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Increase of brain pyridoxal phosphokinase activity following melatonin administration
Printé~~in~reât ~TitâinP~
II' ~' 1283-1288, 1970 .
Pergamon Press
INCREASE Of BRAIN PYRIDOXAL PHOSPHOACIIUSE ACTIVITY FOLLOWING MELATOlfIN ADMINISTRATION . F . AntSn~ay, J . Sepulveda and S . Gonaâles Departamento de Neurobiologia, Instituto de Investigaciones Biosié dicaa, l1NAM . Apdo . Postal 70228, Mézico 20, D .F .
(Received 13 August 19?0; in final form 2 October 1970)
MELATONIN (N-acetil-5-rethozytryptaaiae) is synthetised uniquely in the pineala of eemmals through the action of the ~syme hydrozyindole-0~ethyltranaferase (HIOMT) 1 ' 2 .
Sin~e the activity of this enzyse varies
diurnally aaong rats kept under cyclic lighting conditions, it has been suggested that aelatonin secretion follows a similar rhyth~lc pattern 3 . Melatonin has been identified in urine and peripheral nerve 4 , however, neither the kidney nor the nervous tissue contain measurable HIOMT actiThis has been taken as evidence that the indola ia, in fact,
vity .
2'S. secreted from the pineal i ' The administration of melatonin to ezperi~ental anieals has been shown to influence gonadas ' 7 , thyroid 8 ' 9 ' 10 smooth musc1e1 5 '
16,17
sleep and EEG
+ 11,12
18,19,20,21 ~
anterior pituitary 3'14
Recently, it has also
been demonstrated that the intraperitoneal administration of 4elatonin increases brain serotonin concentration 22 .
The greatest increase of
serotonin is found in the oidbrain and the hypothalauus and it is here where circulating H 3-aelatonin is also most highly concentrated 23 .
It
has been suggested that both the neural and endocrine effects of ad,inisrated melatonin might be related to the changes that this compound produces in the functional activity of the central serotonin-containing neurons
22
Pyridozal phosphokinase (PL-kinase) catalyses the formation of pyridozal phosphate (PL) in the brnin24 .
1283
PL is a cofactor for many
BRAIN PYRIDOXAL PHOBPHOKIIQA3E
1284
Vol . 9, No. 22
reactions : as the prosthetic group of the ensy~e "aromatic L-wino acid decarboxylase" PL is essential for the synthesis of dopamine, 5hydroxytryptaaina (serotonin) and game-amino-butyric acid (GABA) 25 ' 26
.
Ye here report that intraperitoneal injections of melatonin are followed by a rapid increase in the activity of brain PL-kinase . Methods For two weeks prior to the experiment faule wister rata weighing 160 to 170 g were caged individually and kept under noiwal lighting (lights on from : 0600 to 1800 hours, with approxirately 27 to 54 mphot at the level of the animals) .
The rats were given Purina Chow and water ad
libitum . The desired amounts of malatonin were dissolved in one per cent ethanol solution .
Final volumes of 0 .5 ml were injected intreperitonea-
lly between 1000 and 1100 hours . nol solution .
Control animals received only the etha
At the times indicated after injection, the rats were de-
capitated and their brains immediately placed in small beakers containing cracked ice ; ali subsequent procedures were carried out at 0-3° C . Brains were homogenised in 1M phosphate buffer pM 6 .5 (1 :6 w/V) . tes were centrifugated at 30,000 g for 30 minutes .
The homogena-
PL-kinase activity
was determined in the supernatants as described by McCormick end Sne11 27 . The amounts of PL formed were determined with the phenylhydrazine reagent 28 .
Protein was determined according to the method of Lowry, Roserbrough,
Farr and Renda11 29 .
The specific activity of PL-kinase is expressed as
units per mg of protein,
(one unit is defined as the amount of protein
that fosns 1 mpM of PL in 1 hour at 37°C) .
Data are presented as mean +
S .D ., and analysed by the t-test . Results and discussion Groups of six rata received each 200 }ig of melatonin and were sacrificed 90 sin later .
The activity of PL-kinase in the brains of treated
rate was 1234 + 112 .6 units/q but the activity in brains of the control
Vol. 9, No. 22
ERAIN PYRmOXAL PHOBPHOKINA3S
group was only 575 + 29 units/g (P t 0 .001) .
1285
This led us to investigate
the changes of PL-kinase activity as a function of the time after ~elato n1n adainistration .
The anieals received the save doses as before and
were killed 45, 90, 180 or 360 ~1n later .
The highest levels of ansy~e
activity were detected 45 ~inutea after intteperiboneel administration melatonin .
The brains of the rats sacrificed 90 and 180 minutes after
the injection of melatonin showed an et wated PL-kinase activity . After 360 minutes the eruyme activity returned to aorwal values . (Table 1) . TABLE
1
Changes in brain Pyridozal-Phosphokinase (PL-kinase) activity in rats treated with melatonin . Groups of five rats received each 200 uq of melatonin intraperitoneaily, and were killed after various intervals . Ds4 are given as units per grae _+ standard dwiation . Tige after melatonin ad~inistration . 0 min .
633 + 110
45 mia .
2786 + 244 (X)
90 min .
1395 + 113 (X)
180 min .
1414 + 157 (X)
360 min . (X)
PL-kinase activity .
692 + 52
Differs from that of control rata : P < 0 .001 It has been reported that intraperitoneal aàiinistration of large
doses of melatonin appear to be less effective in raising brain serotonin concentration than smaller doses 22 .
Ye therefore studisd the effect
of various doses of melatoain on the activity of brain PL-kinase . Groups of 5 rats were injected rith varying doses of melatonin and sacrificed 90 minutes later .
As shown in Table 2, there was a direct relationship
between the amount of melabonin administered sad the elevation in PLkinase activity .
1288
HRAIN PYRiDOXAL PH05PHOKINASE TABLE
Vol . 9, No. 22
z
Effect of various intraperitoneal doses of meletonin on PyridoxalPhosphokinase (PL-kinase) activity of rat brain . Data are given as units per grar + standard deviation .
(x)
Melatonin
PL-kinase
rg/kq
Activity
Control
590 + 39
0 .25
666 + 113
0 .50
779 + 61 (x)
1 .0
1085 + 35(x)
2 .0
1203 + 95(x)
4 .0
1953 _+ 81(x)
Differs fror that of control rats : P c 0 .001 Experirents in which various concentrations of relatonin were added
to brain horogenates in vitro showed that the indole had no direct effect on the activity of the enayre .
Hence,
it appears likely that
the rsehanisr by which reletonin increases brain PL-kinase activity in vivo involves enhancing the de novo synthesis of the enzyme . 91nce PL is a cofactor for decarboxylases and tranaerinases in the brain24'25, it sight be anticipated that increases in the concentration of this cofactor following aelatonin adrinistration would cause changes
in
several brain functions .
The increases in brain serotonin 22 and
GABA 30 concentrations after relatonin adriniatration ray thus be rediatad by increases in the activities of 5-hydroxytryptophan and glutaric decarboxylases respectively .
It is suggested that relatonin modifies
the retabolic activity of the brain by increasing the availability of PL . Suoserv The intraperitoneal adainistration of relatonin to rata is followed by an increase in the activity of brain Pyridoxal phoaphokinase .
This
vol. 9, No. 22
BRAIN PYRIDOXAL PH05PHOKINASE
1287
effect is saximal 45 mlnutea after oelatonin administration .
Brain
pyridoxal phosphokinase activity shows a direct relationship to oelatonin in the range of 0 .25 - 4 .0 mg/kg body weight . References 1.
J . AXELROD, P .D . MACLEAN, R .W . ALBERE and H .W . WEISSBACH . Regional Neurocheeistrv p . 307 . 5 .3 . Kety and J . Elkes, Eds . Pergason Press, Oxford (1961) .
2.
R .J . WURTMAN, J . AXELROD and D .E . KELLY . mic Press, New York (1968) .
3.
J . AXELROD, R .J . WURTMAN and S .H . SNYDER . (1965) .
4.
J .D . BARCHAS and A .B . LERNER . J . Neurochen . 11, 489 (1964) .
5.
R .J . WURTMAN and F . ANTON-TAY . Recent Pro r . in Honaone Rea . 25, 493 . Acedeaic Press, New York, London 1969 . -
6.
R .J . WURTMAN, J . AXELROD and E .W . CHU .
Science 141, 277 (1963) .
7.
E .W . CHU, R .J . WURTMAN and J . AXELROD .
Endocrinology 75, 238 (1964) .
8.
T . ISHIBASHI, D . W . HAHN, L . SRIVASTA, P . KUMARESAN and C .W . TURNER . Proc . Soc . Exp . Biol . Med . 122, 644 (1966) .
9.
A .B . HOUSSAY, J .H . PAZO and C .E . EPPER . 16, 202 (1966) .
The Pineal p . 115 . AcadeJ . Biol . Chem . 240, 949 --
Acta Physiol . Latinoamer .
10 .
N . CABASSA and R . SCIASCIA . 17 (1965) .
11 .
N .D . DE PROSPO, L .J . DE MARTINO and E .T . MCGRUINNESS . 183 (1968) .
12 .
G .D . NARANG, D . V . SINGH and C .W . TURNER . 125, 184 (1967) .
13 .
A .J. KASTIN end A . V . SCHALLY .
14 .
A .J . KASTIN, T .W . REDDING and A . SCHALLY . Med . 124, 1275 (1967) .
15 .
M .R . QUASTEL and R . RAHAMIMOFF .
16 .
R . RAHAMIMOFF, I . BRUDERMAN and G . GOLSHAMI . (1965) .
17 .
J . HERTZ-ESCHEL and R . RAHAMIMOFF .
18 .
G .S . ARUTYUNYAN, M .D . MASHKOUSKII and L .F . ROSHCHINA . T1330 (1964) .
19 .
T .J . MARCZYNSKI, N . YAMAGUCHI, G .M . LING and L . GRODZINSKA . Experientia 20, 435 (1964) .
Radioterap . Radiobiol . Fis . Med . 20, Life Sci . 7,
Proc . Soc . Exp . Hiol . Med .
Nature 213, 1238 (1967) . Proc . Soc . Exp . Biol .
Srit . J . Phanoacol . 24, 455 (1965) . Life Scien . 4, 2731
Life Sci . 4,
(1965) . Fed . Proc . 23,
128$
BRAIN PYRIDOXAL PH06PHOHINASE
20 .
J . BARCHAS, F . DA COSTA and S . SPECTOR .
21 .
E . ROLDAN and F . ANTON-TAY .
Vol. 9, No . 22
Nature 214, 919 (1967) .
Brain Res . il, 238 (1968) .
22 .
F . ANTON-TAY, CH . CHOU, S . ANTON and R .J . MURTNAN . 277 (1968) .
23 .
F . ANTON-TAY and R .J . MURTNAN .
Nature 221, 474 (1969) .
24 .
D .B . NCCORlIICK and E .E . SHELL . 1371 (1959) .
Proc . Nat . Acad . Sci . (Mash .) 45, -
25 .
M . LOVENBERG, H . WEISSBACH and S . UDENFRIED . 89 (1962) .
26 .
E . ROBERTS, K . ROTHSTEIN and C .F . BAXTER . Wed . 97, 796 (1958) .
27 .
D .B . !lCCORNICIC, M . E . GREGORY and E .E . SHELL . 2026 (1961) .
28 .
H . MADA and E .E . SHELL .
29 .
O .H . LOYRY, N .J . ROSERBROUGH, A .L . FARR and R .J . RANDALL . Chea . 193, 265 (1951) .
30 .
F . ANTON-TAY, B . ORTEGA and M . CRUZ . (1966) .
Stience 162,
J . Biol . Chew . 237, -
Proc . Soc . E7[D . Biol . J . Biol . Chew . 236, -
J . Biol . Chem . 236, 2089 (1961) . J . Biol .
Unpublished Observations
II' ~' 1283-1288, 1970 .
Pergamon Press
INCREASE Of BRAIN PYRIDOXAL PHOSPHOACIIUSE ACTIVITY FOLLOWING MELATOlfIN ADMINISTRATION . F . AntSn~ay, J . Sepulveda and S . Gonaâles Departamento de Neurobiologia, Instituto de Investigaciones Biosié dicaa, l1NAM . Apdo . Postal 70228, Mézico 20, D .F .
(Received 13 August 19?0; in final form 2 October 1970)
MELATONIN (N-acetil-5-rethozytryptaaiae) is synthetised uniquely in the pineala of eemmals through the action of the ~syme hydrozyindole-0~ethyltranaferase (HIOMT) 1 ' 2 .
Sin~e the activity of this enzyse varies
diurnally aaong rats kept under cyclic lighting conditions, it has been suggested that aelatonin secretion follows a similar rhyth~lc pattern 3 . Melatonin has been identified in urine and peripheral nerve 4 , however, neither the kidney nor the nervous tissue contain measurable HIOMT actiThis has been taken as evidence that the indola ia, in fact,
vity .
2'S. secreted from the pineal i ' The administration of melatonin to ezperi~ental anieals has been shown to influence gonadas ' 7 , thyroid 8 ' 9 ' 10 smooth musc1e1 5 '
16,17
sleep and EEG
+ 11,12
18,19,20,21 ~
anterior pituitary 3'14
Recently, it has also
been demonstrated that the intraperitoneal administration of 4elatonin increases brain serotonin concentration 22 .
The greatest increase of
serotonin is found in the oidbrain and the hypothalauus and it is here where circulating H 3-aelatonin is also most highly concentrated 23 .
It
has been suggested that both the neural and endocrine effects of ad,inisrated melatonin might be related to the changes that this compound produces in the functional activity of the central serotonin-containing neurons
22
Pyridozal phosphokinase (PL-kinase) catalyses the formation of pyridozal phosphate (PL) in the brnin24 .
1283
PL is a cofactor for many
BRAIN PYRIDOXAL PHOBPHOKIIQA3E
1284
Vol . 9, No. 22
reactions : as the prosthetic group of the ensy~e "aromatic L-wino acid decarboxylase" PL is essential for the synthesis of dopamine, 5hydroxytryptaaina (serotonin) and game-amino-butyric acid (GABA) 25 ' 26
.
Ye here report that intraperitoneal injections of melatonin are followed by a rapid increase in the activity of brain PL-kinase . Methods For two weeks prior to the experiment faule wister rata weighing 160 to 170 g were caged individually and kept under noiwal lighting (lights on from : 0600 to 1800 hours, with approxirately 27 to 54 mphot at the level of the animals) .
The rats were given Purina Chow and water ad
libitum . The desired amounts of malatonin were dissolved in one per cent ethanol solution .
Final volumes of 0 .5 ml were injected intreperitonea-
lly between 1000 and 1100 hours . nol solution .
Control animals received only the etha
At the times indicated after injection, the rats were de-
capitated and their brains immediately placed in small beakers containing cracked ice ; ali subsequent procedures were carried out at 0-3° C . Brains were homogenised in 1M phosphate buffer pM 6 .5 (1 :6 w/V) . tes were centrifugated at 30,000 g for 30 minutes .
The homogena-
PL-kinase activity
was determined in the supernatants as described by McCormick end Sne11 27 . The amounts of PL formed were determined with the phenylhydrazine reagent 28 .
Protein was determined according to the method of Lowry, Roserbrough,
Farr and Renda11 29 .
The specific activity of PL-kinase is expressed as
units per mg of protein,
(one unit is defined as the amount of protein
that fosns 1 mpM of PL in 1 hour at 37°C) .
Data are presented as mean +
S .D ., and analysed by the t-test . Results and discussion Groups of six rata received each 200 }ig of melatonin and were sacrificed 90 sin later .
The activity of PL-kinase in the brains of treated
rate was 1234 + 112 .6 units/q but the activity in brains of the control
Vol. 9, No. 22
ERAIN PYRmOXAL PHOBPHOKINA3S
group was only 575 + 29 units/g (P t 0 .001) .
1285
This led us to investigate
the changes of PL-kinase activity as a function of the time after ~elato n1n adainistration .
The anieals received the save doses as before and
were killed 45, 90, 180 or 360 ~1n later .
The highest levels of ansy~e
activity were detected 45 ~inutea after intteperiboneel administration melatonin .
The brains of the rats sacrificed 90 and 180 minutes after
the injection of melatonin showed an et wated PL-kinase activity . After 360 minutes the eruyme activity returned to aorwal values . (Table 1) . TABLE
1
Changes in brain Pyridozal-Phosphokinase (PL-kinase) activity in rats treated with melatonin . Groups of five rats received each 200 uq of melatonin intraperitoneaily, and were killed after various intervals . Ds4 are given as units per grae _+ standard dwiation . Tige after melatonin ad~inistration . 0 min .
633 + 110
45 mia .
2786 + 244 (X)
90 min .
1395 + 113 (X)
180 min .
1414 + 157 (X)
360 min . (X)
PL-kinase activity .
692 + 52
Differs from that of control rata : P < 0 .001 It has been reported that intraperitoneal aàiinistration of large
doses of melatonin appear to be less effective in raising brain serotonin concentration than smaller doses 22 .
Ye therefore studisd the effect
of various doses of melatoain on the activity of brain PL-kinase . Groups of 5 rats were injected rith varying doses of melatonin and sacrificed 90 minutes later .
As shown in Table 2, there was a direct relationship
between the amount of melabonin administered sad the elevation in PLkinase activity .
1288
HRAIN PYRiDOXAL PH05PHOKINASE TABLE
Vol . 9, No. 22
z
Effect of various intraperitoneal doses of meletonin on PyridoxalPhosphokinase (PL-kinase) activity of rat brain . Data are given as units per grar + standard deviation .
(x)
Melatonin
PL-kinase
rg/kq
Activity
Control
590 + 39
0 .25
666 + 113
0 .50
779 + 61 (x)
1 .0
1085 + 35(x)
2 .0
1203 + 95(x)
4 .0
1953 _+ 81(x)
Differs fror that of control rats : P c 0 .001 Experirents in which various concentrations of relatonin were added
to brain horogenates in vitro showed that the indole had no direct effect on the activity of the enayre .
Hence,
it appears likely that
the rsehanisr by which reletonin increases brain PL-kinase activity in vivo involves enhancing the de novo synthesis of the enzyme . 91nce PL is a cofactor for decarboxylases and tranaerinases in the brain24'25, it sight be anticipated that increases in the concentration of this cofactor following aelatonin adrinistration would cause changes
in
several brain functions .
The increases in brain serotonin 22 and
GABA 30 concentrations after relatonin adriniatration ray thus be rediatad by increases in the activities of 5-hydroxytryptophan and glutaric decarboxylases respectively .
It is suggested that relatonin modifies
the retabolic activity of the brain by increasing the availability of PL . Suoserv The intraperitoneal adainistration of relatonin to rata is followed by an increase in the activity of brain Pyridoxal phoaphokinase .
This
vol. 9, No. 22
BRAIN PYRIDOXAL PH05PHOKINASE
1287
effect is saximal 45 mlnutea after oelatonin administration .
Brain
pyridoxal phosphokinase activity shows a direct relationship to oelatonin in the range of 0 .25 - 4 .0 mg/kg body weight . References 1.
J . AXELROD, P .D . MACLEAN, R .W . ALBERE and H .W . WEISSBACH . Regional Neurocheeistrv p . 307 . 5 .3 . Kety and J . Elkes, Eds . Pergason Press, Oxford (1961) .
2.
R .J . WURTMAN, J . AXELROD and D .E . KELLY . mic Press, New York (1968) .
3.
J . AXELROD, R .J . WURTMAN and S .H . SNYDER . (1965) .
4.
J .D . BARCHAS and A .B . LERNER . J . Neurochen . 11, 489 (1964) .
5.
R .J . WURTMAN and F . ANTON-TAY . Recent Pro r . in Honaone Rea . 25, 493 . Acedeaic Press, New York, London 1969 . -
6.
R .J . WURTMAN, J . AXELROD and E .W . CHU .
Science 141, 277 (1963) .
7.
E .W . CHU, R .J . WURTMAN and J . AXELROD .
Endocrinology 75, 238 (1964) .
8.
T . ISHIBASHI, D . W . HAHN, L . SRIVASTA, P . KUMARESAN and C .W . TURNER . Proc . Soc . Exp . Biol . Med . 122, 644 (1966) .
9.
A .B . HOUSSAY, J .H . PAZO and C .E . EPPER . 16, 202 (1966) .
The Pineal p . 115 . AcadeJ . Biol . Chem . 240, 949 --
Acta Physiol . Latinoamer .
10 .
N . CABASSA and R . SCIASCIA . 17 (1965) .
11 .
N .D . DE PROSPO, L .J . DE MARTINO and E .T . MCGRUINNESS . 183 (1968) .
12 .
G .D . NARANG, D . V . SINGH and C .W . TURNER . 125, 184 (1967) .
13 .
A .J. KASTIN end A . V . SCHALLY .
14 .
A .J . KASTIN, T .W . REDDING and A . SCHALLY . Med . 124, 1275 (1967) .
15 .
M .R . QUASTEL and R . RAHAMIMOFF .
16 .
R . RAHAMIMOFF, I . BRUDERMAN and G . GOLSHAMI . (1965) .
17 .
J . HERTZ-ESCHEL and R . RAHAMIMOFF .
18 .
G .S . ARUTYUNYAN, M .D . MASHKOUSKII and L .F . ROSHCHINA . T1330 (1964) .
19 .
T .J . MARCZYNSKI, N . YAMAGUCHI, G .M . LING and L . GRODZINSKA . Experientia 20, 435 (1964) .
Radioterap . Radiobiol . Fis . Med . 20, Life Sci . 7,
Proc . Soc . Exp . Hiol . Med .
Nature 213, 1238 (1967) . Proc . Soc . Exp . Biol .
Srit . J . Phanoacol . 24, 455 (1965) . Life Scien . 4, 2731
Life Sci . 4,
(1965) . Fed . Proc . 23,
128$
BRAIN PYRIDOXAL PH06PHOHINASE
20 .
J . BARCHAS, F . DA COSTA and S . SPECTOR .
21 .
E . ROLDAN and F . ANTON-TAY .
Vol. 9, No . 22
Nature 214, 919 (1967) .
Brain Res . il, 238 (1968) .
22 .
F . ANTON-TAY, CH . CHOU, S . ANTON and R .J . MURTNAN . 277 (1968) .
23 .
F . ANTON-TAY and R .J . MURTNAN .
Nature 221, 474 (1969) .
24 .
D .B . NCCORlIICK and E .E . SHELL . 1371 (1959) .
Proc . Nat . Acad . Sci . (Mash .) 45, -
25 .
M . LOVENBERG, H . WEISSBACH and S . UDENFRIED . 89 (1962) .
26 .
E . ROBERTS, K . ROTHSTEIN and C .F . BAXTER . Wed . 97, 796 (1958) .
27 .
D .B . !lCCORNICIC, M . E . GREGORY and E .E . SHELL . 2026 (1961) .
28 .
H . MADA and E .E . SHELL .
29 .
O .H . LOYRY, N .J . ROSERBROUGH, A .L . FARR and R .J . RANDALL . Chea . 193, 265 (1951) .
30 .
F . ANTON-TAY, B . ORTEGA and M . CRUZ . (1966) .
Stience 162,
J . Biol . Chew . 237, -
Proc . Soc . E7[D . Biol . J . Biol . Chew . 236, -
J . Biol . Chem . 236, 2089 (1961) . J . Biol .
Unpublished Observations