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Hormonal dependence of actidione (cycloheximide) action
699
SHORT COMMUNICATIONS
SC 93071
Hormonal dependence of actidione (cycloheximide) action Actidione (cycloheximide), an antibiotic from Streptomyces griseus, is a potent inhibitor of protein synthesis. Information concerning the mechanism of action of this antibiotic has been obtained from the study of the inhibition of growth and protein synthesis in yeasts1, 2, in human epidermoid carcinoma 3 and in tissue cultured L-cells4. Less attention has been given so far to the study of the action of actidione at the level of the metazoan organismS, 6, where complicated interrelationships, especially those of hormonal nature, m a y profoundly influence its mode of action. We have obtained clear evidence for a hormone-dependent action of actidione in vivo from experiments originally designed to study its effects on rat-liver regeneration. Male Sprague-Dawley rats (average weight 200 g) were used. The first intraperitoneal injection of actidione (0.2 mg) was given 8 h after a 30-5 ° % hepatectomy and following injections of the same amount were given every 24 h for a period of seven days. Groups of animals were killed each 24 h, the livers homogenized in io vol. of 0.25 M sucrose and the subcellular fractions isolated by differential centrifugation. After spinning down the nuclei (Io min at 600 xg), mitochondria (IO min at 13 ooo xg), the microsomes were sedimented b y centrifuging for 9 ° min at 59 ooo xg. SCHNEIDER'S method 7 was used for determination of RNA and DNA; protein was determined b y weighing the residuum after the last hot 5 % trichloroacetic acid
TABLE
I
THE EFFECT OF ACTIDIONE ON THE IRNA CONTENT OF RAT (EXPTS. I AND 2) AND MOUSE LIVER
(ExvT.
3)
A n i m a l s ( r a t s a n d m i c e ) i n j e c t e d w i t h a d a i l y d o s e o f 0.2 m g a c t i d i o n e . T i m e r e f e r s t o i n t e r v a l between first injection of actidione and sacrifice of the animal. Data refer to single rats and to pooled liver tissue from four control and four actidione injected mice.
Expl. No. Hepatectomy (%)
Actidione Time (rng) (h)
I
37 31 46 45 33 36
-0.2 -o.8 -1. 4
2
--
--
-0,8 -1. 4
168
---
-1. 4
-168
- -
- -
3
24 24 96 96 168 168 96
D N A (rag) R N A (rag) Total
~/licrosomal
RNA DNA (rag) Total
Microsomal
8.5 9.0 lO.7 lO. 7 13.46 I2.O
69.7 79.9 8o.1 96.2 115.o 151. 4
32.0 39.4 32.5 50.4 31.o 42.o
8.2 8.9 8.0 9.0 8.5 12.6
3.7 4.4 2.94 4.7 2.3 3.5
18.6 18.9 22.0 18.6
132.o 183-2 154.o 215
35.0
7.1 9.7 7.0 11. 5
1.88 2-5 1.57 3.00
16.9 17.6
5.8 6.0
7.7
2-65 2.37
2.19 2.53
48.2 34-5 55.0
7.0
Biochim. Biophys. Acta, lO 3 ( 1 9 6 5 ) 6 9 9 - 7 O l
700
SHORT COMMUNICATIONS
extraction, followed by drying with acetone. An increase of RNA, greatest in the microsomal fraction, was consistently found in actidione-treated animals. DNA and protein contents seemed not to be affected b y actidione. The microsomal accumulation of RNA was also observed in livers of actidione-treated intact rats, but it did not occur in mice (male Swiss albino, average weight 30 g) after injecting for 7 days with a daily dose of 0.2 mg actidione. A further difference was disappearance in the rats, but not the mice, of glycogen precipitated b y 2 vol. of alcohol from the acid-soluble liver fraction. The depletion of glycogen in rat liver did not occur when administration of actidione was followed I h later b y intraperitoneal injection of 5 mg hydrocortisone sodium succinate (Upjohn, Kalamazoo, Mich.), which was reported to act as an antidote in actidione poisoning 5. This observation together with the fact that cortical principles are known to promote the conversion of glucose into glycogen in liver s, suggested that adrenal function influences and is influenced by, actidione. Indeed, it was seen (Table II) that the administration of actidione to intact rats leads to an TABLE II THE EFFECT OF ACTIDIONE ON THE R ~ T A CONTENT OF THE ADRENALS IN INTACT ( E x P T S . AND HYPOPHYSECTOMIZED RATS ( E x P T . 3)
I AND 2)
I n t a c t r a t s were injected w i t h a daily dose of o,2 m g actidione a n d / o r 20 u n i t s ACTH for five consecutive days. H y p o p h y s e c t o m i z e d r a t s were injected w i t h a daily dose of o.I mg actidione for 3 days or w i t h 2o u n i t s ACTH for five days. F r o m four h y p o p h y s e c t o m i z e d , aetidione injected rats, only two survived.
Expt. Treatment No.
Number o[ Av. weight o[ Total D N A animals adrenals (rag) (/zg)
Total R N A (/~g)
RNA DN-A
1
Control Actidione ACTH ACTH+ actidione
2 2 2 2
43 62 71 67
403 385 462 4o4
805 1292 1529 168o
2.0 3.35 3.3 ° 4.15
2
Control Actidione
4 4
4° 52
395 385
81o 13o8
2.05 3.39
Control Actidione ACTH
2 2 2
37.7 28.0 52
285 175 290
26o 154 43o
o.91 o.88 1.98
3
Mierosomal R N A
52o
88o
increase of total level of adrenal RNA, especially in the microsomal fraction, quite similar to the effect of ACTH on the adrenal tissueg, 1°. The increase of DNA, which occurs in adrenals after repeated administration of ACTH 9,1°, was blocked by actidione. The administration of actidione to hypophysectomized rats did not stimulate the increase of adrenal RNA, indicating that this effect is ACTH-mediated. A rapid disappearance of ascorbic acid from the adrenal glands, their increase in weight and the increased production of glucocorticoids manifested in the depletion of thymus tissue, were also observed following repeated administration of actidione. When hydrocortisone (5 rag) was given simultaneously with o.2 mg actidione, these effects were not observed. These observations suggested that increased adrenal function protects against actidione poisoning in rats and the greater resistance of mice against actidione Biochim. Biophys. Acta, lO 3 (1965) 699-7Ol
701
SHORT COMMUNICATIONS
poisoning 5 is also adrenal dependent. The data in Table n I show, indeed, that hypophysectomized rats, which cannot respond to administration of actidione b y an ACTH-stimulated adrenal function, are more sensitive to actidione poisoning than the intact rats, while the adrenalectomized rats are even more sensitive than hypophysectomized rats. Table n I also shows that adrenalectomized mice, while still TABLE III EFFECT OF ACTIDIONE ON SURVIVAL RATE OF INTACT, HYPOPHYSI~CTOMIZED AND ADRENALECTOMIZED ANIMALS
Actidione
(rag)
Intact
Hypophysectomized
Adrenalectomized
Number o/ animals
% survival
Number o/ animals
% survival
Number o/ animals
% survival
Rats
o.I 0.2 0"5
4/4 4/4 4/4
IOO IOO IOO
2/4 2/4 0/3
5° 5° o
I/ 4 0/2 o/3
25 o o
Mice
0.5 I.O 2.0
3/3 3/3 3/3
IOO IOO IOO
--
--
I/3 1/3 0/2
33 33 o
--
--
-
-
--
less sensitive than adrenalectomized rats, became sensitive to actidione poisoning. The data here presented demonstrate that in the mammalian organism the mechanism of actidione action is influenced by adrenal secretion. They do not provide yet any insight into the nature of the interaction between the hormone (presumably hydrocortisone) and actidione. The accumulation of RNA in the microsomal fraction of rat liver due to actidione, could be detected also in actidione-treated hypophysectomized rats and could not be counteracted by hydrocortisone. The nature of this effect, which m a y be of significance for the reaction of the cell in contact with an inhibitor of protein synthesis, must remain a subject of further investigation. This investigation was supported by Grant GB-I828 of the National Science Foundation.
Veterans Administration Hospital, San Fernando and Department o/ Biochemistry, University o/ Southern Cali[ornia, Los Angeles, Caii]. (U.S.A.) Fels Research Institute, Temple University School o/ Medicine, Philadelphia, Pa. (U.S'.A.) I 2 3 4 5 6 7 8 9 io
SILVIO F I A L A
EMERICH FIALA
D. I ~ E R R I D G E , J. Gen. Microbiol., 19 (1958) 497M. R. SIEGEL AND H. G. SISLER, Biochim. Biophys. Acta, 87 (1964) 7o; 83. L. L. BENNETT, JR., D. SMITHERS AND C. T. WARD, Biochim. Biophys. zicta, 87 (1964) 60. H. L. ENNIS AND 1~¢[. LUBIN, Federation Proc., 23 (1964) 269. M. E. GREIG AND A. J . GIBBONS, Toxicol. Appl. Pharmacol., I (I959) 598. J. GoRs•i AND M. C. AXMAN, Arch. Biochem. Biophys., lO5 (1964) 517 . W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 203. C. N. H. LONG, B. KATZlN AND E. G. FRY, Endocrinology, 26 (194 o) 3o9 . S. FIALA, E. E. SPROUL AND A. FIALA, J. Biophys. Biochem. Cytol., 2 (1956) 115. S. FIALA, E. E. SPROUL AND A. FIALA, Proc. Soc. Exptl. Biol. Med., 94 (1957) 517.
Received February 5th, 1965 Biochim. Biophys. Acta, lO 3 (1965) 699-7Ol
SHORT COMMUNICATIONS
SC 93071
Hormonal dependence of actidione (cycloheximide) action Actidione (cycloheximide), an antibiotic from Streptomyces griseus, is a potent inhibitor of protein synthesis. Information concerning the mechanism of action of this antibiotic has been obtained from the study of the inhibition of growth and protein synthesis in yeasts1, 2, in human epidermoid carcinoma 3 and in tissue cultured L-cells4. Less attention has been given so far to the study of the action of actidione at the level of the metazoan organismS, 6, where complicated interrelationships, especially those of hormonal nature, m a y profoundly influence its mode of action. We have obtained clear evidence for a hormone-dependent action of actidione in vivo from experiments originally designed to study its effects on rat-liver regeneration. Male Sprague-Dawley rats (average weight 200 g) were used. The first intraperitoneal injection of actidione (0.2 mg) was given 8 h after a 30-5 ° % hepatectomy and following injections of the same amount were given every 24 h for a period of seven days. Groups of animals were killed each 24 h, the livers homogenized in io vol. of 0.25 M sucrose and the subcellular fractions isolated by differential centrifugation. After spinning down the nuclei (Io min at 600 xg), mitochondria (IO min at 13 ooo xg), the microsomes were sedimented b y centrifuging for 9 ° min at 59 ooo xg. SCHNEIDER'S method 7 was used for determination of RNA and DNA; protein was determined b y weighing the residuum after the last hot 5 % trichloroacetic acid
TABLE
I
THE EFFECT OF ACTIDIONE ON THE IRNA CONTENT OF RAT (EXPTS. I AND 2) AND MOUSE LIVER
(ExvT.
3)
A n i m a l s ( r a t s a n d m i c e ) i n j e c t e d w i t h a d a i l y d o s e o f 0.2 m g a c t i d i o n e . T i m e r e f e r s t o i n t e r v a l between first injection of actidione and sacrifice of the animal. Data refer to single rats and to pooled liver tissue from four control and four actidione injected mice.
Expl. No. Hepatectomy (%)
Actidione Time (rng) (h)
I
37 31 46 45 33 36
-0.2 -o.8 -1. 4
2
--
--
-0,8 -1. 4
168
---
-1. 4
-168
- -
- -
3
24 24 96 96 168 168 96
D N A (rag) R N A (rag) Total
~/licrosomal
RNA DNA (rag) Total
Microsomal
8.5 9.0 lO.7 lO. 7 13.46 I2.O
69.7 79.9 8o.1 96.2 115.o 151. 4
32.0 39.4 32.5 50.4 31.o 42.o
8.2 8.9 8.0 9.0 8.5 12.6
3.7 4.4 2.94 4.7 2.3 3.5
18.6 18.9 22.0 18.6
132.o 183-2 154.o 215
35.0
7.1 9.7 7.0 11. 5
1.88 2-5 1.57 3.00
16.9 17.6
5.8 6.0
7.7
2-65 2.37
2.19 2.53
48.2 34-5 55.0
7.0
Biochim. Biophys. Acta, lO 3 ( 1 9 6 5 ) 6 9 9 - 7 O l
700
SHORT COMMUNICATIONS
extraction, followed by drying with acetone. An increase of RNA, greatest in the microsomal fraction, was consistently found in actidione-treated animals. DNA and protein contents seemed not to be affected b y actidione. The microsomal accumulation of RNA was also observed in livers of actidione-treated intact rats, but it did not occur in mice (male Swiss albino, average weight 30 g) after injecting for 7 days with a daily dose of 0.2 mg actidione. A further difference was disappearance in the rats, but not the mice, of glycogen precipitated b y 2 vol. of alcohol from the acid-soluble liver fraction. The depletion of glycogen in rat liver did not occur when administration of actidione was followed I h later b y intraperitoneal injection of 5 mg hydrocortisone sodium succinate (Upjohn, Kalamazoo, Mich.), which was reported to act as an antidote in actidione poisoning 5. This observation together with the fact that cortical principles are known to promote the conversion of glucose into glycogen in liver s, suggested that adrenal function influences and is influenced by, actidione. Indeed, it was seen (Table II) that the administration of actidione to intact rats leads to an TABLE II THE EFFECT OF ACTIDIONE ON THE R ~ T A CONTENT OF THE ADRENALS IN INTACT ( E x P T S . AND HYPOPHYSECTOMIZED RATS ( E x P T . 3)
I AND 2)
I n t a c t r a t s were injected w i t h a daily dose of o,2 m g actidione a n d / o r 20 u n i t s ACTH for five consecutive days. H y p o p h y s e c t o m i z e d r a t s were injected w i t h a daily dose of o.I mg actidione for 3 days or w i t h 2o u n i t s ACTH for five days. F r o m four h y p o p h y s e c t o m i z e d , aetidione injected rats, only two survived.
Expt. Treatment No.
Number o[ Av. weight o[ Total D N A animals adrenals (rag) (/zg)
Total R N A (/~g)
RNA DN-A
1
Control Actidione ACTH ACTH+ actidione
2 2 2 2
43 62 71 67
403 385 462 4o4
805 1292 1529 168o
2.0 3.35 3.3 ° 4.15
2
Control Actidione
4 4
4° 52
395 385
81o 13o8
2.05 3.39
Control Actidione ACTH
2 2 2
37.7 28.0 52
285 175 290
26o 154 43o
o.91 o.88 1.98
3
Mierosomal R N A
52o
88o
increase of total level of adrenal RNA, especially in the microsomal fraction, quite similar to the effect of ACTH on the adrenal tissueg, 1°. The increase of DNA, which occurs in adrenals after repeated administration of ACTH 9,1°, was blocked by actidione. The administration of actidione to hypophysectomized rats did not stimulate the increase of adrenal RNA, indicating that this effect is ACTH-mediated. A rapid disappearance of ascorbic acid from the adrenal glands, their increase in weight and the increased production of glucocorticoids manifested in the depletion of thymus tissue, were also observed following repeated administration of actidione. When hydrocortisone (5 rag) was given simultaneously with o.2 mg actidione, these effects were not observed. These observations suggested that increased adrenal function protects against actidione poisoning in rats and the greater resistance of mice against actidione Biochim. Biophys. Acta, lO 3 (1965) 699-7Ol
701
SHORT COMMUNICATIONS
poisoning 5 is also adrenal dependent. The data in Table n I show, indeed, that hypophysectomized rats, which cannot respond to administration of actidione b y an ACTH-stimulated adrenal function, are more sensitive to actidione poisoning than the intact rats, while the adrenalectomized rats are even more sensitive than hypophysectomized rats. Table n I also shows that adrenalectomized mice, while still TABLE III EFFECT OF ACTIDIONE ON SURVIVAL RATE OF INTACT, HYPOPHYSI~CTOMIZED AND ADRENALECTOMIZED ANIMALS
Actidione
(rag)
Intact
Hypophysectomized
Adrenalectomized
Number o/ animals
% survival
Number o/ animals
% survival
Number o/ animals
% survival
Rats
o.I 0.2 0"5
4/4 4/4 4/4
IOO IOO IOO
2/4 2/4 0/3
5° 5° o
I/ 4 0/2 o/3
25 o o
Mice
0.5 I.O 2.0
3/3 3/3 3/3
IOO IOO IOO
--
--
I/3 1/3 0/2
33 33 o
--
--
-
-
--
less sensitive than adrenalectomized rats, became sensitive to actidione poisoning. The data here presented demonstrate that in the mammalian organism the mechanism of actidione action is influenced by adrenal secretion. They do not provide yet any insight into the nature of the interaction between the hormone (presumably hydrocortisone) and actidione. The accumulation of RNA in the microsomal fraction of rat liver due to actidione, could be detected also in actidione-treated hypophysectomized rats and could not be counteracted by hydrocortisone. The nature of this effect, which m a y be of significance for the reaction of the cell in contact with an inhibitor of protein synthesis, must remain a subject of further investigation. This investigation was supported by Grant GB-I828 of the National Science Foundation.
Veterans Administration Hospital, San Fernando and Department o/ Biochemistry, University o/ Southern Cali[ornia, Los Angeles, Caii]. (U.S.A.) Fels Research Institute, Temple University School o/ Medicine, Philadelphia, Pa. (U.S'.A.) I 2 3 4 5 6 7 8 9 io
SILVIO F I A L A
EMERICH FIALA
D. I ~ E R R I D G E , J. Gen. Microbiol., 19 (1958) 497M. R. SIEGEL AND H. G. SISLER, Biochim. Biophys. Acta, 87 (1964) 7o; 83. L. L. BENNETT, JR., D. SMITHERS AND C. T. WARD, Biochim. Biophys. zicta, 87 (1964) 60. H. L. ENNIS AND 1~¢[. LUBIN, Federation Proc., 23 (1964) 269. M. E. GREIG AND A. J . GIBBONS, Toxicol. Appl. Pharmacol., I (I959) 598. J. GoRs•i AND M. C. AXMAN, Arch. Biochem. Biophys., lO5 (1964) 517 . W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 203. C. N. H. LONG, B. KATZlN AND E. G. FRY, Endocrinology, 26 (194 o) 3o9 . S. FIALA, E. E. SPROUL AND A. FIALA, J. Biophys. Biochem. Cytol., 2 (1956) 115. S. FIALA, E. E. SPROUL AND A. FIALA, Proc. Soc. Exptl. Biol. Med., 94 (1957) 517.
Received February 5th, 1965 Biochim. Biophys. Acta, lO 3 (1965) 699-7Ol