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Ncurocndocrinology 15: 99 105 (1974)
L-Dopa as a Stimulus for LH Release in the Proestrous Rat:
Blockade of its Action by Pentobarbital Anesthesia1
J.H. W edig 3 and V.L. G ay 3
Interdepartmcm Toxicology Program and Reproductive Endocrinology Program,
Department of Pathology, University of Michigan, Ann Arbor, Mich.
Abstract. Intra-arterial injcctions(8 75mg/kg)of l-3,4 . dihydroxyKey Words
phenylalanine (t.-dopa) at I p.m. on the afternoon of proestrus proL-dopa
duced a rapid increase in scrum LH in unanesthetized female rats.
LH release
Maximum responses were observed following doses of 25 mg/kg alPentobarbital
though the smallest (2.7 mg/kg) and largest (225 mg/kg) doses were
Proestrous rat
ineffective in stimulating LH release. Pentobarbital anesthesia (40mg/kg,
i.p.) at 12.30 p.m. on proestrous prevented the responses to L-dopa. The 25 mg/kg dose
of t.-dopa, which produced maximum effects in the unanesthetized proestrous rat, caused
no detectable LH release in unanesthetized dicstrous rats. These results support the con­
cept that dopaminergic mechanisms are involved in the pre-ovulatory release of LH and
indicate that two of the conditions which prevent spontaneous pre-ovulatory LH release
(pentobarbital anesthesia or a lack of circulating estrogens) may similarly inhibit L-dopamediated LH release.
In rats, the incidence of ovulation is markedly reduced following
treatments which alter brain catecholamine levels [R ubinstein and
S awyer , 1970; K ordon and G lowinski, 1969; Coppola el al., 1966;
Barraclough and S awyer , 1957], Conversely, ovulation may be
Received: July 6th, 1973: revised MS accepted: December 13th, 1973.
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1 Supported by USPHS grants ES-0016 and HD-05318.
2 Recipient of an NIH Toxicology Training Fellowship. Present address: Environ­
mental Hygiene and Toxicology Dept., Olin Research Center, 275 Winchester Ave., New
Haven, Connecticut.
3 Career Development Awardee of the National Institute of Child Health and Human
Development. Present address: Department of Physiology, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania.
W edig/CJay
induced or reinstated by treatments which increase the availability
of endogenous or exogenous catecholamines [ R u b in s t e in and S a w y e r ,
1970; M e y er so n and S a w y e r , 1968; R a z ia n o el u L, 1971], Apparently
the catecholamines do not produce these effects by a direct action
on the pituitary gland [S c h n e id e r and M c C a n n , 1969] but, rather,
initiate a sequence of events involving the release of both LRH
[K a m b e r i et al., 1969; S c h n e id e r and M c C a n n , 1970b] and LH
[S c h n e id e r and M c C a n n , 1970a], The physiological importance of
the LH-releasing action of dopamine (DA) or one precursor of DA,
l-3,4, dihydroxyphenylalanine (L-dopa), has been suggested by the
observation that the release of LH induced by these compounds,
like the spontaneous pre-ovulatory LH release of the cyclic female
rat, is considerably reduced in the absence of circulating estrogens
[ R a z ia n o et al., 1971; S c h n e id e r and M c C a n n , 1970a], We have
sought to extend this correlation by demonstrating that pentobarbital
anesthesia, a potent blocker of the spontaneous pre-ovulatory LH
release [C r a m e r and B a r r a c l o u g h , 1971; C r a m e r et al., 1970],
also prevents L-dopa-induced LH release in the proestrous rat.
Mature female Holtzmann rats (240 260 g) were housed in air-conditioned quarters.
All animals were exposed to the lighting schedule of lights on 5 a.m. to 7 p.m. for a period
of 10 days after which vaginal smears were obtained daily at 9 a.m. for an additional 2
weeks. The responses of rats with regular 4- or 5-day cycles were assessed separately and
were combined only after it was determined that responses were similar. At 9 a.m. on the
morning of functional proestrous (as predicted by vaginal cytology) each rat was anes­
thetized with ether, laparotomized to confirm uterine 'ballooning' and cannulated via the
carotid artery [G ay et at., 1969], Approximately 4 h after these surgical procedures each
unanesthetized rat received 100 units of heparin via the cannula. Blood samples of 50 //I
were obtained at 20-min intervals prior to and following injections of L-dopa. The cannula
was flushed with 40 /d of warm physiological saline immediately after each 50 /d blood
sample had been obtained and after the injection of 0.5 n HCI or L-dopa (Calbiochem, lot
no. 979028). Each blood sample was added directly to an assay tube containing 475 /d of
phosphate-buffered saline (PBS) with I % egg albumin (Sigma Client. Co.) and assayed for
LH by radioimmunoassay [N iswender cl al. 1968] as described previously [G ay et at.,
1969]. Serum LH concentrations have been expressed in terms of B160, a partially purified
rat anterior pituitary gland extract, 1 mg of which is equivalent to 0.17 mg of NIH-LH-Si
or 5 mg of NIAMD-LH-RP-I, as determined by bioassay.
A total of 42 proestrous rats (6/group) were injected via the carotid cannula with
100 //I of 0.5 n HCI containing varying concentrations of L-dopa or, in the case of the
control group, with an equal volume of HCI alone. Doses of L-dopa were 2.7, 8, 25. 75 or
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Materials and Methods
L-Dopa and Pentobarbital on LH
225 mg/kg and were injected over a 2.5-min interval beginning at approximately I p.m.
One group of proestrous rats was injected with pentobarbital (40 mg/kg, i.p.) 30 min
prior to the injection of L-dopa (25 mg/kg). An additional 6 rats were cannulated on the
morning of diestrous and injected with L-dopa (25 mg/kg) at I p.m. On the morning
following the injection each rat was anesthetized with ether. The ovaries and oviducts
were examined and, in those animals in which they were present, tubal ova were counted.
No increase in serum LH concentration was observed during the
40-min interval following injection of acid into unanesthetized
proestrous rats. However, L-dopa (25 mg/kg) caused a significant
increase in circulating LH in such animals (table I), producing serum
LH levels comparable to those measured in castrated and intact
proestrous rats [G ay and M idgley, 1969; G ay et at., 1970]. The
8 and 75 mg/kg doses of L-dopa caused only a moderate increase
in serum LH concentration, and the smallest (2.7 mg/kg ) and largest
(225 mg/kg) doses of L-dopa were totally ineffective in inducing
LH release. Large doses of L-dopa which failed to stimulate LH
release did not prevent subsequent spontaneous LH release as
indicated by the occurrence of ovulation in such rats. In proestrous
rats pentobarbital anesthesia abolished the increase in serum LH
following L-dopa (25 mg/kg) injections, abolished the spontaneous
pre-ovulatory LH release, and prevented ovulation. Unanesthetized
diestrous rats failed to respond to the dose of L-dopa (25 mg/kg)
which was most effective in stimulating LH release in unanesthetized
proestrous animals.
In proestrous rats anesthetized with ether, 3rd ventricle injections
of DA, L-dopa or epinephrine are effective stimuli for LH release
or ovulation [S c h n e id e r and M c C a n n , 1970a; R a z ia n o et al., 1971],
Systemic (i.p.) injections of L-dopa also produce ovulation in
unanesthetized immature rats in which PMS-induced ovulation
has been prevented by treatment with «-methyl-/?-tyrosine [K o r d o n
and G l o w in s k i , 1969], Under these experimental conditions, 3rd
ventricular injections of norepinephrine and serotonin, or systemic
injections of a precursor of norepinephrine (DOPS) were ineffective
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Table 1. Plasma LH concentrations and incidence of ovulation in anesthetized and untreated cyclic female rats following systemic injections
of various doses o f L-dopa
Type of rat
Plasma LH relative to time of day
and time of injection, ng B160/mI
12.45 p.m.
LOO p.m.
1.40 p.m.
-15 min
0 min
-f40 min
Proestrous + HCI
4.00 p.m.
+ 180 min
Number Number Number Tubal ova,
ovumean ±
elevated lating
LH at
LH at
1.40 p.m. 4.00 p.m.
72.6 ± 18.0
14.1 ±0.9
10.8 ± 1.1
11.0 ±1.6
13.4 ± 2.6
88.8 ±15.6
12.1 ±0.9
12.6 ± 1.5
52.8 ±19.5
117.0 ±37.7
13.0 ± 1.9
I2.0± 1.7
11.0 ±2.1
158.6 ±65.1
265.2 ±70.5
14.1 ±1.5
14.4 ± 11.0
12.8 ±6.9
25.2 ± 8.7
89.0 ±20.8
13.1 ±1.7
10.5 ± 3.5
10.1 ±4.4
16.0 ± 6.4
13.0 ± 1.6
+ pentobarbital-'
17.8 ± 7.2
Wedig/G ay
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1 L-dopa dissolved in 100 /d o f 0.5 n HCI injected at 1.00 p.m .: animals receiving no L-dopa were injected with 100 /d o f 0.5 n HCI.
- ND = LH not detectable in quantity of blood (50 /d) used in each assay tube. In untreated proestrous rats, at times other than the
critical period, serum LH normally ranges from 5 to 10 ng/ml.
3 Pentobarbital (40 mg/kg, i.p.) given at 12.30 p.m.
4 Diestrous = day before functional proestrous and pre-ovulatory LH release.
in stimulating LH release. By contrast, R u b in s t e in and S a w y e r
[1970] found epinephrine to be the most effective stimulus for
ovulation in the pentobarbital-anesthetized proestrous rat. a condition
which, in light of the results presented here, may be attributed to
the inhibitory action of pentobarbital on the LH-reieasing action of
DA. Although R a z ia n o et al. [1971] also used pentobarbital-blocked
proestrous rats, they administered DA 105 min prior to the anesthesia
and the results presented here, as well as the data of S c h n e id e r
and M c C a n n [1970b], suggest that DA-induced LH release would
have occurred during the interval between injections.
Attempts have been made to correlate dopaminergic neurons with
the areas of the brain thought to control LH release and it is now
clear that biogenic amines are not uniformly distributed in the brain
but are segregated into distinct tracts and nuclei. The tuberoinfundibular tract which originates in the arcuate nucleus of the hypothalamus
and terminates in the median eminence region just above the pituitary
may be dopaminergic, as indicated by histochemical fluorescence
studies of the cellular localization of catecholamines in these neurons
[C a r l s so n et at., 1962], This tuberoinfundibular tract has been
implicated as the final pathway in the release of LH [F u x e el al.,
1969], However, the most direct evidence suggesting that DA can
function as a neural trigger (transmitter) for the ovulatory mechanism
is provided by injections of DA into the 3rd ventricle.
In our experiments we chose to inject L-dopa systemically rather
than to infuse DA into the 3rd ventricle. t.-dopa, which crosses
the blood brain barrier, is the immediate precursor of DA. Unanesthe­
tized proestrous rats responded to the systemic injections of L-dopa
with a rapid increase in serum LH concentrations. 25 mg/kg of
L-dopa was found to be the optimal dose: it induced a prompt
release of LH in 100% of the unanesthetized proestrous rats.
Because L-dopa was capable of inducing LH release in our unanestheti­
zed proestrous rats we tested the ability of pentobarbital anesthesia
to block this response and found that this anesthesia blocked the
L-dopa-induced increase in serum LH. Although it is not clear
why the larger doses of L-dopa failed to induce LH release in our
rats, it is evident that L-dopa may be converted to norepinephrine
within the animal and may act at various sites within the brain.
K a m b er i et al. [1970] have reported a similar diminished effect of
higher doses of DA injected into the 3rd ventricle of male rats.
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u-Dopa and Pentobarbital on LH
W edig/G ay
In both PMS-treated immature and mature cyclic rats spontaneous
pre-ovulatory LH release is prevented by systemic injections of an
antiserum which neutralizes endogenous estrogens [Ferin et a!.,
1969a, b]. This antiserum is also effective in preventing ovulation
induced by injections of L-dopa into the 3rd ventricle of anesthetized
proestrous rats [R aziano et al., 1971]. Schneider and McC ann
[1970a] have also reported that the diestrous rat is relatively insensitive
to injections of DA (4 /ig) into the 3rd ventricle. These data, combined
with our observation that the unanesthetized diestrous rat failed to
respond to the 25 mg/kg dose of i.-dopa, suggest that the mechanisms
of L-dopa-induced LH release are similar to those which cause LH
release in the proestrous rat. Spontaneous LH release is dependent
upon the presence of serum estrogen and on the activities of the
unanesthetized brain, and it is now clear that similar conditions are
required if L-dopa injections arc to produce optimal release of LH.
Barraclough , C.A. and S awyer, C.H.: Blockade of the release of pituitary ovulating
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Request reprints from : J ohn H. W edig, Department of Pathology. University of Michigan,
Ann Arbor, M l 48104 (USA)
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