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Effect of two ACTH-analogs on noradrenaline metabolism in rat brain
Brain Reaearch, 49 (1973) 483-,185
zl8 3
,{) Elsewer Sctentlfic Pubhshmg Company, Amsterdam - Printed m The Netherlands
Effect of two ACTH-analogs on noradrenaline metabolism in rat brain
D1RK H G V E R S T E E G Rudolf Magnus hlstttute for Pharmacology, ]~ledual Faculty, Umverszty of UtJe~ht, Utledtt ( The Netherlands )
(Accepted October 16th, 1972)
ACTH inhibits extinction of a conditioned avoidance response in shuttle box and pole jump conditioning. This effect is shared by a number of ACTH-analogs, which have the amino sequence 4-10 of A C T H m common and which are virtually devoid of corticotrophic activities, vlz. ct-MSH, fl-MSH, ACTH1 10 and ACTH4 10. ACTH4 lo (7Dphe), an ACTH-analog in which the phenylalanine residue m the 7th position is in the D-form, has effects opposite to that of the L-phe containing peptldes (for a review see de Wledl°). It has been shown by van Wimersma Greldanus and de Wled ll that these peptides exert thmr effect on behavior via an interaction with structures m the brain stem, viz the centrum medianum in the dlencephalon. Since it has been demonstrated that ACTH increases noradrenallne turnover in the hypothalamus, cortex and other parts of the brain 5, it was deemed of interest to investigate the effects of ACTH~-10 and ACTH4 10 (7Dphe) on noradrenahne metabolism in the brain. Male rats of an mbred Wistar strain, weighing 170-180 g, were injected s c. with a single dose of 80/~g ACTH4 10 or ACTH4-10 (7Dphe) as a long acting zinc phosphate preparation. Control animals were mjected with vehicle. Twenty-four hours later a-methyltyroslne methylester (H/4/68~ 300 mg/kg, l p ) was injected and the rate constant of amine loss was determined from the decline of the endogenous noradrenaline concentration after synthesis inhibition, according to Brodie et al.". The rate of synthesis of noradrenahne was calculated from the product of the rate constant of amine loss and the steady state levels of noradrenahne '~. Whole brain and brain parts were taken out and frozen in hquld nitrogen within 60 sec after decapitation of the rat and homogenized in 0.4 N HCIO4 Noradrenahne was adsorbed at pH 8.6 on aluminum oxide and eluted with 0.05 N HC10 ~according to Anton and Sayre 1, and assayed following the method of Laverty and Taylor 6. The brain stem consisted of hypothalamus, thalamus, mesencephalon and medulla oblongata. Cortex stands for whole brain minus brain stem and cerebellum. ACTH4 lo caused a significant increase in the rate of synthesis of noradrenahne in whole brain (17°o, P < 0.005) and brain stem (24 °o, P < 0 05) compared with the rate of synthesis of noradrenahne in brain and brain stem of rats treated with placebo. No significant effect was found in the cortex. ACTH~ 10 (7Dphe) did not materially
484
SHORq
t
t~MMU\tt ~+~+,
TABI_F I fr~f<'l o~ A ( T H . t it~ ,,,Nt) A~. tH4 W H O I I- B R A I N , B R A I N S T [ M
.~ ( 7 [ ) p H I )
~:'N I;tl RAT[ (H- S'~'NIIIFSIS (.q ~R.\DR~NA~,
,
~\l~ (fIRT[\
Siead3 .~tate level :n;',',k" ,
Rare ~on~taat o[ a l l l l l t e /0~ ,k,
,S F M
~h '
i
Rate o/ ~)ntheM~ (ng/g,h)
Number o~ atumals
",, chan~,e
S'L~,nlB,'a,~ l-los,
S L ~I)
Whole brain
334 * 9 ACTH4 lo placebo 348 - 10 ACTHa 10(7ophe) 336 ~ 9
0248 : 0 0 l l 0 204 ~ 0 009 0 t98 1-0010
83 71 67
42 29 18
0207 . 0011 0.170 _ 0.012 0 168 ~ 0015
114 92 89
42 29 18
0 242 ~ 0.019 0 226 q- 0 015 0233 • 0.015
64 62 62
42 29 18
17
p
--~
0005
N S
Braltl 3 l e m
ACTH4 ,, 544 ~ 12 placebo 550 19 ACTH4 lo(7Dphe) 531 21
, 24
P
{}05
--3
N.S
~
N.S
0
N S.
Cortex
ACTH4 -~, 263 t 11 placebo 273 t 11 ACTH ~ lo(7Dphe) 268 t 9
affect the rate of synthesis of noradrenahne in either part. Although lower mean values w e r e f o u n d , n o r a d r e n a l i n e levels w e r e n o t s i g n i f i c a n t l y a l t e r e d (see T a b l e 1). The existence of a correlatmn has been postulated between the i ate of extmcuon of a conditioned avoidance response (CAR) and central noradrenahne
turnover, to
t h e e x t e n t t h a t a n i n c r e a s e d e x t i n c t i o n r a t e is c o r r e l a t e d w i t h a d e c r e a s e in c e n t r a l noradrenahne
t u r n o v e r a n d a d e c r e a s e d r a t e o f e x t i n c t i o n w i t h a n e n h a n c e m e n t 5,'.
A c c o r d i n g t o thxs c o n c e p t s u b s t a n c e s t h a t i n h i b i t e x t i n c t i o n o f a C A R i n i n t a c t ~ats, hke the whole ACTH
molecule and ACTH-analogs
s u c h as A C T H ~ - 1 0 , s h o u l d c a u s e
a n i n c r e a s e m t h e r a t e o f s y n t h e s i s o f n o r a d r e n a l i n e in t h e c e n t r a l n e r v o u s s y s t e m A c t u a l l y , it h a s b e e n r e p o r t e d t h a t A C T H
d o e s so s. A C T H 4 - 1 0 , a n A C T H - a n a l o g
w h i c h l a c k s t h e c o r t i c o t r o p h l c a c t i v i t y o f A C T H 1° a n d w h i c h e x e r t s its b e h a v i o r a l effects u p o n ~ m p l a n t a t m n m c ~ r c u m s c r t b e d a r e a s m t h e b r a i n s t e m 11, i n c r e a s e s t h e r a t e of synthesis of noradrenahne ACTH
on noradrenahne
m the brain stem. This indicates that the influence of
m e t a b o l i s m , h k e its b e h a v i o r a l effects, is n o t m e d m t e d by
the adrenal cortex and seems to corroborate however, ACTH4 of ACTH4
the above-mentioned
p o s t u l a t e . Since,
~0 ( 7 o p h e ) h a s a n effect o n e x t m c t m n o f a C A R
o p p o s i t e to ~hat
i0 b u t fails t o a l t e r t h e r a t e o f s y n t h e s i s o f n o r a d r e n a h n e
in t h e b r a i n , It
f o l l o w s t h a t t h e p o s t u l a t e as s u c h is n o t t e n a b l e . T h e m o r e so as it h a s b e e n f o u n d t h a t ACTH a and ACTH4
10s d o n o t affect t h e l m p m r e d t u r n o v e r o f n o r a d r e n a h n e
brains of hypophysectomized
in t h e
rats, a l t h o u g h t h e d o s e s u s e d w e r e l o w e r t h a n t h o s e
employed m the present experiments. Torda and Wolff v demonstrated
t h a t admml,~-
tration of ACTH does restore the impaired aNhty to synthesize acetylchohne m the brains of hypophysectomized as well. A l t h o u g h
rats and increases this ability in the brains of intact rats
H a d z o v i c a n d d e W l e d ~ f o u n d t h a t t h e effect o f A C T H 1
t. o n
SHORT COMMUNICATIONS
485
extinction o f a pole j u m p i n g a v o i d a n c e response co u l d n o t be p r e v e n t e d by the previous a d m i n i s t r a t i o n o f a t r o p i n e sulfate, the results o f T o r d a and W o l f f p o i n t at the possibility that factors o t h e r t h a n n o r a d r e n e r g i c are also i n v o l v e d in the co n t r o l o f c o n d i t i o n e d behavior. In view o f this a study o f the effects o f A C T H - a n a l o g s on the m e t a b o l i s m o f biogenic amines in m o r e discrete brain regions seems to be required. T h e g e n e r o u s supply o f A C T H - a n a l o g s by O r g a n o n C o m p . , Oss, T h e N e t h e r lands, is gratefully a c k n o w l e d g e d . T h e a u t h o r is very m u c h i n d e b t e d to Miss Ed i t h van Boekel for technical assistance.
I ANTON, A. H , AND SAYRE, D F., A study of the factors affecting the aluminum-oxide trlhydroxylndole procedure for the analysis of catecholammes, J. Pharmacol. exp Ther, 138 (1962) 360-375 2 BRODIE, B B., COSTA, E., DLABAe, A., NEFF, N H , AND SMOOKLER, H H , Apphcatlon of steady state kinetics to the estimation of synthes~s rate and turnover time of tissue catecholamines, J Pharmacol exp. Ther., 154 (1966) 493-498. 3 FUXE,K , CORRODI, H., HOKFELT,T , ANDJONSSON, G., Central monoamine neurons and pituitary -adrenal activity In D. DE WIED AND J. W. A. M. WEIJNEN(Eds.), Pttmtary, Adrenal and the Brain, Progress in Bram Research, Vol 32, Elsevier, Amsterdam, 1970, pp 42-65. 4 HADZOVIC, S., AND DE WIED, D., Central chohnerglc pathways and the inhibitory effect of ACTHI-10 on extinction of a pole jumping avoidance response, Ab~tr. Orgamzmg Committee oJ the fir.st Congr of the Hungarian Sot., Pharmacol Soc., Budapest, O c t , (1971) 5 HOKFFLT,T., AND FUXE,K , On the morphology and the neuroendocrlne role of the hypothalamic catecholamme neurons. In Brain-Endocrine Interaction Median Eminence Structure and Function Int Syrup. Mumch 1971, Karger, Basel 1972, pp 181-223 6 LAVERTY,R., AND TAYLOR,K. M., The fluorometr~c assay of catecholamines and related compounds improvements and extension to the hydroxyindole technique, Anal. Btochem, 22 (1968) 269-279. 7 TORDA, C , AND WOLFF, H G., Effect of pituitary hormones, cortisone and adrenalectomy on some aspects of neuromuscular systems and acetylchohne synthesis, Amer J. Phy~tol, 169 (1952) 140-149 8 VERSTEEG, D H. G , GISPEN, W. H , SCHOTMAN, P , WITTER, A , AND DE WIED, D , Hypophysectomy and rat brain metabohsm, effect of synthetic ACTH analogs, Advane Btochem Psychopharmacol., 6 (1972) 219-239. 9 WFISS,J. M , McEWEN, B S., SILVA, M T., AND KALKUT, M., Pituitary-adrenal alterations and fear responding, Amer J. Phystol, 218 (1970) 864-868. ]0 WIED,D. DE, Effects of peptlde hormones on behavior In W F. GANONGAND L. MARTINI(Eds), Frontiers m Neuroendocrmology, Oxford Univ. Press, New York, 1969, pp 97-140. | l WIMERSMA GREIDANUS, TJ. B. VAN, AND DE WIED, D., Effects of systemic and mtracerebral administration of two opposite acting ACTH-related peptldes on extinction of conditioned avoidance behavior, Neuroendocrmology, 7 (1971) 291-301.
zl8 3
,{) Elsewer Sctentlfic Pubhshmg Company, Amsterdam - Printed m The Netherlands
Effect of two ACTH-analogs on noradrenaline metabolism in rat brain
D1RK H G V E R S T E E G Rudolf Magnus hlstttute for Pharmacology, ]~ledual Faculty, Umverszty of UtJe~ht, Utledtt ( The Netherlands )
(Accepted October 16th, 1972)
ACTH inhibits extinction of a conditioned avoidance response in shuttle box and pole jump conditioning. This effect is shared by a number of ACTH-analogs, which have the amino sequence 4-10 of A C T H m common and which are virtually devoid of corticotrophic activities, vlz. ct-MSH, fl-MSH, ACTH1 10 and ACTH4 10. ACTH4 lo (7Dphe), an ACTH-analog in which the phenylalanine residue m the 7th position is in the D-form, has effects opposite to that of the L-phe containing peptldes (for a review see de Wledl°). It has been shown by van Wimersma Greldanus and de Wled ll that these peptides exert thmr effect on behavior via an interaction with structures m the brain stem, viz the centrum medianum in the dlencephalon. Since it has been demonstrated that ACTH increases noradrenallne turnover in the hypothalamus, cortex and other parts of the brain 5, it was deemed of interest to investigate the effects of ACTH~-10 and ACTH4 10 (7Dphe) on noradrenahne metabolism in the brain. Male rats of an mbred Wistar strain, weighing 170-180 g, were injected s c. with a single dose of 80/~g ACTH4 10 or ACTH4-10 (7Dphe) as a long acting zinc phosphate preparation. Control animals were mjected with vehicle. Twenty-four hours later a-methyltyroslne methylester (H/4/68~ 300 mg/kg, l p ) was injected and the rate constant of amine loss was determined from the decline of the endogenous noradrenaline concentration after synthesis inhibition, according to Brodie et al.". The rate of synthesis of noradrenahne was calculated from the product of the rate constant of amine loss and the steady state levels of noradrenahne '~. Whole brain and brain parts were taken out and frozen in hquld nitrogen within 60 sec after decapitation of the rat and homogenized in 0.4 N HCIO4 Noradrenahne was adsorbed at pH 8.6 on aluminum oxide and eluted with 0.05 N HC10 ~according to Anton and Sayre 1, and assayed following the method of Laverty and Taylor 6. The brain stem consisted of hypothalamus, thalamus, mesencephalon and medulla oblongata. Cortex stands for whole brain minus brain stem and cerebellum. ACTH4 lo caused a significant increase in the rate of synthesis of noradrenahne in whole brain (17°o, P < 0.005) and brain stem (24 °o, P < 0 05) compared with the rate of synthesis of noradrenahne in brain and brain stem of rats treated with placebo. No significant effect was found in the cortex. ACTH~ 10 (7Dphe) did not materially
484
SHORq
t
t~MMU\tt ~+~+,
TABI_F I fr~f<'l o~ A ( T H . t it~ ,,,Nt) A~. tH4 W H O I I- B R A I N , B R A I N S T [ M
.~ ( 7 [ ) p H I )
~:'N I;tl RAT[ (H- S'~'NIIIFSIS (.q ~R.\DR~NA~,
,
~\l~ (fIRT[\
Siead3 .~tate level :n;',',k" ,
Rare ~on~taat o[ a l l l l l t e /0~ ,k,
,S F M
~h '
i
Rate o/ ~)ntheM~ (ng/g,h)
Number o~ atumals
",, chan~,e
S'L~,nlB,'a,~ l-los,
S L ~I)
Whole brain
334 * 9 ACTH4 lo placebo 348 - 10 ACTHa 10(7ophe) 336 ~ 9
0248 : 0 0 l l 0 204 ~ 0 009 0 t98 1-0010
83 71 67
42 29 18
0207 . 0011 0.170 _ 0.012 0 168 ~ 0015
114 92 89
42 29 18
0 242 ~ 0.019 0 226 q- 0 015 0233 • 0.015
64 62 62
42 29 18
17
p
--~
0005
N S
Braltl 3 l e m
ACTH4 ,, 544 ~ 12 placebo 550 19 ACTH4 lo(7Dphe) 531 21
, 24
P
{}05
--3
N.S
~
N.S
0
N S.
Cortex
ACTH4 -~, 263 t 11 placebo 273 t 11 ACTH ~ lo(7Dphe) 268 t 9
affect the rate of synthesis of noradrenahne in either part. Although lower mean values w e r e f o u n d , n o r a d r e n a l i n e levels w e r e n o t s i g n i f i c a n t l y a l t e r e d (see T a b l e 1). The existence of a correlatmn has been postulated between the i ate of extmcuon of a conditioned avoidance response (CAR) and central noradrenahne
turnover, to
t h e e x t e n t t h a t a n i n c r e a s e d e x t i n c t i o n r a t e is c o r r e l a t e d w i t h a d e c r e a s e in c e n t r a l noradrenahne
t u r n o v e r a n d a d e c r e a s e d r a t e o f e x t i n c t i o n w i t h a n e n h a n c e m e n t 5,'.
A c c o r d i n g t o thxs c o n c e p t s u b s t a n c e s t h a t i n h i b i t e x t i n c t i o n o f a C A R i n i n t a c t ~ats, hke the whole ACTH
molecule and ACTH-analogs
s u c h as A C T H ~ - 1 0 , s h o u l d c a u s e
a n i n c r e a s e m t h e r a t e o f s y n t h e s i s o f n o r a d r e n a l i n e in t h e c e n t r a l n e r v o u s s y s t e m A c t u a l l y , it h a s b e e n r e p o r t e d t h a t A C T H
d o e s so s. A C T H 4 - 1 0 , a n A C T H - a n a l o g
w h i c h l a c k s t h e c o r t i c o t r o p h l c a c t i v i t y o f A C T H 1° a n d w h i c h e x e r t s its b e h a v i o r a l effects u p o n ~ m p l a n t a t m n m c ~ r c u m s c r t b e d a r e a s m t h e b r a i n s t e m 11, i n c r e a s e s t h e r a t e of synthesis of noradrenahne ACTH
on noradrenahne
m the brain stem. This indicates that the influence of
m e t a b o l i s m , h k e its b e h a v i o r a l effects, is n o t m e d m t e d by
the adrenal cortex and seems to corroborate however, ACTH4 of ACTH4
the above-mentioned
p o s t u l a t e . Since,
~0 ( 7 o p h e ) h a s a n effect o n e x t m c t m n o f a C A R
o p p o s i t e to ~hat
i0 b u t fails t o a l t e r t h e r a t e o f s y n t h e s i s o f n o r a d r e n a h n e
in t h e b r a i n , It
f o l l o w s t h a t t h e p o s t u l a t e as s u c h is n o t t e n a b l e . T h e m o r e so as it h a s b e e n f o u n d t h a t ACTH a and ACTH4
10s d o n o t affect t h e l m p m r e d t u r n o v e r o f n o r a d r e n a h n e
brains of hypophysectomized
in t h e
rats, a l t h o u g h t h e d o s e s u s e d w e r e l o w e r t h a n t h o s e
employed m the present experiments. Torda and Wolff v demonstrated
t h a t admml,~-
tration of ACTH does restore the impaired aNhty to synthesize acetylchohne m the brains of hypophysectomized as well. A l t h o u g h
rats and increases this ability in the brains of intact rats
H a d z o v i c a n d d e W l e d ~ f o u n d t h a t t h e effect o f A C T H 1
t. o n
SHORT COMMUNICATIONS
485
extinction o f a pole j u m p i n g a v o i d a n c e response co u l d n o t be p r e v e n t e d by the previous a d m i n i s t r a t i o n o f a t r o p i n e sulfate, the results o f T o r d a and W o l f f p o i n t at the possibility that factors o t h e r t h a n n o r a d r e n e r g i c are also i n v o l v e d in the co n t r o l o f c o n d i t i o n e d behavior. In view o f this a study o f the effects o f A C T H - a n a l o g s on the m e t a b o l i s m o f biogenic amines in m o r e discrete brain regions seems to be required. T h e g e n e r o u s supply o f A C T H - a n a l o g s by O r g a n o n C o m p . , Oss, T h e N e t h e r lands, is gratefully a c k n o w l e d g e d . T h e a u t h o r is very m u c h i n d e b t e d to Miss Ed i t h van Boekel for technical assistance.
I ANTON, A. H , AND SAYRE, D F., A study of the factors affecting the aluminum-oxide trlhydroxylndole procedure for the analysis of catecholammes, J. Pharmacol. exp Ther, 138 (1962) 360-375 2 BRODIE, B B., COSTA, E., DLABAe, A., NEFF, N H , AND SMOOKLER, H H , Apphcatlon of steady state kinetics to the estimation of synthes~s rate and turnover time of tissue catecholamines, J Pharmacol exp. Ther., 154 (1966) 493-498. 3 FUXE,K , CORRODI, H., HOKFELT,T , ANDJONSSON, G., Central monoamine neurons and pituitary -adrenal activity In D. DE WIED AND J. W. A. M. WEIJNEN(Eds.), Pttmtary, Adrenal and the Brain, Progress in Bram Research, Vol 32, Elsevier, Amsterdam, 1970, pp 42-65. 4 HADZOVIC, S., AND DE WIED, D., Central chohnerglc pathways and the inhibitory effect of ACTHI-10 on extinction of a pole jumping avoidance response, Ab~tr. Orgamzmg Committee oJ the fir.st Congr of the Hungarian Sot., Pharmacol Soc., Budapest, O c t , (1971) 5 HOKFFLT,T., AND FUXE,K , On the morphology and the neuroendocrlne role of the hypothalamic catecholamme neurons. In Brain-Endocrine Interaction Median Eminence Structure and Function Int Syrup. Mumch 1971, Karger, Basel 1972, pp 181-223 6 LAVERTY,R., AND TAYLOR,K. M., The fluorometr~c assay of catecholamines and related compounds improvements and extension to the hydroxyindole technique, Anal. Btochem, 22 (1968) 269-279. 7 TORDA, C , AND WOLFF, H G., Effect of pituitary hormones, cortisone and adrenalectomy on some aspects of neuromuscular systems and acetylchohne synthesis, Amer J. Phy~tol, 169 (1952) 140-149 8 VERSTEEG, D H. G , GISPEN, W. H , SCHOTMAN, P , WITTER, A , AND DE WIED, D , Hypophysectomy and rat brain metabohsm, effect of synthetic ACTH analogs, Advane Btochem Psychopharmacol., 6 (1972) 219-239. 9 WFISS,J. M , McEWEN, B S., SILVA, M T., AND KALKUT, M., Pituitary-adrenal alterations and fear responding, Amer J. Phystol, 218 (1970) 864-868. ]0 WIED,D. DE, Effects of peptlde hormones on behavior In W F. GANONGAND L. MARTINI(Eds), Frontiers m Neuroendocrmology, Oxford Univ. Press, New York, 1969, pp 97-140. | l WIMERSMA GREIDANUS, TJ. B. VAN, AND DE WIED, D., Effects of systemic and mtracerebral administration of two opposite acting ACTH-related peptldes on extinction of conditioned avoidance behavior, Neuroendocrmology, 7 (1971) 291-301.