem. Physiol., 1965, Vol. 15, pp. 263 to 266. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION THYROXINE AND INTESTINAL MITOCHONDRIA P. J. STANBURY School of Biological Sciences, Zoology Building, University of Sydney, New South Wales, Australia • (Rtc~ved
29 D e c e m b e r 1964)
A b s t r a c t - - 1 . Hepatic mitochondria of rats, guinea pigs, chicken, toads and
earp swell in 1 x 10-s-1 x 10-~M thyroxine as do the intestinal mitochondria of guinea pigs and toads. 2. The intestinal mitochondria of rats appear neither to swell nor to contract after isolation in Trisbuffered sucrose. 3. The intestinal mitochondria of chicken and carp swell in 1 x 10-aM thyroxine; but in 1 x 10-4 and 1 x 10-SM thyroxine the intestinal mitochondria of these animals appear to swell less than they do spontaneously. THE swelling in various concentrations of thyroxine of the isolated intestinal mitochondria of the rat, guinea-pig, chicken, toad and carp is reported below. Mitochondria isolated from the intestine of the rat did not appear to swell either spontaneously or in the presence of up to 1 x 10-SM thyroxine (Fig. 1). Stanbury (1961) has shown that when the intestinal cells of the rat are homogenized an inhibitor of cytochrome oxidase is released which contains a high proportion of oleic acid. This inhibitor is analogous to Lehninger's "/5-factor", a mixture of fatty acids which causes mitochondria to swell, and which is released in greater amounts upon the addition of thyroxine (Lehninger & Remmert, 1959). I suggest that the reason why the intestinal mitochondria of the rat did not appear to swell is that the fatty acid released during the preparation of the mitochondria causes them to swell so much that further swelling is impossible even upon the addition of thyroxine. The behaviour of the other mitochondria examined is shown in Fig. 2. All the hepatic mitochondria and the intestinal mitochondria of the guinea-pigs and toads swelled in thyroxine. Lehninger (1962; 1963) has suggested that the enzymes associated with the mitochondrial membrane are responsible for the transformation of oxidationreduction energy not only into phosphate-bond energy, but also into mechanical energy. According to the state of the intermediates involved in oxidative phosphorylation the "mechano-enzymes" change their shape or arrangement within the membrane and so alter the permeability of the mitochondrion. The energy 263
P.J. STANBURY
264
obtained from electron transport and oxidative phosphorylation is said to be held in an intermediate ~ P compound. If thyroxine inhibits transfer of this energy to ADP, then an accumulation of intermediate ~ P might be expected. This would lead usually, as in the results above, to the swelling of the mitochondrion.
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10 15 20 MINUTES FIG. 1. T h e effect of thyroxine on the swelling of mitochondria isolated from the liver (L) and the intestine (I) of the rat. L s = spontaneous swelling; Lb = swelling in the presence of 1 x 10 -~ M thyroxine ; La = swelling in the presence of 1 x 10 -a M thyroxine. T h e intestinal mitochondria did not swell either spontaneously or in the presence of up to 10 -a M t h y r o x i n e . T h e mitochondria were isolated by differential centrifugation in 0"3 M sucrose containing 0-01 M Tris (hydroxymethylaminom e t h a n e ) - H C l buffer, p H 7'4. Aliquots of the resuspended mitochondria were added to cuvettes containing buffered sucrose (the control) and various concentrations of thyroxine in buffered sucrose. Mitochondrial swelling was estimated by recording changes in optical density at 520 m/~.
The intestinal mitochondria of the chicken and the carp appeared to respond differently. Under the experimental conditions the lower concentrations of thyroxine seemed to confer some protection against swelling to these mitochondria. In some circumstances the small amount of intermediate ~ P accumulated under the influence of the lower concentrations of thyroxine may be removed by reactions other than the A T P and swelling ones. For example, the nitrogen retention brought about by thyroxine in fish may indicate that in some circumstances the accumulated intermediate ~ P is channelled preferentially into protein synthesis rather than into the mechanical changes of swelling.
THYROXINE AND INT~TINAL MITOCHONDRIA
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FZG. 2. T h e effect of thyroxine on the swelling of mitochondria isolated from the liver (L) and the intestine (I) of the guinea-pig, chicken, toad and carp. T h e subscript S denotes spontaneous swelling; the subscripts 7, 5, 4 and 3 denote swelling in the presence of 1 x 10 -7, 1 x 10 -s, 1 x 10 -4 and 1 x 10 -3 M thyroxine respectively.
266
P . J . STANBURY
It seems unlikely that the " p r o t e c t i o n - f r o m - s w e l l i n g " effect of thyroxine is directly connected with the evolutionara' state of the animal as m i t o c h o n d r i a isolated f r o m the flight muscles of the bee do not show this effect. REFERENCES LEHNINGERA. L. (1962) Water uptake and extrusion by mitochondria in relation to oxidative phosphorylation. Physiol. Rev. 42, 467-517. LEHNINGERA. L. (1963) Respiration-linked mechanochemical changes in mitochondria. In Horizons in Biochemistry (Edited by KASHA M. & PULMAN B.) pp. 421-435. Academic Press, New York. LEHNINGER A. L. & REMMERT L. F. (1959) An endogenous uncoupling and swelling agent in liver mitochondria and its enz.~Tnatic formation. J. Biol. Chem. 234, 2459-2464. STANBURYP. J. (1961) Comparison of the mitochondria of the small intestine of vertebrates. Nature, Lond. 192, 67.
SHORT COMMUNICATION THYROXINE AND INTESTINAL MITOCHONDRIA P. J. STANBURY School of Biological Sciences, Zoology Building, University of Sydney, New South Wales, Australia • (Rtc~ved
29 D e c e m b e r 1964)
A b s t r a c t - - 1 . Hepatic mitochondria of rats, guinea pigs, chicken, toads and
earp swell in 1 x 10-s-1 x 10-~M thyroxine as do the intestinal mitochondria of guinea pigs and toads. 2. The intestinal mitochondria of rats appear neither to swell nor to contract after isolation in Trisbuffered sucrose. 3. The intestinal mitochondria of chicken and carp swell in 1 x 10-aM thyroxine; but in 1 x 10-4 and 1 x 10-SM thyroxine the intestinal mitochondria of these animals appear to swell less than they do spontaneously. THE swelling in various concentrations of thyroxine of the isolated intestinal mitochondria of the rat, guinea-pig, chicken, toad and carp is reported below. Mitochondria isolated from the intestine of the rat did not appear to swell either spontaneously or in the presence of up to 1 x 10-SM thyroxine (Fig. 1). Stanbury (1961) has shown that when the intestinal cells of the rat are homogenized an inhibitor of cytochrome oxidase is released which contains a high proportion of oleic acid. This inhibitor is analogous to Lehninger's "/5-factor", a mixture of fatty acids which causes mitochondria to swell, and which is released in greater amounts upon the addition of thyroxine (Lehninger & Remmert, 1959). I suggest that the reason why the intestinal mitochondria of the rat did not appear to swell is that the fatty acid released during the preparation of the mitochondria causes them to swell so much that further swelling is impossible even upon the addition of thyroxine. The behaviour of the other mitochondria examined is shown in Fig. 2. All the hepatic mitochondria and the intestinal mitochondria of the guinea-pigs and toads swelled in thyroxine. Lehninger (1962; 1963) has suggested that the enzymes associated with the mitochondrial membrane are responsible for the transformation of oxidationreduction energy not only into phosphate-bond energy, but also into mechanical energy. According to the state of the intermediates involved in oxidative phosphorylation the "mechano-enzymes" change their shape or arrangement within the membrane and so alter the permeability of the mitochondrion. The energy 263
P.J. STANBURY
264
obtained from electron transport and oxidative phosphorylation is said to be held in an intermediate ~ P compound. If thyroxine inhibits transfer of this energy to ADP, then an accumulation of intermediate ~ P might be expected. This would lead usually, as in the results above, to the swelling of the mitochondrion.
0"61
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0.40
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0.25
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5
10 15 20 MINUTES FIG. 1. T h e effect of thyroxine on the swelling of mitochondria isolated from the liver (L) and the intestine (I) of the rat. L s = spontaneous swelling; Lb = swelling in the presence of 1 x 10 -~ M thyroxine ; La = swelling in the presence of 1 x 10 -a M thyroxine. T h e intestinal mitochondria did not swell either spontaneously or in the presence of up to 10 -a M t h y r o x i n e . T h e mitochondria were isolated by differential centrifugation in 0"3 M sucrose containing 0-01 M Tris (hydroxymethylaminom e t h a n e ) - H C l buffer, p H 7'4. Aliquots of the resuspended mitochondria were added to cuvettes containing buffered sucrose (the control) and various concentrations of thyroxine in buffered sucrose. Mitochondrial swelling was estimated by recording changes in optical density at 520 m/~.
The intestinal mitochondria of the chicken and the carp appeared to respond differently. Under the experimental conditions the lower concentrations of thyroxine seemed to confer some protection against swelling to these mitochondria. In some circumstances the small amount of intermediate ~ P accumulated under the influence of the lower concentrations of thyroxine may be removed by reactions other than the A T P and swelling ones. For example, the nitrogen retention brought about by thyroxine in fish may indicate that in some circumstances the accumulated intermediate ~ P is channelled preferentially into protein synthesis rather than into the mechanical changes of swelling.
THYROXINE AND INT~TINAL MITOCHONDRIA
0.65[
265
o-6s
k~,,,,.,"
GUII',EA PIG
0-60 l - k ' , . . _ - -
"
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LS
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15 20 CARP
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10
15
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20
0
-L3
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5
10
15
20
M~NUTES
FZG. 2. T h e effect of thyroxine on the swelling of mitochondria isolated from the liver (L) and the intestine (I) of the guinea-pig, chicken, toad and carp. T h e subscript S denotes spontaneous swelling; the subscripts 7, 5, 4 and 3 denote swelling in the presence of 1 x 10 -7, 1 x 10 -s, 1 x 10 -4 and 1 x 10 -3 M thyroxine respectively.
266
P . J . STANBURY
It seems unlikely that the " p r o t e c t i o n - f r o m - s w e l l i n g " effect of thyroxine is directly connected with the evolutionara' state of the animal as m i t o c h o n d r i a isolated f r o m the flight muscles of the bee do not show this effect. REFERENCES LEHNINGERA. L. (1962) Water uptake and extrusion by mitochondria in relation to oxidative phosphorylation. Physiol. Rev. 42, 467-517. LEHNINGERA. L. (1963) Respiration-linked mechanochemical changes in mitochondria. In Horizons in Biochemistry (Edited by KASHA M. & PULMAN B.) pp. 421-435. Academic Press, New York. LEHNINGER A. L. & REMMERT L. F. (1959) An endogenous uncoupling and swelling agent in liver mitochondria and its enz.~Tnatic formation. J. Biol. Chem. 234, 2459-2464. STANBURYP. J. (1961) Comparison of the mitochondria of the small intestine of vertebrates. Nature, Lond. 192, 67.