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Trytophan hydroxylase changes in midbrain of the rat after chronic morphine administration
I~ife Sc~ences Vol. 9 Part I, pp. 633-837, 1970. tint in Great Hrita~n
Pergamon Press
TRYPTOPfiAN HYDRO7CYLASE CHANGES IN MIDBRAIN OF THE RAT AFTER CHRONIC MORPHINE ADMINISTRATION ) Efrain C . Azmitia, Jr . The Rockefeller University, New York, N . Y .
10021
Paul Hess and Donald Reis Department of Neurology Cornell University Medical College, New York, N . Y .
10021
(Received 14 January 1970; in final form 23 March 1970) Recently it ha~ been proposed that the physcial dependence and tolerance associated with chronic administration of morphine are related to an increase in turnover of serotonin in the brain (1) .
Furthermore, the evidence that the
development of tolerance requires de novo protein synthesis and that tolerance can be partially abolished by pretreatment with pats-chlorophenylalanine, a specific inhibitor of tryptophan hydroxylase (2), suggests that the increase in serotonin turnover induced by morphine might be due to a de novo synthesis of tryptophan hydroxylase, the rate-limiting enzyme in serotonin metabolism (3) . In the present report we have studied the changes in tryptophan hydroxylase activity during a tolerance-withdrawal cycle to morphine in the mldbrain of the rat .
We shall show that the predicted changes in this enzyme occur .
The re-
port that corticoeterone levels in the rat can regulate tryptophan hydroxylase activity and our finding that chronic injection of saline led to a significant increase in this enzyme, permit us to speculate on a possible hormonal mechanram for explaining the action of morphine on the serotonin system .
1 This investigation was supported by USPHS grants MH 13189, GM 01789, NB 06911, and NB 31756 .
633
834
TRYPTOPHAN HYDRORYLA3E ACTIVITY
Vol. 9, No. 11
Method Female Sprague-Dawley rats, weighing between 230 and 260 gm, were housed 4 - 6 to a cage in a thermally controlled room with a 12-hour light-dark cycle, and maintained on standard lab chow ad libitum . into four equal groups for the study . during the course of the experiment .
Forty animals were divided
Unhandlad controls were not disturbed Tolerant and withdrawal groups received
morphine sulphate injected eubcutaneously over a 16-day period, beginning with a 5 mg/kg dose administered twice a day (8 :00 a .m. and 4 :00 p .m .), and increasing up to a done of 95 mg/kg given three times a day (8 :'00 a,m ., noon, 4 :00 p .m .) .
Tha tolerant animals were killed on the morning of the sixteenth day,
2 hours after an injection of 130 mg/kg morphine .
Tha withdrawal group re-
ceived a similar dose of morphine 48 hours before they were killed .
A saline
control group received injections of equal volume of isotonic saline on the same schedule ae the tolerant group . The rats were decapitated and the midbrain was dissected from the brain . Thin region was selected for assay because the cell bodies of moat ascending eerotonin-containing neurone are localized here (4) and changee in enzyme activity are easily detected in this region (5) .
Tryptophan hydroxylase acti-
vity was measured on whole homogenate by a modification of 14 C-02 trapping from tryptophan-1- 14C (Calbiochem, 9 .6 mCi/mmole)
(6) .
The final concentration
of tryptophan in the reaction mixture was 4 x 10- 6 M which would eliminate any direct decarboxylaticn of tyrptophan since the Km for the decarboxylase enzyme is much lower for S-OÜ-tryptophan (2 x 10 -5 M) than for tr~ptophan (3 x 10 -3 M) (7) . Results The levels of tryptophan hydroxylase activity measured in the midbrain in the four groups tested are ahawn in graphic forna in Figure 1 .
The saline -
injected group demonstrated a significant increase in this enzyme as compared with the unhandled controls .
This change can be attributed to the known stress
caused in the animal by hai.n g handled and injected subcutaneously while
Vol. 9, No. 11
835
TRYPTOPHAN HYDRORYLA3E ACTIVITY
0.8 v~ 0.6
a~
0 .4
0
E a 0.2 0
P<0 .01
P<0.02
FIG .
w P<0.05
1
Tryptophan hpdroxylase activity levels . The enzyme activity is expressed as the amount of 14C-02 released in one hour's incubation at 37°C from tryptophan-l-l~+C at pH 8 .2 . The p values and the standard error were determined by using Student's t-test on 8 determinations for each group . constrained in a Plexiglas tube, since it has been shown that streasora which cause an activation of the pituitary-adrenal axis will elevate the level of tryptophan hydroxylase in the midbrain of normal rata (8) .
The enzyme level
in tolerant animals showed a 40% increase when compared with unhandled controls and a 17% increase when compared with the saline group .
Thus, tolerance to
morphine is accompanied by an enzyme increase in the midbrain which is consistent with the reported increase of aerotonin turnover .
After 48 hours of
896
TRYPTOPAAN AYDROXYLASE ACTIVITY
Vol. 9, No. 11
withdrawal, the enzyme level has dropped from its tolerant level but still remains elevated when compared with unhandled controls (E < .O1) though not with saline controls . Discussion The increase in tryptophan hydroxylase levels during chronic administration of morphine is consistent with the report that serotonin turnover is also increased .
However, the magnitude of this increase in enzymatic activity ie
not comparable to the 200 - 500% increase in aerotonin turnover found in whole brains of mice following morphine pellet implantation (1) . possible explanations for thin discrepancy : morphine administration differ,
There are several
(a) the species and method of
(b) the in vitro enzyme-activity assay may
reflect only a fraction of the actual in vivo synthesis rate,
This seems
unlikely since a recent study has shown a good correlation between enzyme levels and turnover rates in rats (9),
(c) the enzyme activity measures in the
midbrain may not reflect the activity in nerve terminals, since the newly synthesized enzyme may be rapidly transported to nerve terminals where it may be allowed to accumulate .
Therefore, although the midbrain is a sensitive indi-
cator of de novo synthesis, problems may arise if an attempt is made to compare results from this area with those on whole brain . The increase in enzyme activity induced by the injection of saline alone is of particular interest gince this treatment would be sufficient to stimulate production of corticosterone in normal rats .
It has been shown that, in adrenal-
ectomized rats, exogenously administered corticoaterone raises the level of tryptophan hydroxylase in the midbrain by a mechanism dependent on protein synthesis (5) .
Moreover, activity of this enzyme can be elevated in normal rat
midbrain by various stimuli, such as electric foot shock, cold exposure, and ether anesthesia, which activate the pituitary-adrenal axis as judged by the levels of plasma corticosterone (8) .
The question is therefore raised whether
the effects of morphine in increasing brain tryptophan hydroaylase activity might be partially mediated by hormonal induction,aince
it is well known that
Vol . 9, No. 11
TRYPTOPHAN HYDROXYLA3E ACTIVITY
637
morphine is a powerful activator of the pituitary-adrenal axis (10) . References 1.
E, L . WAY, H . H, LOii, and F . SHBN, Science 162, 1290 (1968) .
2.
K . KOE and A . J . WEISSMAN, J . Pharmacol . Sxy . TheraD . 154, 499 (1969) .
3.
H . GREEN and J, L . SAWYER, Aaalyt . Biochem . 15, 53 (1966) .
4.
K . FI1%~E, Z . Zellforech . 61, 710 (1964) .
5.
E, C . A?!~??'?A , Jr . and B, S . McEWEN, Science 166, 1274 (1969) .
6.
A . ICHIYAMA, S . NARAMIïRA, Y . NISHIZURA, and 0 . HAYAISEiI, Adv . Pharmacol . 6A, 5 (1968) .
7.
W. LOVENBERG, H, iJSISSBACH, and S, iJDENFRIEND, J . Biol . Chem . 237, 89 (1962) .
8.
E, C . AZlffTIA, Jr . and B . S . McSWEN, in preparation .
9.
S, C . AZI+IITIA, Jr ., S . AhGERI, and E . COSTA, in preparation .
10 .
R, GEORGE and E, L . FLAY, Brit . J . Phasmacol . 10, 260 (1955) .
Pergamon Press
TRYPTOPfiAN HYDRO7CYLASE CHANGES IN MIDBRAIN OF THE RAT AFTER CHRONIC MORPHINE ADMINISTRATION ) Efrain C . Azmitia, Jr . The Rockefeller University, New York, N . Y .
10021
Paul Hess and Donald Reis Department of Neurology Cornell University Medical College, New York, N . Y .
10021
(Received 14 January 1970; in final form 23 March 1970) Recently it ha~ been proposed that the physcial dependence and tolerance associated with chronic administration of morphine are related to an increase in turnover of serotonin in the brain (1) .
Furthermore, the evidence that the
development of tolerance requires de novo protein synthesis and that tolerance can be partially abolished by pretreatment with pats-chlorophenylalanine, a specific inhibitor of tryptophan hydroxylase (2), suggests that the increase in serotonin turnover induced by morphine might be due to a de novo synthesis of tryptophan hydroxylase, the rate-limiting enzyme in serotonin metabolism (3) . In the present report we have studied the changes in tryptophan hydroxylase activity during a tolerance-withdrawal cycle to morphine in the mldbrain of the rat .
We shall show that the predicted changes in this enzyme occur .
The re-
port that corticoeterone levels in the rat can regulate tryptophan hydroxylase activity and our finding that chronic injection of saline led to a significant increase in this enzyme, permit us to speculate on a possible hormonal mechanram for explaining the action of morphine on the serotonin system .
1 This investigation was supported by USPHS grants MH 13189, GM 01789, NB 06911, and NB 31756 .
633
834
TRYPTOPHAN HYDRORYLA3E ACTIVITY
Vol. 9, No. 11
Method Female Sprague-Dawley rats, weighing between 230 and 260 gm, were housed 4 - 6 to a cage in a thermally controlled room with a 12-hour light-dark cycle, and maintained on standard lab chow ad libitum . into four equal groups for the study . during the course of the experiment .
Forty animals were divided
Unhandlad controls were not disturbed Tolerant and withdrawal groups received
morphine sulphate injected eubcutaneously over a 16-day period, beginning with a 5 mg/kg dose administered twice a day (8 :00 a .m. and 4 :00 p .m .), and increasing up to a done of 95 mg/kg given three times a day (8 :'00 a,m ., noon, 4 :00 p .m .) .
Tha tolerant animals were killed on the morning of the sixteenth day,
2 hours after an injection of 130 mg/kg morphine .
Tha withdrawal group re-
ceived a similar dose of morphine 48 hours before they were killed .
A saline
control group received injections of equal volume of isotonic saline on the same schedule ae the tolerant group . The rats were decapitated and the midbrain was dissected from the brain . Thin region was selected for assay because the cell bodies of moat ascending eerotonin-containing neurone are localized here (4) and changee in enzyme activity are easily detected in this region (5) .
Tryptophan hydroxylase acti-
vity was measured on whole homogenate by a modification of 14 C-02 trapping from tryptophan-1- 14C (Calbiochem, 9 .6 mCi/mmole)
(6) .
The final concentration
of tryptophan in the reaction mixture was 4 x 10- 6 M which would eliminate any direct decarboxylaticn of tyrptophan since the Km for the decarboxylase enzyme is much lower for S-OÜ-tryptophan (2 x 10 -5 M) than for tr~ptophan (3 x 10 -3 M) (7) . Results The levels of tryptophan hydroxylase activity measured in the midbrain in the four groups tested are ahawn in graphic forna in Figure 1 .
The saline -
injected group demonstrated a significant increase in this enzyme as compared with the unhandled controls .
This change can be attributed to the known stress
caused in the animal by hai.n g handled and injected subcutaneously while
Vol. 9, No. 11
835
TRYPTOPHAN HYDRORYLA3E ACTIVITY
0.8 v~ 0.6
a~
0 .4
0
E a 0.2 0
P<0 .01
P<0.02
FIG .
w P<0.05
1
Tryptophan hpdroxylase activity levels . The enzyme activity is expressed as the amount of 14C-02 released in one hour's incubation at 37°C from tryptophan-l-l~+C at pH 8 .2 . The p values and the standard error were determined by using Student's t-test on 8 determinations for each group . constrained in a Plexiglas tube, since it has been shown that streasora which cause an activation of the pituitary-adrenal axis will elevate the level of tryptophan hydroxylase in the midbrain of normal rata (8) .
The enzyme level
in tolerant animals showed a 40% increase when compared with unhandled controls and a 17% increase when compared with the saline group .
Thus, tolerance to
morphine is accompanied by an enzyme increase in the midbrain which is consistent with the reported increase of aerotonin turnover .
After 48 hours of
896
TRYPTOPAAN AYDROXYLASE ACTIVITY
Vol. 9, No. 11
withdrawal, the enzyme level has dropped from its tolerant level but still remains elevated when compared with unhandled controls (E < .O1) though not with saline controls . Discussion The increase in tryptophan hydroxylase levels during chronic administration of morphine is consistent with the report that serotonin turnover is also increased .
However, the magnitude of this increase in enzymatic activity ie
not comparable to the 200 - 500% increase in aerotonin turnover found in whole brains of mice following morphine pellet implantation (1) . possible explanations for thin discrepancy : morphine administration differ,
There are several
(a) the species and method of
(b) the in vitro enzyme-activity assay may
reflect only a fraction of the actual in vivo synthesis rate,
This seems
unlikely since a recent study has shown a good correlation between enzyme levels and turnover rates in rats (9),
(c) the enzyme activity measures in the
midbrain may not reflect the activity in nerve terminals, since the newly synthesized enzyme may be rapidly transported to nerve terminals where it may be allowed to accumulate .
Therefore, although the midbrain is a sensitive indi-
cator of de novo synthesis, problems may arise if an attempt is made to compare results from this area with those on whole brain . The increase in enzyme activity induced by the injection of saline alone is of particular interest gince this treatment would be sufficient to stimulate production of corticosterone in normal rats .
It has been shown that, in adrenal-
ectomized rats, exogenously administered corticoaterone raises the level of tryptophan hydroxylase in the midbrain by a mechanism dependent on protein synthesis (5) .
Moreover, activity of this enzyme can be elevated in normal rat
midbrain by various stimuli, such as electric foot shock, cold exposure, and ether anesthesia, which activate the pituitary-adrenal axis as judged by the levels of plasma corticosterone (8) .
The question is therefore raised whether
the effects of morphine in increasing brain tryptophan hydroaylase activity might be partially mediated by hormonal induction,aince
it is well known that
Vol . 9, No. 11
TRYPTOPHAN HYDROXYLA3E ACTIVITY
637
morphine is a powerful activator of the pituitary-adrenal axis (10) . References 1.
E, L . WAY, H . H, LOii, and F . SHBN, Science 162, 1290 (1968) .
2.
K . KOE and A . J . WEISSMAN, J . Pharmacol . Sxy . TheraD . 154, 499 (1969) .
3.
H . GREEN and J, L . SAWYER, Aaalyt . Biochem . 15, 53 (1966) .
4.
K . FI1%~E, Z . Zellforech . 61, 710 (1964) .
5.
E, C . A?!~??'?A , Jr . and B, S . McEWEN, Science 166, 1274 (1969) .
6.
A . ICHIYAMA, S . NARAMIïRA, Y . NISHIZURA, and 0 . HAYAISEiI, Adv . Pharmacol . 6A, 5 (1968) .
7.
W. LOVENBERG, H, iJSISSBACH, and S, iJDENFRIEND, J . Biol . Chem . 237, 89 (1962) .
8.
E, C . AZlffTIA, Jr . and B . S . McSWEN, in preparation .
9.
S, C . AZI+IITIA, Jr ., S . AhGERI, and E . COSTA, in preparation .
10 .
R, GEORGE and E, L . FLAY, Brit . J . Phasmacol . 10, 260 (1955) .