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Serotonin oxidation by rat brain monoamine oxidase: Inhibition by 4-chloroamphetamine
Life Sciences Yol . 5, pp . 2247-2252, 1966 . Printed in Great Britain.
Pergamon Press Ltd .
SSROTONIN OXIDATION BY RAT BRAIN MONOAMINS OXIDASE : INHIBITION HY 4-CHLOROAMPHSTAMINS Ray W. Fuller The Lilly Research Laboratories, Eli Lilly and Compeay Indianapolis 6, Indiana (Received 1 September 1966) 4-Chloroamphetamine (4CAmph), 4-chloromethamphetamine and related compounds lower aerotonin levels in the brain of rata (1-4) .
This could occur by inhibition of aerotonin biosynthesis
or by release of bound aerotonin . Considerable evidence indicates that chloroamphetaminea do not inhibit aerotonin synthesis . Chloroamphetamines did not decrease the activity of tryptophan hydroxylas .e or of 5-hydroxytryptophan decarboxylase in vitro (2, 4), the uptake of tracer amounts of 5-hydroxytryptophan into rat brain (4), or the increase in brain aerotonin levels produced by administration of large doses of tryptophan or 5-hydroxytryptophan to rats (2) . Serotonin that is bound is protected from oxidation by moaoamine oxidase (MAO) ; when it is released, it is exposed to MAO . Roos and Werdinius found that when aerotonin was released by reserpine, brain 5-hydroxyindoleacetic acid levels increased . However, Pletscher (1,2) reported that brain 5-hydroxyindoleacetic acid levels were decreased along with administration of 4CAmph .
aerotonin after
This finding would be consistent with
decreased aerotonin formation ( 6 ,7) .
It is not explained if
4CAmph releases bound aerotonin, unless the drug simultaneously blocks aerotonin conversion to 5-hydroxyindoleacetic acid by inhibiting MAO (8) . 2247
2248
BEROTOIPIIQ OZIDATION
Yol . 5, No . 23
4CAmph, like amphetamine, is known to be a reversible inhibitor of MAO in vitro (9 ) .
Earlier studies, using liver MAO with
kynursaiae as substrate, did not indicate that the potency of 4CAmph as ea MAO inhibitor vas sufficient to produce significant MAO inhibition in vivo (9) .
We have investigated the inhibition
by 4CAaph of the ozidation of serotonin by rat brain mitochoadrisl MAO, sad have obtained indirect evidence that 4CAmph may inhibit rat brain MAO in vivo alaost coapletely .
These studies, described
is this paper, reaove an important argument against release as the mechanism of serotoaia depletion by 4CAmph .
.
Experimeatal Rat brain mitochondria were prepared, using the fractionation methods of Hogebooa (10) .
5-Hydro:ytryptamine-3'-C14 creatiniae
sulfate, 32 .0 Wc~iraole, vas obtained from Nuclear Chicago .
For
MAO assay, incubation mixtures were prepared according to the aethod of Wurtaen and Axelrod (11), ezcept that serotoain vas substrate sad the eztracting solvent vas ethyl acetate (12) .
Radio-
activity in acid-Washed ethyl acetate extracts vas determined by liquid scintillation spectrometry . For the in vivo ezperiaeats, male albino rate Weighing 130160 g were used .
4CAmph vas ia~ected i .p . at a dose of 0 .1 mmole~
kg (20 .6 ag~kg) .
The rata were decapitated and Whole brains Were
reaoved, frozen imaediately on dry ice, mad stored at -15' . Serotonin in the brains vas determined by the aethod of Mead and Finger (13) .
Drug levels Were measured by the methyl orange
spectrophotoaetric method of Azelrod (14) as modified by Dubnick et al . (15) .
Since 4CAmph is a reversible inhibitor of MAO,
in vivo inhibition cannot be measured directly by removing the brain sad analyzing for MAO activity, as this procedure dilutes the inhibitor.
SEROTONIN 0%IDATION
Yol . 5, No . 23
2249
Results and Discussion The in vitro inhibition of rat brain mitochoadrial MAO xas studied, using aerotonia as substrate st e concentration of 8 .3 x 10 -5 M .
Fifty per cent inhibition xas found to occur with
a concentration oP 1 .2 x 10'6 M 4CAnph . substrate concentration xas varied .
Ia another naperimeat,
Fig. 1 is a Liaexeaver-Burk
plot oP the resulting data, shoxing 4 CAmph to be a competitive inhibitor. Fig . 1 .
In vitro inhibition by 4-CAmph oP aerotonia oxidation by rat brain mü,ochondria . Reciprocal plot of velocity in mWmoles~min versus millimolar substrate concentration.
From this plot, the Km Por serotoain xas determined to be 65 .8 WM . The Ki for 4CA~ph xas 1.31 NM, calculated from the equation K1 =
1
- 1
(16) .
The ratio of .02.tor 2Ci~Km ahoxs that 4CAmph is as unexpectedly potent inhibitor is this system .
Vol . 5, No . 23
SEROTONIN OxIDATION
2250
idith these constants, Km and Ki, it is possible to calculate the theoretical per cent inhibition of serotoain oxidation in the brain by determining the concentrations of serotoain and 4CAmph . The equation ie
Y~i = Km +
s
(1~)
where Vi is the velocity in the presence of inhibitor, Vo ie the velocity in the absence of inhibitor, s is the substrate (serotoain) concentration in brain, and áp is calculated ae ri + Kil Km, 1 being the inhibitor K1 (4CAmp) concentration in brain . Table 1 chows serotoain and 4CAmph concentrations in brain after administration of 4CAmph .
With this information, V1~Vo was
calculated, and from this the theoretical per cent inhibition was determined . Table l . Hrs . after 4-CAmph Iaj . 0
These are oleo shown in Table 1 . Calculation of per teat inhibition of serotoain oxidation by 4-CAmph in rat brain . Concentration is brain, wM Berotonin 4-CAmph
Calculated values Vi~Vo ~ Inhibition
3.2
0
2
2 .8
131
.010
4
2.3
114
.012
16
i .2
68
.019
l .o
0
99 99 98
For ae long as 16 hrs after injection of 4CAmph, virtually complete inhibition of serotoain oxidation in the brain would be expected . It ws~ earlier found that, after 16 hrs, brain 5-hydroayindoleacetic acid and serotoain levels begin to return to normal (1,2,3) . Thus it appears that the lowering of brain 5-hydroxyindoleacetic acid levels by 4CAmph could be entirely accounted for by inhibition of l(AO oxidation of serotoain .
Vol . 5, No . 23
SEROTONIId OBIDATION
2251
Summary 4CAmph xas found to be a potent inhibitor of the oxidation of serotonin by rat brain mitochondrial MAO in vitro .
On the bneis
of the levels of 4CAmph and of eerotonin in braia, nearly cosplete inhibition of serotonia oxidation in vivo would be expected .
This
may account Por the loxered 5-hydroxyindoleacetic acid levels in brain after 4CAmph administration .
4CAmph probably exerts a dual
action on brain serotonin : release oP bound amine and inhibition of its metabolism by MAO . Ackaoxledgmente The assistance of Mr . C . Wesley Hines sad Miss Monna Jo Poole is acknoxledged xith appreciation . References 1.
A . PLETSCHER, W. P. BURKARD, H . BRUDERER sad K. F . Qey, Life Sciences 11, 828 (1963) .
2.
A . PLETSCHER, a . BARTHOLIAI, H . BRUDERER, W. P . HURKARD sad K. F . OEY, J. Pharmacol . 14~, 344 (1964) .
3.
R . W . FULLER, C . W. HIRES and J. MILLS, Fed . Proc . 2~, 146 (1964) .
4.
R . W . FULLER, C . W. HIKES sad J. MILLS, Biochem. Pharmacol . 14, 483 (1965) .
5.
H . E . ROOS sad H. WERDINIUS, Life Sciences 2, 92 (1963) .
6.
B . E. ROOS and H . WERDINIUS, Life Sciences ~, 105 (1962) .
7.
D . F. SHARMAR and S. E . SMITH, J . Neurochem. ~, 403 (1962),
8.
A . PLETSCHER, M . DA PRADA, a. HARTHOLINI~ W. P . HURKARD and H . BRUDERER, Life Sciences 4, 2301 (1965) .
9.
R. W. FULLER and C . P. WALTERS, Biochem. Pharnacol . 14, 159 (1965) "
10 .
a. H . HOQEBOOM, ia Methods in Enzymology 1, 16 (1955) "
2252 11 .
SF.ROTONIN OBIDATION
Vol . 5, No . 23
R . J. WURTMAR sad J. AXELROD, Biochem . Pharmacol. _12, 1439 (1963) "
12 .
R . B . McCAI~lAR, M . W. McCAMAR, J . M. HURT and M. S . SMITH, J. Reurochem. 12, 15 (1965) .
13 .
J. A . R . MEAD and K. F . FIRaER, Biochem . Pharmacol. 6,
52
(1961) . 14 .
J. AXELROD, J . Pharmacol. 110, 315 (1954) .
15 .
B . DUBRICK, a . A . LESSOR, R . LEVERETT, D . F . MORGAN Gad
16 .
d. E . PHILLIPS, J . Pharmacol . ~, 8g (1963) .
M. DIXOR aad S . C . WEBB, Eazvmes . Academic Press, Rew York (1964) .
lq .
S . UDERF3IERD, Pharmacol . Rev . ~ 43 (1966) .
Pergamon Press Ltd .
SSROTONIN OXIDATION BY RAT BRAIN MONOAMINS OXIDASE : INHIBITION HY 4-CHLOROAMPHSTAMINS Ray W. Fuller The Lilly Research Laboratories, Eli Lilly and Compeay Indianapolis 6, Indiana (Received 1 September 1966) 4-Chloroamphetamine (4CAmph), 4-chloromethamphetamine and related compounds lower aerotonin levels in the brain of rata (1-4) .
This could occur by inhibition of aerotonin biosynthesis
or by release of bound aerotonin . Considerable evidence indicates that chloroamphetaminea do not inhibit aerotonin synthesis . Chloroamphetamines did not decrease the activity of tryptophan hydroxylas .e or of 5-hydroxytryptophan decarboxylase in vitro (2, 4), the uptake of tracer amounts of 5-hydroxytryptophan into rat brain (4), or the increase in brain aerotonin levels produced by administration of large doses of tryptophan or 5-hydroxytryptophan to rats (2) . Serotonin that is bound is protected from oxidation by moaoamine oxidase (MAO) ; when it is released, it is exposed to MAO . Roos and Werdinius found that when aerotonin was released by reserpine, brain 5-hydroxyindoleacetic acid levels increased . However, Pletscher (1,2) reported that brain 5-hydroxyindoleacetic acid levels were decreased along with administration of 4CAmph .
aerotonin after
This finding would be consistent with
decreased aerotonin formation ( 6 ,7) .
It is not explained if
4CAmph releases bound aerotonin, unless the drug simultaneously blocks aerotonin conversion to 5-hydroxyindoleacetic acid by inhibiting MAO (8) . 2247
2248
BEROTOIPIIQ OZIDATION
Yol . 5, No . 23
4CAmph, like amphetamine, is known to be a reversible inhibitor of MAO in vitro (9 ) .
Earlier studies, using liver MAO with
kynursaiae as substrate, did not indicate that the potency of 4CAmph as ea MAO inhibitor vas sufficient to produce significant MAO inhibition in vivo (9) .
We have investigated the inhibition
by 4CAaph of the ozidation of serotonin by rat brain mitochoadrisl MAO, sad have obtained indirect evidence that 4CAmph may inhibit rat brain MAO in vivo alaost coapletely .
These studies, described
is this paper, reaove an important argument against release as the mechanism of serotoaia depletion by 4CAmph .
.
Experimeatal Rat brain mitochondria were prepared, using the fractionation methods of Hogebooa (10) .
5-Hydro:ytryptamine-3'-C14 creatiniae
sulfate, 32 .0 Wc~iraole, vas obtained from Nuclear Chicago .
For
MAO assay, incubation mixtures were prepared according to the aethod of Wurtaen and Axelrod (11), ezcept that serotoain vas substrate sad the eztracting solvent vas ethyl acetate (12) .
Radio-
activity in acid-Washed ethyl acetate extracts vas determined by liquid scintillation spectrometry . For the in vivo ezperiaeats, male albino rate Weighing 130160 g were used .
4CAmph vas ia~ected i .p . at a dose of 0 .1 mmole~
kg (20 .6 ag~kg) .
The rata were decapitated and Whole brains Were
reaoved, frozen imaediately on dry ice, mad stored at -15' . Serotonin in the brains vas determined by the aethod of Mead and Finger (13) .
Drug levels Were measured by the methyl orange
spectrophotoaetric method of Azelrod (14) as modified by Dubnick et al . (15) .
Since 4CAmph is a reversible inhibitor of MAO,
in vivo inhibition cannot be measured directly by removing the brain sad analyzing for MAO activity, as this procedure dilutes the inhibitor.
SEROTONIN 0%IDATION
Yol . 5, No . 23
2249
Results and Discussion The in vitro inhibition of rat brain mitochoadrial MAO xas studied, using aerotonia as substrate st e concentration of 8 .3 x 10 -5 M .
Fifty per cent inhibition xas found to occur with
a concentration oP 1 .2 x 10'6 M 4CAnph . substrate concentration xas varied .
Ia another naperimeat,
Fig. 1 is a Liaexeaver-Burk
plot oP the resulting data, shoxing 4 CAmph to be a competitive inhibitor. Fig . 1 .
In vitro inhibition by 4-CAmph oP aerotonia oxidation by rat brain mü,ochondria . Reciprocal plot of velocity in mWmoles~min versus millimolar substrate concentration.
From this plot, the Km Por serotoain xas determined to be 65 .8 WM . The Ki for 4CA~ph xas 1.31 NM, calculated from the equation K1 =
1
- 1
(16) .
The ratio of .02.tor 2Ci~Km ahoxs that 4CAmph is as unexpectedly potent inhibitor is this system .
Vol . 5, No . 23
SEROTONIN OxIDATION
2250
idith these constants, Km and Ki, it is possible to calculate the theoretical per cent inhibition of serotoain oxidation in the brain by determining the concentrations of serotoain and 4CAmph . The equation ie
Y~i = Km +
s
(1~)
where Vi is the velocity in the presence of inhibitor, Vo ie the velocity in the absence of inhibitor, s is the substrate (serotoain) concentration in brain, and áp is calculated ae ri + Kil Km, 1 being the inhibitor K1 (4CAmp) concentration in brain . Table 1 chows serotoain and 4CAmph concentrations in brain after administration of 4CAmph .
With this information, V1~Vo was
calculated, and from this the theoretical per cent inhibition was determined . Table l . Hrs . after 4-CAmph Iaj . 0
These are oleo shown in Table 1 . Calculation of per teat inhibition of serotoain oxidation by 4-CAmph in rat brain . Concentration is brain, wM Berotonin 4-CAmph
Calculated values Vi~Vo ~ Inhibition
3.2
0
2
2 .8
131
.010
4
2.3
114
.012
16
i .2
68
.019
l .o
0
99 99 98
For ae long as 16 hrs after injection of 4CAmph, virtually complete inhibition of serotoain oxidation in the brain would be expected . It ws~ earlier found that, after 16 hrs, brain 5-hydroayindoleacetic acid and serotoain levels begin to return to normal (1,2,3) . Thus it appears that the lowering of brain 5-hydroxyindoleacetic acid levels by 4CAmph could be entirely accounted for by inhibition of l(AO oxidation of serotoain .
Vol . 5, No . 23
SEROTONIId OBIDATION
2251
Summary 4CAmph xas found to be a potent inhibitor of the oxidation of serotonin by rat brain mitochondrial MAO in vitro .
On the bneis
of the levels of 4CAmph and of eerotonin in braia, nearly cosplete inhibition of serotonia oxidation in vivo would be expected .
This
may account Por the loxered 5-hydroxyindoleacetic acid levels in brain after 4CAmph administration .
4CAmph probably exerts a dual
action on brain serotonin : release oP bound amine and inhibition of its metabolism by MAO . Ackaoxledgmente The assistance of Mr . C . Wesley Hines sad Miss Monna Jo Poole is acknoxledged xith appreciation . References 1.
A . PLETSCHER, W. P. BURKARD, H . BRUDERER sad K. F . Qey, Life Sciences 11, 828 (1963) .
2.
A . PLETSCHER, a . BARTHOLIAI, H . BRUDERER, W. P . HURKARD sad K. F . OEY, J. Pharmacol . 14~, 344 (1964) .
3.
R . W . FULLER, C . W. HIRES and J. MILLS, Fed . Proc . 2~, 146 (1964) .
4.
R . W . FULLER, C . W. HIKES sad J. MILLS, Biochem. Pharmacol . 14, 483 (1965) .
5.
H . E . ROOS sad H. WERDINIUS, Life Sciences 2, 92 (1963) .
6.
B . E. ROOS and H . WERDINIUS, Life Sciences ~, 105 (1962) .
7.
D . F. SHARMAR and S. E . SMITH, J . Neurochem. ~, 403 (1962),
8.
A . PLETSCHER, M . DA PRADA, a. HARTHOLINI~ W. P . HURKARD and H . BRUDERER, Life Sciences 4, 2301 (1965) .
9.
R. W. FULLER and C . P. WALTERS, Biochem. Pharnacol . 14, 159 (1965) "
10 .
a. H . HOQEBOOM, ia Methods in Enzymology 1, 16 (1955) "
2252 11 .
SF.ROTONIN OBIDATION
Vol . 5, No . 23
R . J. WURTMAR sad J. AXELROD, Biochem . Pharmacol. _12, 1439 (1963) "
12 .
R . B . McCAI~lAR, M . W. McCAMAR, J . M. HURT and M. S . SMITH, J. Reurochem. 12, 15 (1965) .
13 .
J. A . R . MEAD and K. F . FIRaER, Biochem . Pharmacol. 6,
52
(1961) . 14 .
J. AXELROD, J . Pharmacol. 110, 315 (1954) .
15 .
B . DUBRICK, a . A . LESSOR, R . LEVERETT, D . F . MORGAN Gad
16 .
d. E . PHILLIPS, J . Pharmacol . ~, 8g (1963) .
M. DIXOR aad S . C . WEBB, Eazvmes . Academic Press, Rew York (1964) .
lq .
S . UDERF3IERD, Pharmacol . Rev . ~ 43 (1966) .