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Stimulation increases incorporation of [3H]lysine into rat brain and liver proteins
Brain Research
Bulletin,
All rights of reproduction
Vol. 2, pp. 243-245, 1977. Copyright 0 ANKHO in any form reserved, Printed in the U.S.A.
InternationaI
Inc.
Stimulation Increases Incorporation of [3H]Lysine into Rat Brain and Liver Proteins HOWARD D. REES,l DANIEL J. ENTINCH2 AND ADRIAN J. DUNN”
(Received 22 April 1977) REES, H. D., D. 3. ENTINGH AND A. J. DUNN. ~t~~~~utio~~ imreases ~~~~~)~~#~ati~~z of [“Hf&%‘r?e irrtn mt hruir? and liver proteins. BRAIN RES. BULL. 2(4f 243-245, i917. - Exposure of rats to electric footshocks or merely to the footshock apparatus increased the incorporation of [ 3H] lysine into brain and liver protein. The effect was present in both
fore- and hindbrain. The footshock treatment was the more effective stimulus, producing larger and more significant changes. These results resemble those previously observed in C57B1/6J mice. and thus suggest that altered protein or amino acid metabolism is a general response to stress in rodents. ( 3H] lysine
Liver proteins
Stimulation
Stress
Footshock
-
__--__--
.-.---..
[’ H] lysine injections were given both intracranially and subcutaneously to label both brain and liver proteins. Ten PCi of L-1 4,5-3 H] lysine monohydrochloride ( 19 Ci/mmole, Amersham-S~arle) was injected through the skull holes into each lateral ventricle at a depth of 3 mm and 30 gti was injected subcutaneously in the back of the neck immediately afterwards. Rats were sacrificed by decapitation precisely 10 min after the first injection, and the brain and a sample of the right lobe of the liver rapidly excised. The brain was separated into two parts by a midcollicuiar cut. The tissues were homogenized in 5 ml of 0.05 M borate buffer and the radioactivity in the dried homogenate and in protein determined by a modification of the paper disc method of Mans and Novelli 191 as previously described [ill.
IN PREVIOUS studies we detected an increased incorporation of [ 3H] lysine into proteins of brain and liver when male mice of the C57B1/6J strain were trained in a jump-box one-way active avoidance task [ 1I]. The biochemical changes were not specific to the training experienee, since they also occurred when mice were exposed to a series of footshocks or buzzers, or merely to the training apparatus. To test the generality of these observations we have performed similar experiments in the rat. METHOD Male Wistar rats weighing 290-320 g were obtained from Hilltop Laboratory Animals, Scottdale, PA, and were housed in individual cages for 7 days prior to the experiment. Two 1 mm holes were drilled in the skulls of the rats under ether anesthesia on the day before the experiment. The hates were placed 2 mm on either side of the midline 2 mm posterior to bregma to allow injection of the isotope into the lateral ventricles. The scalp incision was closed loosely with a single wound clip. Behavior procedures were modeled on those used previously for mice. Three groups were used. Quiet rats were left undisturbed until [ 3 H] lysine injections. Apparatusexposed rats were placed in a step-down avoidance apparatus [8] and lef’t undisturbed for 15 min. They were then returned to their home cages and injected with [” H] lysine 20 min later. Footshocked rats were treated as the apparatus-exposed ones except that 20 footshocks (0.6 mA, 1 set duration) were delivered to the grid floor of the apparatus at random intervals throughout the 15 min period.
RESULTS We have previously shown that the radioactivity precipitated on the paper disc is essentially all in protein [ 1 11 and that the difference between the total dried radioactivity and the precipitate is essentially all in free lysine [ 41. Thus we have computed a relative radioactivity, equal to protein radioactivity divided by free lysine radioactivity, to correct for variations in uptake of the precursor. The results are presented in Table 1. As expected from the studies on mice, footshock proved to be a more effective stimulus than mere exposure to the apparatus. Footshock significantly increased the relative radio-activity of protein in both parts of brain. .4pparatus+xposure produced a similar increase that was significant only in the hindbrain: In the liver, both treatments increased the relative
i Present address: Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, Utrecht, The Netherlands. 2 Present address: Department of Resources Planning, W266 1, The MITRE Corporation, Westgate Research Park, McLean, VA 22101. 3Present address: Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL 32610 (to which correspondence should be addressed). 243
REES,
244
TABLE EFFECTS
OF APPARATUS-EX~SURE
‘Treatment
Mean -r SEM
UPTAKE
Protein radioactivity % change
AND DUNN
I
AND FOOTSHOCK ON t?HlLYStNE PROTEIN OF BRAIN AND LIVER
Acid-soluble radioactivity
ENTINGH
AND
INCORPORATION
I NT0
Relative radioactivity
Mean f SEM
% change
Mean + SEM
% change
Forebrain Quiet Apparatus-exposure Footshocks Hindbr~iin Quiet Apparatus-exposure Footshocks Liver Quiet Apparatus-exposure Footshocks *Significantly different from Quiet, pCO.05, ip
but this was significant
only
with
footshock.
While the free lysine radioactivity was essentially unchanged, footshock significantly increased the protein radioactivity. These results are in good agreement with those obtained in mice, despite technical alterations which included a different route of injection. In rats. apparatus-exposure and footshocks increased the relative radioactivity of hind-brain protein by 14 and i 6?6_respectively: our previous studies of mice found 12 ;and 14% increases in these two measures [I I]. In rats. apparatus-exposure and footshocks. respectiveiy, produced 6 and 12% increases in relative radioactivity of protein in the forebrain, the larger of the two brain parts; in mice these treatments produced 7 and 12% increases in the relative radioactivity of whole brain protein. The relative radioactivity of liver protein in rats responded to apparatus-exposure and footshocks with increases of 23 and 58%; the corresponding changes in mice were 30 and 61 Q, Thus the effects of environmental stimulation on the incorporation of [ 3H] lysine in to brain and liver proteins is probably a general phenomenon in rodents. DISCUSSION It is notable that others have observed increases in the incorporation of labelled amino acids into brain proteins when rats were exposed to novel stimuli. Appel er at’. [ 21 demonstrated an increased incorporation of f 3H] ieucine into cortical protein when rats that had been confined to darkness for 3 days were reexposed to light for 15 min. Rose [ 1.51 showed that t3 HI lysine incorporation into
cerebral
cortex protein was increased when rats were first exposed to light. All-man and Das [ 11 observed that stimulation in an activity wheel likewise stimulated
[ 3Hf leucine incorporation into brain proteins. These data suggest that the stress of novel situations may be sufficient to cause a change of the incorporation of amino acids into protein. Very mild novel stimulation can alter incorporation of lysine in brain without affecting the liver [ 71. Whether these changes of precursor incorporation into protein reflect changes in the rate of protein synthesis remains to be determined. A change of the rate of protein synthesis is the most likely explanation, but changes of precursor metabolism cannot be excluded and the small magnitude of the changes render further analysis difficult. Changes have been observed with two dissimilar amino acid precursors, [’ H] lysine and [ 7H] leucine [ 5, I I 1. The mechanisms of the response also remains to be determined. Our investigations in mice suggest that the response may at least in part be mediated by ACTH [6]. This would be consistent with other observations that ACTH increases the rate of incorporation of amino acids into brain proteins 1.3, 6, 10. 13 14. 16, 17, 181. Nevertheless, there is probably also a direct neural cornponent [ 121.
This research was supported by grants f’rom the U.S. Public Health Service (MH18136, NS074$7), the National Science foundation (GB35634X), and the t&a-Geigy Corporation. The technical assistance of Dr. Terri Damstra. Mary Denny and Glen Thursby is gratefully appreciated. We are also grateful for the provision of facilities by Drs. Edward Glassman and John E. Wilson.
REFERENCES 1. Altman,
J. and G. D. Das. Behavioral manipulations’ and protein metabolism of the .brain: Effects of motor exercise an the utilization of’ Ieucine-3H. Plr_vsiol. Behav. 1: 105-108, 1960.
2.
Appel.
S. H.,
Response
to
836.-838,
1967.
W. Davis and S. Scott.
environmental
stimulation.
Nrain polysomes:
S&nue
157:
STIMULATION
AND PROTEIN
SYNTHESIS
IN RATS
-3. Dunn. A. J. The chemistry of learning and the formation of 4.
5.
6.
7.
8.
9.
10.
memory. In: Molecular and Functional Neurobiology, edited by W. G. &pen. Amsterdam: Elsevier, 1976, pp. 347-387. Dunn, A. J. and B. J. Begert. Effects of electroconvulsive shock and cyclohe~imide on the incorporation of amino acids into proteins of mouse brain subcellular fractions. f !iQrtrocirem, 26: 369-375, 1976. Dunn, A. J.. D. Entingh. T. Entingh, W. H. Gispen, B. Ma&us, R. Perumal, H. D. Rees and L. Borgan. Biochemical correlates of brief behavioral experiences. In: The ~el~~osc~ences. Third Study Prop-an?, edited by F. 0. Schmitt and F. G. Worden. Cambridge: IL1.I.T.Press, 1974, pp. 679-684. Dunn, A. J., P. IM. Iuvone and H. D. Rees. Neurochemical responses of mice to ACTH and lysine vasopressin. Pkartnac. ~~[~cj~e?~t. Bekav. 5: (Suppl. I), 139-145, 1976. Entingh. D. V. and 7. Damstra. Effects of handling and etherization on incorporation of [‘H] lysine into protein of mouse brain and liver. PIzarmuc. Biochrm. Behav. 5: 11 l-l 16, 1976. Machlus, B., J. E. Wilson and E. Classman. Brain phosphoproteins: The effect of short experiences on the phosphorylation of nuclear proteins of rat brain. Behav. Biol. 10: 43-62,1974. Mans. R. J. and G. D. Novelli. Measurement of the incorporation of radioactive amino acids into protein by a filterpaper disc method. Archs Biochem. 94: 48.--53, 196 I. Reading, H. W. Effects of some adrenocorticotropin analogues on protein synthesis in brain. Biwkerrr. J. 127: 7P, 1972.
245 11. Rees, H. D., L. I,. Brogan, D. J. Entingh, A. J. Dunn. P. G. Shinkman, T. Damstra-Entingh, J. E. Wilson and G. Glassman. Effect of sensory stimulation on the uptake and incorporation of radioactive lysine into protein of mouse brain and liver. Braif? Rex 68: 143--l%, 1974. 12. Rees, H. D. and A. J. Dunn. The role of the pittlitary-adrenal system in the footshock-induced increase of [“HI lysine incorporation into mouse brain and liver proteins. Braiu Rex 120: 317-320.1977. 13. Rees, H. I)., A. J. Dunn and P. M. luvone. Behavioral biochemical responses of mice to the intraventricular administration of ACTH analogs and lysine vasopressin. I,@, Sci 8: 1333-1340, 1976. 14. Reith, M. E. A., P. Schotman and W. H. Gispen. Hypophysectomy, ACTH and in vitro protein synthesis in rat brain stem slices. Bruin Res. 81: 571-575, 1974. 15. Rose. S. P. R. Changes in visual cortex on first exposure of rats to light: Effect on incorporation of tritiated lysinr into protein. Nature 215: 252-255, 1967. 16. Rudman, D.. S. W. Scott. A. E. Del Rio. D. H. Houser and S. Sheen. Effect of meianotropic peptides on protein synthesis in mouse brain. AIM.J. !?+siol. 226: 681-692, 1974. 17. Schotman. P., W. H. Gispen, W. S. Janz and D. de Wied. I-ffects of ACTH analogues on macromolecule metabolism in the brain stem of ll~~pophysectomi~ed rats. Brain Rcr. 47: 347--362, 1972. 18. Semiginovsky, B. and B. Jakoubek. Effects of AC’TH on the incorporation of I-(U-‘4C)leucine into the brain and cpinal cord of mice. &air? Rrs. 35: 3 19-323, 197 1.
Bulletin,
All rights of reproduction
Vol. 2, pp. 243-245, 1977. Copyright 0 ANKHO in any form reserved, Printed in the U.S.A.
InternationaI
Inc.
Stimulation Increases Incorporation of [3H]Lysine into Rat Brain and Liver Proteins HOWARD D. REES,l DANIEL J. ENTINCH2 AND ADRIAN J. DUNN”
(Received 22 April 1977) REES, H. D., D. 3. ENTINGH AND A. J. DUNN. ~t~~~~utio~~ imreases ~~~~~)~~#~ati~~z of [“Hf&%‘r?e irrtn mt hruir? and liver proteins. BRAIN RES. BULL. 2(4f 243-245, i917. - Exposure of rats to electric footshocks or merely to the footshock apparatus increased the incorporation of [ 3H] lysine into brain and liver protein. The effect was present in both
fore- and hindbrain. The footshock treatment was the more effective stimulus, producing larger and more significant changes. These results resemble those previously observed in C57B1/6J mice. and thus suggest that altered protein or amino acid metabolism is a general response to stress in rodents. ( 3H] lysine
Liver proteins
Stimulation
Stress
Footshock
-
__--__--
.-.---..
[’ H] lysine injections were given both intracranially and subcutaneously to label both brain and liver proteins. Ten PCi of L-1 4,5-3 H] lysine monohydrochloride ( 19 Ci/mmole, Amersham-S~arle) was injected through the skull holes into each lateral ventricle at a depth of 3 mm and 30 gti was injected subcutaneously in the back of the neck immediately afterwards. Rats were sacrificed by decapitation precisely 10 min after the first injection, and the brain and a sample of the right lobe of the liver rapidly excised. The brain was separated into two parts by a midcollicuiar cut. The tissues were homogenized in 5 ml of 0.05 M borate buffer and the radioactivity in the dried homogenate and in protein determined by a modification of the paper disc method of Mans and Novelli 191 as previously described [ill.
IN PREVIOUS studies we detected an increased incorporation of [ 3H] lysine into proteins of brain and liver when male mice of the C57B1/6J strain were trained in a jump-box one-way active avoidance task [ 1I]. The biochemical changes were not specific to the training experienee, since they also occurred when mice were exposed to a series of footshocks or buzzers, or merely to the training apparatus. To test the generality of these observations we have performed similar experiments in the rat. METHOD Male Wistar rats weighing 290-320 g were obtained from Hilltop Laboratory Animals, Scottdale, PA, and were housed in individual cages for 7 days prior to the experiment. Two 1 mm holes were drilled in the skulls of the rats under ether anesthesia on the day before the experiment. The hates were placed 2 mm on either side of the midline 2 mm posterior to bregma to allow injection of the isotope into the lateral ventricles. The scalp incision was closed loosely with a single wound clip. Behavior procedures were modeled on those used previously for mice. Three groups were used. Quiet rats were left undisturbed until [ 3 H] lysine injections. Apparatusexposed rats were placed in a step-down avoidance apparatus [8] and lef’t undisturbed for 15 min. They were then returned to their home cages and injected with [” H] lysine 20 min later. Footshocked rats were treated as the apparatus-exposed ones except that 20 footshocks (0.6 mA, 1 set duration) were delivered to the grid floor of the apparatus at random intervals throughout the 15 min period.
RESULTS We have previously shown that the radioactivity precipitated on the paper disc is essentially all in protein [ 1 11 and that the difference between the total dried radioactivity and the precipitate is essentially all in free lysine [ 41. Thus we have computed a relative radioactivity, equal to protein radioactivity divided by free lysine radioactivity, to correct for variations in uptake of the precursor. The results are presented in Table 1. As expected from the studies on mice, footshock proved to be a more effective stimulus than mere exposure to the apparatus. Footshock significantly increased the relative radio-activity of protein in both parts of brain. .4pparatus+xposure produced a similar increase that was significant only in the hindbrain: In the liver, both treatments increased the relative
i Present address: Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, Utrecht, The Netherlands. 2 Present address: Department of Resources Planning, W266 1, The MITRE Corporation, Westgate Research Park, McLean, VA 22101. 3Present address: Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL 32610 (to which correspondence should be addressed). 243
REES,
244
TABLE EFFECTS
OF APPARATUS-EX~SURE
‘Treatment
Mean -r SEM
UPTAKE
Protein radioactivity % change
AND DUNN
I
AND FOOTSHOCK ON t?HlLYStNE PROTEIN OF BRAIN AND LIVER
Acid-soluble radioactivity
ENTINGH
AND
INCORPORATION
I NT0
Relative radioactivity
Mean f SEM
% change
Mean + SEM
% change
Forebrain Quiet Apparatus-exposure Footshocks Hindbr~iin Quiet Apparatus-exposure Footshocks Liver Quiet Apparatus-exposure Footshocks *Significantly different from Quiet, pCO.05, ip
but this was significant
only
with
footshock.
While the free lysine radioactivity was essentially unchanged, footshock significantly increased the protein radioactivity. These results are in good agreement with those obtained in mice, despite technical alterations which included a different route of injection. In rats. apparatus-exposure and footshocks increased the relative radioactivity of hind-brain protein by 14 and i 6?6_respectively: our previous studies of mice found 12 ;and 14% increases in these two measures [I I]. In rats. apparatus-exposure and footshocks. respectiveiy, produced 6 and 12% increases in relative radioactivity of protein in the forebrain, the larger of the two brain parts; in mice these treatments produced 7 and 12% increases in the relative radioactivity of whole brain protein. The relative radioactivity of liver protein in rats responded to apparatus-exposure and footshocks with increases of 23 and 58%; the corresponding changes in mice were 30 and 61 Q, Thus the effects of environmental stimulation on the incorporation of [ 3H] lysine in to brain and liver proteins is probably a general phenomenon in rodents. DISCUSSION It is notable that others have observed increases in the incorporation of labelled amino acids into brain proteins when rats were exposed to novel stimuli. Appel er at’. [ 21 demonstrated an increased incorporation of f 3H] ieucine into cortical protein when rats that had been confined to darkness for 3 days were reexposed to light for 15 min. Rose [ 1.51 showed that t3 HI lysine incorporation into
cerebral
cortex protein was increased when rats were first exposed to light. All-man and Das [ 11 observed that stimulation in an activity wheel likewise stimulated
[ 3Hf leucine incorporation into brain proteins. These data suggest that the stress of novel situations may be sufficient to cause a change of the incorporation of amino acids into protein. Very mild novel stimulation can alter incorporation of lysine in brain without affecting the liver [ 71. Whether these changes of precursor incorporation into protein reflect changes in the rate of protein synthesis remains to be determined. A change of the rate of protein synthesis is the most likely explanation, but changes of precursor metabolism cannot be excluded and the small magnitude of the changes render further analysis difficult. Changes have been observed with two dissimilar amino acid precursors, [’ H] lysine and [ 7H] leucine [ 5, I I 1. The mechanisms of the response also remains to be determined. Our investigations in mice suggest that the response may at least in part be mediated by ACTH [6]. This would be consistent with other observations that ACTH increases the rate of incorporation of amino acids into brain proteins 1.3, 6, 10. 13 14. 16, 17, 181. Nevertheless, there is probably also a direct neural cornponent [ 121.
This research was supported by grants f’rom the U.S. Public Health Service (MH18136, NS074$7), the National Science foundation (GB35634X), and the t&a-Geigy Corporation. The technical assistance of Dr. Terri Damstra. Mary Denny and Glen Thursby is gratefully appreciated. We are also grateful for the provision of facilities by Drs. Edward Glassman and John E. Wilson.
REFERENCES 1. Altman,
J. and G. D. Das. Behavioral manipulations’ and protein metabolism of the .brain: Effects of motor exercise an the utilization of’ Ieucine-3H. Plr_vsiol. Behav. 1: 105-108, 1960.
2.
Appel.
S. H.,
Response
to
836.-838,
1967.
W. Davis and S. Scott.
environmental
stimulation.
Nrain polysomes:
S&nue
157:
STIMULATION
AND PROTEIN
SYNTHESIS
IN RATS
-3. Dunn. A. J. The chemistry of learning and the formation of 4.
5.
6.
7.
8.
9.
10.
memory. In: Molecular and Functional Neurobiology, edited by W. G. &pen. Amsterdam: Elsevier, 1976, pp. 347-387. Dunn, A. J. and B. J. Begert. Effects of electroconvulsive shock and cyclohe~imide on the incorporation of amino acids into proteins of mouse brain subcellular fractions. f !iQrtrocirem, 26: 369-375, 1976. Dunn, A. J.. D. Entingh. T. Entingh, W. H. Gispen, B. Ma&us, R. Perumal, H. D. Rees and L. Borgan. Biochemical correlates of brief behavioral experiences. In: The ~el~~osc~ences. Third Study Prop-an?, edited by F. 0. Schmitt and F. G. Worden. Cambridge: IL1.I.T.Press, 1974, pp. 679-684. Dunn, A. J., P. IM. Iuvone and H. D. Rees. Neurochemical responses of mice to ACTH and lysine vasopressin. Pkartnac. ~~[~cj~e?~t. Bekav. 5: (Suppl. I), 139-145, 1976. Entingh. D. V. and 7. Damstra. Effects of handling and etherization on incorporation of [‘H] lysine into protein of mouse brain and liver. PIzarmuc. Biochrm. Behav. 5: 11 l-l 16, 1976. Machlus, B., J. E. Wilson and E. Classman. Brain phosphoproteins: The effect of short experiences on the phosphorylation of nuclear proteins of rat brain. Behav. Biol. 10: 43-62,1974. Mans. R. J. and G. D. Novelli. Measurement of the incorporation of radioactive amino acids into protein by a filterpaper disc method. Archs Biochem. 94: 48.--53, 196 I. Reading, H. W. Effects of some adrenocorticotropin analogues on protein synthesis in brain. Biwkerrr. J. 127: 7P, 1972.
245 11. Rees, H. D., L. I,. Brogan, D. J. Entingh, A. J. Dunn. P. G. Shinkman, T. Damstra-Entingh, J. E. Wilson and G. Glassman. Effect of sensory stimulation on the uptake and incorporation of radioactive lysine into protein of mouse brain and liver. Braif? Rex 68: 143--l%, 1974. 12. Rees, H. D. and A. J. Dunn. The role of the pittlitary-adrenal system in the footshock-induced increase of [“HI lysine incorporation into mouse brain and liver proteins. Braiu Rex 120: 317-320.1977. 13. Rees, H. I)., A. J. Dunn and P. M. luvone. Behavioral biochemical responses of mice to the intraventricular administration of ACTH analogs and lysine vasopressin. I,@, Sci 8: 1333-1340, 1976. 14. Reith, M. E. A., P. Schotman and W. H. Gispen. Hypophysectomy, ACTH and in vitro protein synthesis in rat brain stem slices. Bruin Res. 81: 571-575, 1974. 15. Rose. S. P. R. Changes in visual cortex on first exposure of rats to light: Effect on incorporation of tritiated lysinr into protein. Nature 215: 252-255, 1967. 16. Rudman, D.. S. W. Scott. A. E. Del Rio. D. H. Houser and S. Sheen. Effect of meianotropic peptides on protein synthesis in mouse brain. AIM.J. !?+siol. 226: 681-692, 1974. 17. Schotman. P., W. H. Gispen, W. S. Janz and D. de Wied. I-ffects of ACTH analogues on macromolecule metabolism in the brain stem of ll~~pophysectomi~ed rats. Brain Rcr. 47: 347--362, 1972. 18. Semiginovsky, B. and B. Jakoubek. Effects of AC’TH on the incorporation of I-(U-‘4C)leucine into the brain and cpinal cord of mice. &air? Rrs. 35: 3 19-323, 197 1.