Proc. Natl. Acad. Sci.
USA
Vol. 88, pp. 2763-2767, April 1991
Physiology/Pharmacology
Interleukin la inhibits prostaglandin E2 release to suppress
pulsatile release of luteinizing hormone but not follicle-
stimulating hormone
(third-ventricular injection/in vitro hypothalamic incubation/plasma gonadotropins/Iuliberin)
VALERIA RETTORI*, MARTHA F. GIMENOt, ARMANDO KARARAf, M. CARMEN GONZALEZ,
AND SAMUEL M. MCCANN*
*Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX
75235-9040; tCEFYBO, Serrano 665, Buenos Aires 1414, Argentina; tDepartment of Nephrology, Vanderbilt University, Nashville, TN 37232; and
Departamento de Fisiologia, Universidad de La Laguna, Tenerife, E-38320 Spain
Contributed by Samuel M. McCann, December 31, 1990
ABSTRACT Interleukin la (IL-la), a powerful endoge-
nous pyrogen released from monocytes and macrophages by
bacterial endotoxin, stimulates corticotropin, prolactin, and
somatotropin release and inhibits thyrotropin release by hy-
pothalamic action. We injected recombinant human IL-la into
the third cerebral ventricle, to study its effect on the pulsatile
release of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) in conscious, freely moving, ovariectomized
rats. Intraventricular injection of 0.25 pmol of IL-la caused an
almost immediate reduction of plasma LH concentration; this
decrease was statistically significant 20 min after i jection and
occurred through a highly significant reduction in the number
of LH pulses, with no effect on pulse amplitude. In contrast,
there was no change in pulse frequency but a small significant
elevation in amplitude of FSH pulses. Intraventricular iujec-
tion of the diluent had no effect on gonadotropin release. The
results provide further evidence for separate hypothalamic
control mechanisms for FSH and LH release. To determine the
mechanism of the suppression of LH release, mediobasal
hypothalamic fragments were incubated in vitro with IL-1a (10
pM) and the release of LH-releasing hormone (LHRH) and
prostaglandin E2 into the medium was measured by RIA in the
presence or absence of norepinephrine (50 jpM). IL-la reduced
basal LHRH release and blocked LHRH release induced by
norepinephrine. It had no effect on the basal release of pros-
taglandin E2; however, it completely inhibited the release of
PGE2 evoked by norepinephrine. To evaluate the possibility
that IL-la might also interfere with the epoxygenase pathway
of arachidonic acid metabolism, epoxyeicosatrienoic acids were
also measured. IL-la had no effect on the content of epoxy-
eicosatrienoic acids in the hypothalamic fragments as measured
by gas chromatography and mass spectrometry. In conclusion,
IL-la suppresses LH but not FSH release by an almost
complete cessation of pulsatile release of LH in the castrated
rat. The mechanism of this effect appears to be by inhibition of
prostaglandin E2-mediated release of LHRH.
Bacterial infection induces fever and alterations of pituitary
hormone secretion (1). Because bacterial pyrogens induce
fever, it was first believed that these hormonal changes might
be mediated directly by the bacterial pyrogens. However,
early work revealed a delay between the injection of such
pyrogens and the onset of fever, which suggested that an
endogenous pyrogen was produced by the bacterial pyrogens
(2).
Indeed, a purified preparation of bacterial pyrogen injected
intravenously into dogs induced fever, after a delay, that was
The publication costs of this article were defrayed in part by page charge
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2763
accompanied by an increase in corticotropin secretion as
evidenced by a rise in plasma cortisol concentration (3).
Endogenous pyrogen was also shown to evoke corticotropin
release (4). Finally, one of these active substances, interleu-
kin 1 (IL-1), was isolated, its structure was determined, and
it was synthesized (5). It is released from macrophages and
other cells of the immune system following interaction with
bacterial pyrogen, a lipopolysaccharide that reacts with spe-
cific receptors on the cells to induce the release of IL-1,
which is an important endogenous pyrogen (5).
Research with IL-1 showed that it not only induces fever
but also induces the release of corticotropin by a hypotha-
lamic action that involves release of corticotropin-releasing
factor (6, 7) and possibly by a direct pituitary action (8). It
also alters release of other anterior pituitary hormones,
including prolactin and growth hormone, and inhibits thyro-
tropin release (9). The pattern of pituitary hormone release
induced by IL-1 is quite similar to that evoked by stressful
stimuli in humans: release of corticotropin, prolactin, and
growth hormone and inhibition of thyrotropin release (10).
Because stress also inhibits gonadotropin secretion (11), it is
reasonable to evaluate the effects of this monokine on
gonadotropin secretion. IL-la has been shown to suppress
the release of luteinizing hormone (LH) in castrated animals
(12) and to block the preovulatory release of LH (13). Since
LH release is pulsatile in castrated rats (14), the present
experiments were performed to determine the mechanism of
suppression of LH release, to determine whether there was
any effect on release of follicle-stimulating hormone (FSH),
and to examine the subcellular mechanism by which the
release of LH was suppressed.
MATERIALS AND METHODS
Rats. Male (230-270 g) and female (200-230 g) rats (Holtz-
mann, Madison, WI) were used for in vitro and in vivo
experiments, respectively. They were housed in group cages
(10 rats per cage) under controlled conditions of light (14 hr
of light, 10 hr of darkness) and temperature (24 iPC); rat
chow and water were freely available.
In Vivo Experiments. The female rats were ovariectomized
3 weeks prior to the cannulation. A 23-gauge stainless steel
cannula was implanted into the third cerebral ventricle (3V)
as described (15). After cannulation the rats were kept in
individual cages. Five to 8 days after implantation of the
cannula into the 3V, an indwelling catheter was placed into
Abbreviations: IL-1, interleukin 1; LH, luteinizing hormone; LHRH,
LH-releasing hormone; FSH, follicle-stimulating hormone; PGE2,
prostaglandin E2; EET, epoxyeicosatrienoic acid; MBH, medial
basal hypothalamus; 3V, third cerebral ventricle.
ITo whom reprint requests should be addressed.
2764 Physiology/Pharmacology: Rettori et al.
the right atrium of the heart through the right jugular vein
(16). The evening before an experiment the animals were
brought to the laboratory. Between 0900 and 1000, after the
connection of extension tubing to the jugular catheter, the
rats were allowed to rest for at least 1 hr. One heparinized
blood sample (0.3 ml) was withdrawn from the jugular
catheter before 3V injection of IL-la (2.5 A.l, 0.25 pmol) or
vehicle (2.5 1.d of 76 mM Na2HPO4/10 mM NaH2PO4/73 mM
sucrose/0.5% human plasma albumin, pH 7.4). Blood sam-
ples (0.3 ml) were obtained at 10-min intervals for 2 hr after
the injection. After removal of each blood sample, an equal
volume of 0.9% NaCl (saline) was injected to maintain blood
volume. Care was taken not to disturb the animals during the
experiment. Experiments were begun between 1000 and
1100. After centrifugation, plasma samples were stored at
-20bC until assayed for hormones. The IL-1 was recombi-
nant human IL-la produced in Escherichia coli and obtained
from Collaborative Research (catalogue no. 40041, lot 88-
1385). One half-maximal unit was equivalent to 1 ng of IL-1
with a molecular weight of 17,000.
In Vitro Experiments. Rats were decapitated, their brains
were removed, and medial basal hypothalami (MBHs) were
dissected as described (15). The MBH fragments were incu-
bated in 1 ml of Krebs-Ringer bicarbonate/glucose buffer
(pH 7.4) in an atmosphere of 95% 02/5% CO2 with constant
shaking at 60 cycles per min at 370C. In all cases, the tissues
were preincubated for 30 min, after which the medium was
replaced by fresh medium containing IL-1 (10 pM) and/or
norepinephrine (50 ,uM; Sigma). At the end of the incuba-
tions, the media were transferred to centrifuge tubes and
centrifuged for 2 min in a microcentrifuge; Aliquots were
assayed immediately for prostaglandin E2 (PGE2) and the rest
was stored at -200C until assayed for LH-releasing hormone
(LHRH).
RIA of PGE2, LHRH, and Gonadotropins. The amount of
PGE2 released into the incubation medium was determined
by RIA (125I RIA kit, NEN). The sensitivity of the assay was
0.25 pg per tube and the curve was linear up to 25 pg per tube.
The crossreactivity was 3% with PGEI and negligible with
arachidonic acid and other prostaglandins. LHRH was as-
sayed (17) using antiserum R11373 kindly provided by V. D.
Ramirez (Univ. of Illinois, Champaign-Urbana, IL). The
sensitivity of the assay was 0.6 pg per tube and the curve was
linear up to 100 pg of LHRH.
Plasma levels of LH and FSH were assayed according to
the directions in the National Institute of Diabetes and
Digestive and Kidney Diseases kits and results were ex-
pressed in terms of the RP-2 reference preparations for LH
and FSH, respectively.
Pulsatile release of FSH and LH was analyzed as described
(18, 19).
Measurement of Endogenous Epoxyeicosatrienoic Acids
(EETs). MBH fragments were incubated in Krebs-Ringer
bicarbonate/glucose solution at 370C as described above for
PGE2 and LHRH measurements. After preincubation for 30
min, the medium was replaced by fresh medium containing
IL-1 (10 pM) and/or norepinephrine (50 ttM) and incubated
for 1 hr. Four MBHs were pooled from each experimental
group, and after addition of 0.2 ,uCi of [3H8]EET mixture (200
Ci/mmol; 1 Ci - 37 GBq) as recovery standards, the samples
were homogenized in 2 ml of CH30H/H20 (2:3, vol/vol)
containing triphenylphosphine (0.1 mM) and were extracted
into acidified CHC13/CH30H. The combined organic phases
were evaporated under N2 and hydrolyzed, and the free
EETs were purified by reverse-phase HPLC (20). The en-
dogenous EETs were derivatized into pentafluorobenzyl
esters (EET-PFB) and purified by reverse-phase HPLC.
After the biological samples were mixed with known amounts
of [2H8]EET-PFB mixtures as internal standards, they were
analyzed by gas chromatography/mass spectrometry (20).
Proc. Natl. Acad. Sci. USA 88 (1991)
Quantifications were done by gas chromatography/negative-
ion chemical ionization (NICI) mass spectral analysis of the
PFB esters by selected ion monitoring at m/z 319 (loss of PFB
from endogenous EET-PFB) and 327 (loss of PFB from
[2H8]EET-PFB standards). Based on these ion ratios and the
final percent recoveries, the total endogenous EET content
was calculated and expressed as nanograms of EETs per
MBH.
Statistics. Data were analyzed by one-way analysis of
variance and the Student-Newman-Keuls multiple compar-
ison test for unequal replicates. Differences with P < 0.05
were considered significant.
RESULTS
Effect of Intracerebroventricular Injection of IL-la on Pul-
satile Release of FSH and LH in Ovariectomized Rats. To
determine the action of the monokine on pulsatile release of
LH and FSH, either the diluent or IL-la (2.5 A.l, 0.25 pmol)
was microinjected into the 3V after removal of an initial blood
sample from the conscious, freely moving animals. Samples
were then collected every 10 min for 2 hr. In diluent-injected
controls, pulsatile release of LH was unaltered (Figs. 1-3);
there were approximately three pulses during the 2 hr of
observation and plasma LH concentration was unchanged. In
marked contrast, the release of plasma LH declined in all of
the animals microinjected with IL-la, although there were
differences in the delay before a decline occurred. This delay
10 -
-7.5
'5f
-J
3r 5I-
E
OL 2.5
I I i --I I I
0 10 20 30 40 50 60 70 80 90 100 110120
Time (min)
200
0150
I..
(U- 1 00
I1
E
FL 50 I-
3v
0 10 20 30 40 50 60 70 80 90 100110120
Time (min)
FIG. 1. Mean (+ SEM) plasma LH (Upper) and FSH (Lower)
concentrations after microinjection of 0.25 pmol of IL-la (E, n = 10
rats) or 2.5 /Al of diluent (e, n = 7) into the 3V. Asterisks indicate P
< 0.05 (*) or P < 0.01 (**) versus diluent-injected controls.
 Physiology/Pharmacology: Rettori et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2765

CONTROLS Lia LH FSH

PULSE FREQUENCY
20 # 7 200 c4 4

I.
3v
10 .<
#
i||.|. I *.

L
*
,.

*0
e
WVit
r
.Rs
A
oi 3
*..4...is
150
cm

i3
&2t
i>
3
2
F f

so E
z
CONTROL ILla
1st hr 2nd hr 1st hr 2nd hr
.g 3 3
&D2 p<fl05
gE1
zI .(7).
CONTROL
I(7
lILa XONTROL ILla
p<0.001
(10)
2
1

CONTROL ILla CONTROL ILla

II
PULSE AMPLITUDE
1mu I 15
I 150r
100 E10

50 o O< PO

200 E
I
-
2
150 I-->1 I
TROUGH VALUES
tm
1501 I
100
0)

Ea)0. 200 X CEOU

j
50TEC CONTROL p<0.
.

o I

c:
CONTROL IL1a
FIG. 3. Analysis of pulsatile LH and FSH release in animals
injected with IL-lai or diluent illustrated in Fig. 1. P values refer to
differences between IL-la-injected rats (n = 10) and the controls (n
= 7).

Time (min)
FIG. 2. Plasma FSH and LH concentrations of individual animals
injected with IL-la or diluent into the 3V. Numbers (#) are the
identification numbers of the rats. These results are from some of the
animals included in Fig. 1.
ranged from 10 min to 70 min, by which time the release of
LH was suppressed in all animals and it remained depressed
for the remaining 50 min of the experiment. Because of the
variable delay in onset of the inhibition, there was only a
small decrease in mean frequency of pulses during the first
hour (Figs. 2 and 3); however, all pulses were extinguished
during the second hour following injection of IL-la. The
amplitude of the remaining pulses before suppression was
unaltered (Figs. 2 and 3), and for that reason the trough values
were only moderately suppressed when the mean plasma LH
over the entire 2 hr of observation was considered (Fig. 3).
Thus, the effect of IL-1 was on the frequency of pulses, and
the amplitude of remaining pulses was unaltered.
As in the case of LH, pulsatile release of FSH was
unaltered in the animals injected with the diluent, as indicated
by continued pulses and no significant alteration in mean
values throughout the experiment (Figs. 1-3), although the
mean values at the end ofthe experiment were slightly but not
significantly lower than initial concentrations. In the control
animals the frequency of FSH pulses was not significantly
different from that for LH. Pulses of FSH were sometimes
synchronized with those of LH; however, in many instances
they were asynchronous or even completely out of phase
with those of LH. This asynchrony has been noted frequently
in prior studies (21).
In striking contrast to the results with LH, the pulsatile
release of FSH was unaffected by the intraventricular injec-
tion of IL-la (Figs. 1-3). Mean plasma FSH concentrations
decreased slightly during the experiment; however, the val-
ues at the end of the experiment were not significantly
different from those of the diluent-injected controls. Pulsatile
release of FSH continued even though the pulsatile release of
LH was completely inhibited and there was no change in the
frequency of pulses throughout the experiment. Indeed, the
amplitude of FSH pulses in the animals injected with IL-la
was slightly higher than that in those injected with the diluent
(Fig. 3).
Effect of IL-la on LHRH Release from MBH Fragments.
We hypothesized that the reduction in pulsatile LH release
2766 Physiology/Pharmacology: Rettori et al. Proc. NatL Acad Sci. USA 88 (1991)
8r
p<0.01 p<0.01
c7 I
E I

C
0
._
5 w
w

I 4

C IL1a NE lILa

1 o-'
1 M
1011M 5x1 0-5M
5x105 M E+
NE

,34 FIG. 6. Effect of IL-la alone, norepinephrine (NE), or IL-la plus

2 norepinephrine on the endogenous content of EETs in MBH of male
2I rats. Incubations were performed as for measurement of PGE2
I **
release. Values are means SEM.
(12)1. (8)S IL-1 on the levels of EETs in the MBH of male rats. There
C lI a NE ILI a was a slight but not significant decline in the content of EETs
I011 M 5x104 M N+E in MBH fragments incubated with 10 pM IL-la. No change
in EET content followed incubation with 50 ,uM norepineph-
FIG. 4. Effect of IL-la on LHRH release from MBH fragments rine, and those values were not further altered in the presence
incubated in vitro with or without norepinephrine (NE). Numbers in of 10 pM IL-la (Fig. 6).
parentheses represent number of MBH fragments used. **, P < 0.01
versus control (C) release.
DISCUSSION
was caused by the ability of IL-la to suppress the release of The results show that, in agreement with the report of Rivier
LHRH. This was tested in vitro by incubating MBH frag- (12), a minute amount of IL-la injected into the 3V sup-
ments with 10 pM IL-la. This did result in a decrease in basal presses LH release in the castrated rat; This suppression is
LHRH release (Fig. 4). LHRH release is induced by norad- induced by a cessation of the pulsatile release ofthe hormone
renergic terminals that make synaptic contact with the and does not extend to the other gonadotropin, FSH, whose
LHRH neuronal cell bodies and terminals (22). Norepineph- release is only slightly suppressed without alteration in
rine at 50 ,uM (a concentration previously found to be pulsatile release. IL-la is another of many examples of
effective) released LHRH from the MBH fragments (Fig. 4), compounds that can dissociate the release of FSH and LH
and this action was largely blocked by coincubation with acutely by hypothalamic action (18, 24, 25). These findings,
IL-la at the same very low concentration that suppressed plus the frequent asynchrony of pulsations of FSH and LH,
basal release (10 pM). argue strongly for a separate hypothalamic control of FSH
Effect of IL-la on the Release of PGE2 from MBH Frag- release mediated by an FSH-releasing factor (26). This factor
ments. LHRH release induced by norepinephrine is mediated has been partially purified (27), but it has yet to be isolated,
by the release of PGE2 (22). Consequently, we evaluated the its structure determined and its synthesis accomplished.
effect of IL-la on the release of PGE2 from MBH fragments. Selective FSH release can be induced by a portion of the
Although 10 pM IL-la had no effect on basal release of POE2, prepro-LHRH molecule, namely, gonadotropin-releasing
it significantly decreased the release of PGE2 induced by the hormone-associated peptide 1-13 (28).
addition of 50 uM norepinephrine to the medium (Fig. 5). Pulsatile LH release is driven by pulsatile release of LHRH
Effect of IL-la on Endogenous EETs Content in MBH. (14), and indeed IL-la suppresses the release of LHRH from
Since the metabolites of the epoxygenase pathway of ara- MBH fragments incubated in vitro. The pulses are thought to
chidonic acid metabolism have been implicated in the release be generated by release of norepinephrine, which in turn
of hypothalamic peptides (23), we evaluated the action of stimulates LHRH release via a PGE2 step (22). It appears that
IL-la acts by blocking the PGE2 release induced by norepi-
Fp <0.002 P <0.0051 nephrine. The MBH contains many axonal terminals of the
LHRH neuronal system and terminals of ascending norad-
=
cm 15 renergic axons that make axo-axonal (14) contact with the
Cl
-'-
LHRH axons. IL-la presumably acts on its recently de-
ct 1.0
scribed receptors (29) on the LHRH axons to block the
1.0 T IlE release of POE2 in response to noradrenergic input.
T
EETs, products of the recently discovered epoxygenase
I pathway, have also been postulated to be involved in release
cm
Wu 0.5 of hypothalamic peptides (23). Norepinephrine did not alter
a- the content of EETs in the MBH, and there was no effect of
IL-la in the presence or absence of norepinephrine. There-
fore, we postulate that this pathway plays no role in the action
IL a NE of IL-la to block LH release.
o-11M 5x10-5M NE
We thank Judy Scott for her excellent secretarial assistance. We
FIG. 5. Release of PGE2 from MBH fragments incubated in vitro thank Thelma Williams and John Johnson for technical assistance
with IL-la, norepinephrine (NE), or IL-la plus norepinephrine. **, with radioimmunoassay of LH and FSH. We are grateful to the
P < 0.01 versus control release. National Institute of Diabetes and Digestive and Kidney Diseases
 Physiology/Pharmacology: Rettori et al.
Pituitary Hormone Program for providing kits for measurements of
rat LH and FSH. This work was supported by National Institutes of
Health Grants DK40094 and HD09988.
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