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Hypophysectomy, ACTH1–10 andin vitro protein synthesis in rat brain stem slices
Brain Research, 81 (1974) 571-575
571
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Hypophysectomy, ACTHt.lo and in vitro protein synthesis in rat brain stem slices*
M. E. A. REITH, P. SCHOTMAN AND W. H. GISPEN
Division of Molecular Neurobiology, Rudolf Magnus Institute/'or Pharmacology and Laboratory of Physiological Chemistry, Medical Faculty, Institute of Molecular Biology, University of Utrecht Utrecht (The Netherlands)
(Accepted August 27th, 1974)
Hypophysectomy impairs acquisition o f conditioned avoidance behavior in the shuttle-box. Treatment with A C T H or N-terminal fragments o f A C T H (ACTHI-lo and ACTH4-10) restores avoidance performance of hypophysectomized rats towards normal 4. The behavioral effect of these neuropeptides is caused by a direct action on central nervous structures, presumably the posterior thalamus (nucleus parafascicularis) 21. Neurochemical studies have shown that hypophysectomy decreases the incorporation of [aH]leucine into rat brain stem protein in vivo, measured 5 rain after injection of the precursor directly into the diencephalon2L Treatment of hypophysectomized rats with the synthetic N-terminal fragment of ACTH, ACTHI-10-7-L-Phe enhances this incorporation 17. Furthermore, data obtained from experiments on polyacrylamide gel electrophoresis of sequential extracted fractions of brain stem proteins suggest a rather general effect on protein labeling, i.e. a general decrease after hypophysectomy and an overall increase after treatment with ACTH~-~0-7-L-PhelL These experiments, and others on precursor pool metabolism 13,14,17,19, suggest that the observed alterations in leucine incorporation may be explained by a direct influence of the neuropeptide on the mechanism o f protein synthesis. To substantiate this hypothesis, in vitro incorporation of radioactive leucine into proteins in slices from the posterior thalamus was studied. In vitro experiments offer the advantages o f a controlled supply of the radioactive precursor and the possibility of measuring the uptake of the precursor into the tissue. The present communication reports on the effects of hypophysectomy and in vitro addition of ACTHI-10-7-L-Phe in this system. Male Wistar rats, weighing approximately 110-120 g, were used. Hypophysectomy was performed via the transauricular route under anesthesia with ether. Sham-operated rats were o f the same weight. Loss of body weight, adrenal atrophy and macroscopic inspection of the sella turcica were used as indications that hypo* Reprint requests should be sent to: M. E. A. Reith, Division of Molecular Neurobiology, Institute of Molecular Biology, University of Utrecht, Transitorium 3, Padualaan 8, De Uithof, Utrecht, The Netherlands.
572
SHORT COMMUNICATIONS
physectomy had been performed successfully. Three weeks after the operation the animals were sacrificed by decapitation. The brain was rapidly removed and cerebellum and medulla oblongata were cut off. A slice of about 1 mm thickness was taken out of the brain at about 3400 # m from the frontal zero plane 8, using a slicer equipped with razor blades by which frontal slices could be made. The slice so obtained was placed with the caudal site upwards and subjected to further dissection by cutting off all tissue left and right from lines between the fasciculus medialis prosencephali and crus cerebri, and above and under lines through the recessus pinealis and fasciculus mammillothalamicus. In this way a brain stem slice containing posterior thalamic tissue including the nucleus parafascicularis was obtained, weighing approximately 25 mg. The dissection took merely a couple of minutes after which preincubation was started. The slice was preincubated for 30 min at 37 °C in 2 illl Krebs phosphate medium 7 (mM: NaCI, 124; KCI, 6.7; MgSO4, 1.3: CaCle, 0.75; Na2HPO4, 20; glucose, 10; adjusted with 2 M HCI to pH 7.4; equilibrated with pure 02) prior to addition of about l # C i of L-[U-14C]leucine (specific activity 311 and 348 mCi/ mmole; Radiochemical Centre, Amersham, England) and then incubated for 10, 30 or 60 min. At the end of the incubation the slice was blotted with filter paper and washed 3 times with 5 ml of ice-cold medium containing unlabeled leucine (10 mM), each time for 5 rain, to remove extracellular amino acid. The intracellular amino acid is presumed to be preserved under these conditions 2,6.9,1°,'~°. The radioactivity present in the 15 min cold-wash will be referred to as 'extracellular' and the radioactivity remaining in the tissue as 'intracellular'. After the cold-wash, the tissue was homogenized in 1.5 ml of the same medium. In order to measure the incorporation of [14C]leucine into proteins, samples of the homogenate plus 5 mg albumin carrier protein were treated with an equal volume of 1 M perchloric acid at 0 °C and centrifuged. Precipitates were washed 3 times with 0.5 M perchloric acid, transferred to scintillation vials and solubilized using approximately 1 ml Soluene (Packard sample solubilizer) per pellet. Identical results were obtained after washing the precipitates with 0.5 M perchloric acid containing 10 m M leucine, indicating that non-specific absorption of radioactive leucine to precipitable material was unlikely. The uptake of radioactive leucine into the extracellular and intracellular space was measured by counting samples of the pooled cold-washes and the homogenate. Protein content of the homogenate was determined by the method of Lowry et al. 11. Fig. 1 shows the time course of the uptake of [14C]leucine into the slice and the incorporation into proteins. Similar results were obtained when the data were expressed as either per mg tissue or per nag protein. The precursor entered the extracellular space of the tissue very rapidly, the level already being high after 10 min of" incubation (Fig. 1A). A further rise in extracellular uptake was observed, presumably due to tissue swelling 3,'~. However, the concentration of precursor in the extracellular space remained, most likely, at a constant level almost throughout the total incubation period 12. The amount of intracellular label increased gradually during the 60 rain of incubation: the uptake started to level off after about 30 rain (Fig. IA). The incorpo-
SHORT COMMUNICATIONS
573
¢n 800 ¢n
3000
B
"-. 600 E
E
~- 2000
o.
~oo 1000
200
2'0
'
,'0
'
'
6'0
201
L
/,i0
i
610
time of incubation in min.
t i m e o f i n c u b a t i o n in min.
Fig. 1. The in vitro uptake of [U-14C]leucine into intracellular and extracellular space and incorporation into proteins of rat brain stem slices. Slices were preincubated for 30 min before adding 1/~Ci [14C]leucine, and incubation continued for the times indicated. At the end of the incubation, slices were washed 3 times with ice-cold medium containing unlabeled leucine, each time for 5 min. A: • O, uptake of [14C]ieucine by the intracellular space; © . . . . O, uptake of [z4C]leucine by the extracellular tissue space. B: incorporation of [~4C]leucine into proteins. Results are the mean ± S.E.M. (vertical bar) for 5 or 6 slices.
r a t i o n o f r a d i o a c t i v e leucine into p r o t e i n increased linearly u p to at least 60 min o f i n c u b a t i o n (Fig. 1B). A n i n c o r p o r a t i o n p e r i o d o f 30 min was used to d e t e r m i n e the effects o f h y p o p h y s e c t o m y a n d ACTHI-z0-7-L-Phe, the i n c o r p o r a t i o n being linear for this p e r i o d o f time. T a b l e I presents the results o b t a i n e d with slices o f h y p o p h y s e c t o m i z e d a n d s h a m - o p e r a t e d animals. T h r e e weeks after r e m o v a l o f the p i t u i t a r y , a m a r k e d decrease in i n c o r p o r a t i o n was found, a m o u n t i n g to a p p r o x i m a t e l y 30 ~o. This a l t e r a t i o n was n o t due to differences in the p e n e t r a t i o n o f the p r e c u r s o r into the extracellular space n o r to the u p t a k e into cells (Table I). ACTHz-10-7-L-Phe, p r e s e n t d u r i n g p r e i n c u b a t i o n a n d the i n c o r p o r a t i o n p e r i o d , was f o u n d to s t i m u l a t e the rate o f i n c o r p o r a t i o n .
TABLE I EFFECT OF HYPOPHYSECTOMY ON UPTAKE AND INCORPORATION in vitro OF [U-14C]LEUCINE IN RAT BRAIN STEM SLICES*
N = number of rats (incubations). Results given are mean ± S.E.M. N
Hypophysectomized Sham-operated Difference in ~ (sham = 100%)
10 10
Radioactivity ( disint./ min/ mg tissue) 'Extracellular'
'lntracellular'
Protein
1861 ± 61 1890 ± 139
2453 ± 135 2686 ± 146
211 ± 20 300 ± 34 - - 30**
* Preincubation during 30 min and incorporation during 30 min. P < 0.05 (Student t-test).
**
574
SHORI
COMMUNICA lIONS
TABLE I1 EFFECT OF
ACTH1 lo-7-L-Phe ON
UPTAKE AND INCORPORATION ill vitro OF [U-14C]EEUCINE IN BRAIN
STEM SLICES OF HYPOPHYSECTOMIZED RATS*
N = number of rats (incubations). Results are means ±: S.E.M.
Control ACTHx_lo-7-L-Phe 10-8 M 5 × 10 7M 10 5M
N
'Extracellular'
"lntracellular'
Protein
% D(ff'erence
17
1925 ± 116
2030 ± 104
271 ___:14
--
10 10 17
2172 ± 59 1788± 101 1796± 95
2338 ± 78 1913 ± 90 2030± 75
307 ± 20 363i26 3767~39
-t- 13 -t- 34** ~ 39**
* Preincubation during 30 rain and incorporation during 30 min. * * P < 0.05 (Student t-test).
In Table II the combined data of two experiments are presented, indicating a significant increase of 39 and 34~o at a peptide concentration of l0 -a and 5 × l0 -7 M respectively. At a concentration of l0 -s M a tendency of + 13 ~ (5 ~ < P < 1 0 ~ ) was observed. Since the presence of ACTHI-10 in the medium seemed not to alter the uptake of label into the slice, the observed influence on incorporation cannot be accounted for by an effect on precursor uptake into the slice. The observed changes in in vitro incorporation of [t4C]leucine into brain stem proteins as a result of hypophysectomy and in in vitro addition of ACTHI-10-7-L-Phe are in agreement with previous observations in in vivo studies with [aH]leucine. Under these conditions hypophysectomy caused a decrease in incorporation of about 23 ~ , whilst treatment of hypophysectomized rats with ACTH~-10-7-L-Phe had a stimulatory effect amounting to approximately 28 ~o17,1s; in vitro similar effects of hypophysectomy and the peptide (in a concentration of 5 × l0 -7 and l0 -5 M) on leucine incorporation were found (Tables I and II). In the present study no changes were observed in precursor uptake during the incorporation period used; in addition, conversion of [14C]leucine to other metabolites seems negligible 1. Therefore, changes in leucine incorporation as found after hypophysectomy and ACTHI-10-7-L-Phe may reflect changes in protein synthesis. The effect of ACTH1--10 on brain protein synthesis is consistent with the results reported by Rudman et al. 16 on the effects of melanotropic peptides, which share the sequence ACTH4-10, on protein synthesis in mouse brain. Their observations on the penetration of a-aminoisobutyric acid, and the accumulation, the concentration and incorporation of various amino acids also indicate a stimulatory effect of these peptides on protein synthesis. The present in vitro study provides further evidence for a direct action of A C T H I - l o on central nervous structures. The authors wish to thank Mr. J. Brakkee for excellent biotechnical assistance. The supply of ACTHI-10 by Organon, Oss is gratefully acknowledged. Part of this research was sponsored by a grant from F U N G O - Z W O 13-31-03A, The Netherlands.
SHORT COMMUNICATIONS
575
1 BLASBERG, R., AND LAJTHA, A., Substrate specificity of steady-state amino acid transport in mouse brain slices, Arch. Biochem. Biophys., 112 (1965) 361-377. 2 COHEN, S. R., BLASBERG,R., LEVI, G., AND LA~THA, A., Compartmentation of the inulin space in mouse brain slices, J. Neurochem., 15 (1968) 707-720. 3 COHEN,S. R., STAMPLEMAN,P. F., AND LAJTHA,A., The temperature-dependent compartmentation of the 'extracellular space' in mouse brain slices as revealed by the markers: inulin, sucrose, D-mannitol, o-sorbitol and sulfate, Brain Research, 21 (1970) 419-434. 4 DE WILD, D., Pituitary-adrenal system hormones and behavior. In F. O. SCHMITT AND F. G. WORDEN(Eds.), The Neurosciences, Third Study Program, Rockefeller University Press, New York, 1974, pp. 653-666. 5 ELLIOT, K. A. C., The use of brain slices. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 11, Plenum Press, New York, 1969, pp. 103-114. 6 HIDER, R. C., FERN, E. B., AND LONDON, D. R., Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis, Biochem. J., 121 (1971) 817-827. 7 JONES, C. T., AND BANKS, P., The effect of electrical stimulation on the incorporation of L-[14C]valine into the protein of chopped tissue from guinea-pig cerebral cortex, Biochem. J., 118 (1970) 791-800. 8 KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, a Stereotaxic Atlas, Krieger, Huntington, 1963, 25 pp. 9 KosTYO, J. L., Rapid effects of growth hormone on amino acid transport and protein synthesis, Ann. N.Y. Acad. Sci., 148 (1968) 389-407. 10 LEVI, G., CHERAYIL,A., AND LAJTHA,A., Cerebral amino acid transport in vitro. III. heterogeneity of exit, J. Neurochem., 12 (1965) 757-770. 11 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 PAPP1US,H. M., AND ELLIOTT, K. A. C., Water distribution in incubated slices of brain and other tissues, Canad. J. Biochem., 34 (1956) 1007-1022. 13 REITH, M. E. A., SCHOTMAN,P., AND GlSPEN, W. H., Brain protein metabolism and hypophysectomy, 1RCS Med. Sci., (73-3) 3-10-6. 14 REtTH, M. E. A., GISPEN, W. H., AND SCHOTMAN,P., Hypophysectomy and metabolism of brain proteins. In E. GENAZZANIAND H. HERKEN(Eds.), Central Nervous System, Studies on Metabolic Regulation and Function, Springer, Berlin, 1974, pp. 236-240. 15 REITH, M. E. A., GISPEN, W. H., AND SCHOTMAN,P., in preparation. 16 RUDMAN, D., SCOTT, J. W., DEE RIO, A. E., HOUSER, O. H., AND SHEEN, S., Effect of melanotropic peptides on protein synthesis in mouse brain, Amer. J. Physiol., 226 (1974) 687-692. 17 SCHOTMAN,P., GISPEN, W. H., JANSZ, H. S., AND DE WILD, D., Effects of A C T H analogues on macromolecule metabolism in the brain stem of hypophysectomized rats, Brain Research, 46 (1972) 349-362. 18 SCHOTMAN,P., AND GISPEN, W. H., Analogues of ACTH, conditioned avoidance behaviour and metabolism of macromolecules in brain of rat. In E. GENAZZANIAND H. HERKEN(Eds.), Central Nervous System, Studies on Metabolic Regulation and Function, Springer, Berlin, 1974, pp. 231-235. 19 SCHOTMAN, P., GIPON, L., AND GISPEN, W. H., Conversion of [4,5-aH]leucine into aHaO and tritiated metabolites in rat brain tissue. Comparison of a peripheral and intracranial route of administration, Brain Research, 70 (1974) 377-380. 20 VAN VENROOY, W. J., POORT, C., KRAMER, M. F., AND JANSEN, M., Relationship between extracellular amino acids and proteins synthesis in vitro in the rat pancreas, Europ. J. Biochem., 30 (1972) 427-433. 21 VAN WIEMERSMAGREIDANUS,TJ. B., AND DE WILD, D., Effects of systemic and intracerebral administration of two opposite acting ACTH-related peptides on extinction of conditioned avoidance behavior, Neuroendocrinology, 7 (1971) 188-196. 22 VERSTEEG, D. H. G., GISPEN, W. H., SCHOTMAN,P., WITTER,A., AND DE WILD, D., Hypophysectomy and rat brain metabolism: effects of synthetic A C T H analogs. In E. COSTA,L. L. IVERSEN AND R. PAOLETTI(Eds.), Studies of Neurotransmitters at the Synaptic Level, Advanc. Biochem. Psychopharmacology, Vok 6, Raven Press, New York, 1972, pp. 219-239.
571
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Hypophysectomy, ACTHt.lo and in vitro protein synthesis in rat brain stem slices*
M. E. A. REITH, P. SCHOTMAN AND W. H. GISPEN
Division of Molecular Neurobiology, Rudolf Magnus Institute/'or Pharmacology and Laboratory of Physiological Chemistry, Medical Faculty, Institute of Molecular Biology, University of Utrecht Utrecht (The Netherlands)
(Accepted August 27th, 1974)
Hypophysectomy impairs acquisition o f conditioned avoidance behavior in the shuttle-box. Treatment with A C T H or N-terminal fragments o f A C T H (ACTHI-lo and ACTH4-10) restores avoidance performance of hypophysectomized rats towards normal 4. The behavioral effect of these neuropeptides is caused by a direct action on central nervous structures, presumably the posterior thalamus (nucleus parafascicularis) 21. Neurochemical studies have shown that hypophysectomy decreases the incorporation of [aH]leucine into rat brain stem protein in vivo, measured 5 rain after injection of the precursor directly into the diencephalon2L Treatment of hypophysectomized rats with the synthetic N-terminal fragment of ACTH, ACTHI-10-7-L-Phe enhances this incorporation 17. Furthermore, data obtained from experiments on polyacrylamide gel electrophoresis of sequential extracted fractions of brain stem proteins suggest a rather general effect on protein labeling, i.e. a general decrease after hypophysectomy and an overall increase after treatment with ACTH~-~0-7-L-PhelL These experiments, and others on precursor pool metabolism 13,14,17,19, suggest that the observed alterations in leucine incorporation may be explained by a direct influence of the neuropeptide on the mechanism o f protein synthesis. To substantiate this hypothesis, in vitro incorporation of radioactive leucine into proteins in slices from the posterior thalamus was studied. In vitro experiments offer the advantages o f a controlled supply of the radioactive precursor and the possibility of measuring the uptake of the precursor into the tissue. The present communication reports on the effects of hypophysectomy and in vitro addition of ACTHI-10-7-L-Phe in this system. Male Wistar rats, weighing approximately 110-120 g, were used. Hypophysectomy was performed via the transauricular route under anesthesia with ether. Sham-operated rats were o f the same weight. Loss of body weight, adrenal atrophy and macroscopic inspection of the sella turcica were used as indications that hypo* Reprint requests should be sent to: M. E. A. Reith, Division of Molecular Neurobiology, Institute of Molecular Biology, University of Utrecht, Transitorium 3, Padualaan 8, De Uithof, Utrecht, The Netherlands.
572
SHORT COMMUNICATIONS
physectomy had been performed successfully. Three weeks after the operation the animals were sacrificed by decapitation. The brain was rapidly removed and cerebellum and medulla oblongata were cut off. A slice of about 1 mm thickness was taken out of the brain at about 3400 # m from the frontal zero plane 8, using a slicer equipped with razor blades by which frontal slices could be made. The slice so obtained was placed with the caudal site upwards and subjected to further dissection by cutting off all tissue left and right from lines between the fasciculus medialis prosencephali and crus cerebri, and above and under lines through the recessus pinealis and fasciculus mammillothalamicus. In this way a brain stem slice containing posterior thalamic tissue including the nucleus parafascicularis was obtained, weighing approximately 25 mg. The dissection took merely a couple of minutes after which preincubation was started. The slice was preincubated for 30 min at 37 °C in 2 illl Krebs phosphate medium 7 (mM: NaCI, 124; KCI, 6.7; MgSO4, 1.3: CaCle, 0.75; Na2HPO4, 20; glucose, 10; adjusted with 2 M HCI to pH 7.4; equilibrated with pure 02) prior to addition of about l # C i of L-[U-14C]leucine (specific activity 311 and 348 mCi/ mmole; Radiochemical Centre, Amersham, England) and then incubated for 10, 30 or 60 min. At the end of the incubation the slice was blotted with filter paper and washed 3 times with 5 ml of ice-cold medium containing unlabeled leucine (10 mM), each time for 5 rain, to remove extracellular amino acid. The intracellular amino acid is presumed to be preserved under these conditions 2,6.9,1°,'~°. The radioactivity present in the 15 min cold-wash will be referred to as 'extracellular' and the radioactivity remaining in the tissue as 'intracellular'. After the cold-wash, the tissue was homogenized in 1.5 ml of the same medium. In order to measure the incorporation of [14C]leucine into proteins, samples of the homogenate plus 5 mg albumin carrier protein were treated with an equal volume of 1 M perchloric acid at 0 °C and centrifuged. Precipitates were washed 3 times with 0.5 M perchloric acid, transferred to scintillation vials and solubilized using approximately 1 ml Soluene (Packard sample solubilizer) per pellet. Identical results were obtained after washing the precipitates with 0.5 M perchloric acid containing 10 m M leucine, indicating that non-specific absorption of radioactive leucine to precipitable material was unlikely. The uptake of radioactive leucine into the extracellular and intracellular space was measured by counting samples of the pooled cold-washes and the homogenate. Protein content of the homogenate was determined by the method of Lowry et al. 11. Fig. 1 shows the time course of the uptake of [14C]leucine into the slice and the incorporation into proteins. Similar results were obtained when the data were expressed as either per mg tissue or per nag protein. The precursor entered the extracellular space of the tissue very rapidly, the level already being high after 10 min of" incubation (Fig. 1A). A further rise in extracellular uptake was observed, presumably due to tissue swelling 3,'~. However, the concentration of precursor in the extracellular space remained, most likely, at a constant level almost throughout the total incubation period 12. The amount of intracellular label increased gradually during the 60 rain of incubation: the uptake started to level off after about 30 rain (Fig. IA). The incorpo-
SHORT COMMUNICATIONS
573
¢n 800 ¢n
3000
B
"-. 600 E
E
~- 2000
o.
~oo 1000
200
2'0
'
,'0
'
'
6'0
201
L
/,i0
i
610
time of incubation in min.
t i m e o f i n c u b a t i o n in min.
Fig. 1. The in vitro uptake of [U-14C]leucine into intracellular and extracellular space and incorporation into proteins of rat brain stem slices. Slices were preincubated for 30 min before adding 1/~Ci [14C]leucine, and incubation continued for the times indicated. At the end of the incubation, slices were washed 3 times with ice-cold medium containing unlabeled leucine, each time for 5 min. A: • O, uptake of [14C]ieucine by the intracellular space; © . . . . O, uptake of [z4C]leucine by the extracellular tissue space. B: incorporation of [~4C]leucine into proteins. Results are the mean ± S.E.M. (vertical bar) for 5 or 6 slices.
r a t i o n o f r a d i o a c t i v e leucine into p r o t e i n increased linearly u p to at least 60 min o f i n c u b a t i o n (Fig. 1B). A n i n c o r p o r a t i o n p e r i o d o f 30 min was used to d e t e r m i n e the effects o f h y p o p h y s e c t o m y a n d ACTHI-z0-7-L-Phe, the i n c o r p o r a t i o n being linear for this p e r i o d o f time. T a b l e I presents the results o b t a i n e d with slices o f h y p o p h y s e c t o m i z e d a n d s h a m - o p e r a t e d animals. T h r e e weeks after r e m o v a l o f the p i t u i t a r y , a m a r k e d decrease in i n c o r p o r a t i o n was found, a m o u n t i n g to a p p r o x i m a t e l y 30 ~o. This a l t e r a t i o n was n o t due to differences in the p e n e t r a t i o n o f the p r e c u r s o r into the extracellular space n o r to the u p t a k e into cells (Table I). ACTHz-10-7-L-Phe, p r e s e n t d u r i n g p r e i n c u b a t i o n a n d the i n c o r p o r a t i o n p e r i o d , was f o u n d to s t i m u l a t e the rate o f i n c o r p o r a t i o n .
TABLE I EFFECT OF HYPOPHYSECTOMY ON UPTAKE AND INCORPORATION in vitro OF [U-14C]LEUCINE IN RAT BRAIN STEM SLICES*
N = number of rats (incubations). Results given are mean ± S.E.M. N
Hypophysectomized Sham-operated Difference in ~ (sham = 100%)
10 10
Radioactivity ( disint./ min/ mg tissue) 'Extracellular'
'lntracellular'
Protein
1861 ± 61 1890 ± 139
2453 ± 135 2686 ± 146
211 ± 20 300 ± 34 - - 30**
* Preincubation during 30 min and incorporation during 30 min. P < 0.05 (Student t-test).
**
574
SHORI
COMMUNICA lIONS
TABLE I1 EFFECT OF
ACTH1 lo-7-L-Phe ON
UPTAKE AND INCORPORATION ill vitro OF [U-14C]EEUCINE IN BRAIN
STEM SLICES OF HYPOPHYSECTOMIZED RATS*
N = number of rats (incubations). Results are means ±: S.E.M.
Control ACTHx_lo-7-L-Phe 10-8 M 5 × 10 7M 10 5M
N
'Extracellular'
"lntracellular'
Protein
% D(ff'erence
17
1925 ± 116
2030 ± 104
271 ___:14
--
10 10 17
2172 ± 59 1788± 101 1796± 95
2338 ± 78 1913 ± 90 2030± 75
307 ± 20 363i26 3767~39
-t- 13 -t- 34** ~ 39**
* Preincubation during 30 rain and incorporation during 30 min. * * P < 0.05 (Student t-test).
In Table II the combined data of two experiments are presented, indicating a significant increase of 39 and 34~o at a peptide concentration of l0 -a and 5 × l0 -7 M respectively. At a concentration of l0 -s M a tendency of + 13 ~ (5 ~ < P < 1 0 ~ ) was observed. Since the presence of ACTHI-10 in the medium seemed not to alter the uptake of label into the slice, the observed influence on incorporation cannot be accounted for by an effect on precursor uptake into the slice. The observed changes in in vitro incorporation of [t4C]leucine into brain stem proteins as a result of hypophysectomy and in in vitro addition of ACTHI-10-7-L-Phe are in agreement with previous observations in in vivo studies with [aH]leucine. Under these conditions hypophysectomy caused a decrease in incorporation of about 23 ~ , whilst treatment of hypophysectomized rats with ACTH~-10-7-L-Phe had a stimulatory effect amounting to approximately 28 ~o17,1s; in vitro similar effects of hypophysectomy and the peptide (in a concentration of 5 × l0 -7 and l0 -5 M) on leucine incorporation were found (Tables I and II). In the present study no changes were observed in precursor uptake during the incorporation period used; in addition, conversion of [14C]leucine to other metabolites seems negligible 1. Therefore, changes in leucine incorporation as found after hypophysectomy and ACTHI-10-7-L-Phe may reflect changes in protein synthesis. The effect of ACTH1--10 on brain protein synthesis is consistent with the results reported by Rudman et al. 16 on the effects of melanotropic peptides, which share the sequence ACTH4-10, on protein synthesis in mouse brain. Their observations on the penetration of a-aminoisobutyric acid, and the accumulation, the concentration and incorporation of various amino acids also indicate a stimulatory effect of these peptides on protein synthesis. The present in vitro study provides further evidence for a direct action of A C T H I - l o on central nervous structures. The authors wish to thank Mr. J. Brakkee for excellent biotechnical assistance. The supply of ACTHI-10 by Organon, Oss is gratefully acknowledged. Part of this research was sponsored by a grant from F U N G O - Z W O 13-31-03A, The Netherlands.
SHORT COMMUNICATIONS
575
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