Development of novel neurokinin 3 receptor (NK3R) selective agonists with resistance to proteolytic degradation

Article abstract of DOI:10.1021/jm500771w

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.

Full text of DOI:10.1021/jm500771w

Brief Article
pubs.acs.org/jmc
Development of Novel Neurokinin 3 Receptor (NK3R) Selective
Agonists with Resistance to Proteolytic Degradation
Ryosuke Misu,† Shinya Oishi,*,† Ai Yamada,† Takashi Yamamura,‡ Fuko Matsuda,§ Koki Yamamoto,†
Taro Noguchi,† Hiroaki Ohno,† Hiroaki Okamura,‡ Satoshi Ohkura,§ and Nobutaka Fujii*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
‡Animal Physiology Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-0901, Japan
§Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
S
* Supporting Information
ABSTRACT: Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the
neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists
to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The
modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and
prolonged bioactivity.
Table 1. Sequences of NKB, [MePhe7]-NKB, and Senktide
INTRODUCTION
■
Neurokinin B (NKB) is one of the mammalian tachykinin
peptides and an endogenous ligand for the neurokinin-3
receptor (NK3R). NKB regulates dopamine release as a
neurotransmitter in the central nervous system1,2 and induces
contraction of the portal vein, venoconstriction of the
mesenteric bed, and an increase in heart rate in the
periphery.3−5 Recently, it has been demonstrated that NKB-
NK3R signaling is involved in the central control of
reproduction.6−8 A landmark discovery was the identification
of missense mutations in either the Tac3 or Tacr3 genes (which
encode NKB or NK3R, respectively) that cause severe
gonadotropin deficiency in humans.9 In kisspeptin/NKB/
dynorphin A (KNDy) neurons in the hypothalamic arcuate
nucleus (ARC), NKB, with kisspeptin and dynorphin A,
cooperatively modulates the release of gonadotropin-releasing
hormone (GnRH).10
The tonic mode of GnRH release is pulsatile, which is driven
by the hypothalamic neural substrate, the so-called GnRH pulse
generator. Because the GnRH pulse frequency controls baseline
levels of circulating gonadotropins, this neural substrate has
been thought to play a pivotal role in the hypothalamo-
pituitary-gonadal (HPG) axis.11 In goats, a method that
monitors the electrophysiological manifestations of the GnRH
pulse generator as periodic bursts of multiple-unit activity
(MUA volleys) has been established.8,12 Using this method, it
has been demonstrated that intracerebroventricular (icv)
administration of NKB8 or intravenous injection of an
NK3R-selective agonist, senktide,13 immediately induced the
MUA volley, suggesting involvement of NKB-NK3R signaling
in GnRH pulse generation.14
peptide
sequence
neurokinin B
(NKB)
H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2
[MePhe7]-NKB
H-Asp-Met-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH2
succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2
senktide
been employed for a number of in vitro experiments using
primary cultures or cell lines.1,19,20 In vivo studies using NKB or
senktide also provided insights regarding regulation of the HPG
axis function. However, the reported actions of these NK3R
agonists remain controversial. For example, icv treatment with
NKB had no effect in male rats,21 whereas icv administration of
senktide to female rats resulted in decreased plasma LH
levels.22 In contrast, another study reported that senktide
increased plasma LH levels in female rats.23 These apparently
inconsistent data may be attributable to enzymatic degradation
following in vivo administration of the peptides. Indeed, it has
been reported that NKB is degraded by neutral endopeptidase
(NEP) 24.11 (also known as neprilysin or enkephalinase) at
four peptide bonds (e.g., Asp4-Phe5, Phe5-Phe6, Phe6-Val7 and
Gly8-Leu9, Table 1).24 We envisioned that substitution of the
degradable peptide bond(s) in NK3R selective agonists with
appropriate noncleavable isosteres would confer resistance to
enzymatic degradation while maintaining NK3R agonistic
activity. This could potentially provide novel therapeutic
agents, with prolonged effects, for reproductive disorders of
human and domestic animals. This may also prove to be useful
research tool(s) to investigate the role of NKB-NK3R pair in
HPG axis regulation, addressing a major confounding influence
of enzymatic degradation.
In this study, we investigated proteolytic digestion of NKB-
selective agonist peptides in animal serum, hypothalamic
NKB shares the C-terminal common sequence, −Phe-Xaa-
Gly-Leu-Met-NH2, with other tachykinin peptides including
substance P (SP) and neurokinin A (NKA).15−17 NK3R-
selective agonists that have been reported so far include
[MePhe7]-NKB18 and senktide (Table 1). These peptides have
Received: April 30, 2014
Published: September 23, 2014
© 2014 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
extracts, and by recombinant NEP 24.11. In addition, on the
basis of degradation of these peptides, novel peptidomimetic
senktide analogues with resistance against proteinase-mediated
degradation were designed and synthesized using a series of
dipeptide isosteres. In vivo biological activity of the analogues
was assessed using the MUA recording method in conscious
goats.
RESULTS AND DISCUSSION
■
Proteolytic Degradation of NK3R-Selective Agonists.
Initially, we investigated the proteolytic degradation of
[MePhe7]-NKB in rat, pig, goat, and cattle serum. Treatment
of [MePhe7]-NKB with pig serum provided the fragment
peptides 1−5 by N-terminus degradation (Figure 1B). Peptides
1−5 were similarly produced by treatment with sera derived
from the other animal species, although the degradation
patterns were different (Supporting Information). Peptides 2,
3, and 5 were mainly obtained by serum derived from cattle,
pig, and goat, respectively. Fragment 6 was also observed
following 24 h treatment with rat serum. The proteolytic
degradation was terminated at the Phe-MePhe peptide bond in
sera of all animals, and the resistance could be attributed to the
N-methyl group of MePhe. We also analyzed the proteolytic
degradation of [MePhe7]-NKB in pig hypothalamic extracts,
which is the site of action of NKB in the central nervous system
(Figure 1C). [MePhe7]-NKB was immediately degraded by
treatment with hypothalamic extract to generate peptides 5, 6,
and the C-terminal carboxylate 7. Under these conditions,
fragments 1−4 were not observed, probably owing to the rapid
degradation of [MePhe7]-NKB.
Next, we investigated the biological activities of fragment
peptides 1−7, which were synthesized separately by Fmoc-
based solid-phase peptide synthesis (Table 2). Receptor
binding was evaluated by competitive binding assay using
[
125I]-BH-SP for human NK1R, [125I]-NKA for human NK2R,
and ([125I]His3, MePhe7)-NKB for human NK3R, respectively.
The shorter fragments among peptides 1−5 exhibited less
inhibitory activity for NK3R binding, whereas peptides 6 and 7
did not inhibit NK3R binding. In contrast, slightly improved
NK1R binding inhibition was observed for shorter peptide
fragments 1−5 and moderate inhibitory activity for NK2R.
NK3R agonistic activity was assessed by monitoring intra-
cellular Ca2+ flux induced by NK3R activation, with peptides
1−5 exhibiting similar activity (EC50 = 0.091−0.35 nM), which
is apparently inconsistent with the structure−activity relation-
ship data obtained by binding assays. This may be because of
the heterologous binding competition, in which the IC50 values
do not always truly reflect competitor binding, in the binding
assay.25−27
The proteolytic degradation of senktide in serum and pig
hypothalamic extracts was investigated (Supporting Informa-
tion). Senktide was stable in animal sera and pig hypothalamic
extracts over 24 h, indicating that N-terminal succinyl capping
on senktide prevents degradation by exopeptidases in serum
and hypothalamus. We also analyzed senktide degradation by
NEP 24.11. In contrast to treatment in serum and hypothalamic
extracts, more than 80% of senktide was digested by NEP 24.11
within 24 h (Figure 2). Enzymatic hydrolysis occurred at the
Gly8-Leu9 bond, which corresponds to the cleavage site of NKB
by NEP 24.11.24 The resulting senktide N-terminal fragment 8
and C-terminal fragment 9 did not exhibit agonistic activity at
100 nM (data not shown), suggesting that NEP 24.11 is able to
rapidly deactivate the senktide administered in vivo.
Figure 1. Proteolytic degradation of [MePhe7]-NKB in serum or
hypothalamic extracts. (A) Cleavage sites of [MePhe7]-NKB in animal
sera or pig hypothalamic extracts and the sequences of the digested
peptides. The degradation profiles of [MePhe7]-NKB following
treatment with (B) pig serum, (C) pig hypothalamic extracts
[MePhe7]-NKB (closed circles), 1 (opened circles), 2 (closed
triangles), 3 (opened squares), 4 (closed squares), 5 (closed
diamonds), 6 (opened diamonds), and 7 (opened triangles).
[MePhe7]-NKB was incubated in serum or hypothalamic extracts at
37 °C. The sample was analyzed by HPLC (detection at 220 nm).
Data represent the mean SD (n = 3).
We turned our attention to development of novel senktide
derivatives with an increased enzymatic stability. An approach
that has been explored to provide resistance against enzymatic
degradation while maintaining NK3R agonistic activity includes
the use of an appropriate dipeptide isostere at the cleavable
Gly-Leu dipeptide moiety. Because the introduction of a
dipeptide isostere structure to bioactive peptides might lead to
loss of the original bioactivity, we designed several senktide
derivatives containing a variety of dipeptide isostere sub-
structures. The Gly-Leu dipeptide surrogates for the standard
Fmoc-based solid-phase peptide synthesis were synthesized
according to the facile preparation approach that we previously
reported.28 The amidoxime- and amidine-containing peptides
10d,e were also obtained using aldoxime resin.29,30
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Table 2. Biological Activities of [MePhe7]-NKB Degradation Products
c
binding inhibition (%)
a
b
peptide
[MePhe7]-NKB
IC50 (μM)
EC50 (nM)
NK1R
<10
NK2R
0.012 0.004
0.022 0.008
0.043 0.012
0.059 0.022
2.9 1.8
0.064 0.016
0.33 0.014
0.28 0.041
0.091 0.021
0.24 0.054
0.35 0.093
>100
57 1.3
78 1.9
74 1.3
73 0.98
84 1.6
77 0.53
1
2
3
4
5
6
7
22 1.3
18 1.6
15 2.6
49 0.81
70 0.55
2.3 0.34
>10000
d
d
−
−
−
−
d
d
>10000
>100
a
b
IC50 values are the concentrations for 50% inhibition of ([125I]His3, MePhe7)-NKB (0.1 nM) binding to human NK3R (n = 3). EC50 values are the
c
concentrations needed for 50% induction of Ca2+ influx in human NK3R expressing CHO cells (n = 3). Binding inhibition (%) was calculated by
binding inhibition assay using a radioactive ligand (0.1 nM) and each peptide (10 μM). 100% binding inhibition was calculated based on background
d
signal, obtained by measurement of the wells without receptor membrane. Not evaluated.
Table 3. Biological Activities of Senktide Derivatives for
NK3R
Figure 2. Proteolytic degradation of senktide by NEP 24.11. (A)
Cleavage site of senktide and the sequences of the digested peptides.
(B) The degradation profile of senktide following treatment with NEP
24.11: senktide (closed circles), 8 (closed triangles), 9 (open
triangles). Senktide was incubated in NEP 24.11 solution at 37 °C
a
IC50 values indicate the concentration needed for 50% inhibition of
and analyzed by HPLC (detection at 220 nm). Data represent the
receptor binding of ([125I]His3, MePhe7)-NKB to human NK3R.
mean SD (n = 3).
b
EC50 values indicate the concentration needed for 50% of the full
agonistic activity for human NK3R induced by 100 nM senktide.
Design and Biological Evaluations of Senktide
Derivatives with Gly-Leu Dipeptide Isostere. All designed
peptides (10a−e) worked as full NK3R agonists, exhibiting a
good correlation between binding affinity and agonistic activity
(Table 3). Among peptidomimetics 10a−e, (E)-alkene
dipeptide isostere-containing analogue 10a was most potent
It is worth noting in terms of the receptor selectivity of peptides
10a−e that binding to NK1R and NK2R was not observed at
30 μM, indicating that these peptides are NK3R-selective
(Supporting Information).
(IC50 = 0.056
0.012 μM; EC50 = 0.016
0.002 nM),
To estimate the biological activity of 10a in animals, we
evaluated its agonistic activity against rat, cattle, and goat NK3R
(Table 4). Peptide 10a showed potency similar to senktide
comparable to the parent senktide (IC50 = 0.056 0.003 μM;
EC50 = 0.011 0.004 nM).31 The alkene substructure, which
mimics a planar amide bond may contribute to the high affinity
and potent agonistic activity, which are in contrast to the lower
toward rat NK3R (EC50(senktide) = 0.013
0.004 nM;
EC50(10a) = 0.019 0.004 nM), cattle NK3R (EC50(senktide)
= 0.34 0.12 nM; EC50(10a) = 0.36 0.031 nM), and goat
bioactivities of a flexible ethylene-type derivative 10b (IC50
=
19 0.88 μM; EC50 = 0.92 0.13 nM) and α,β-unsaturated
derivative 10c (IC50 = 13 3.9 μM; EC50 = 1.9 0.86 nM).
Amidoxime 10d and amidine derivative 10e showed 34- and 9-
times lower bioactivity compared with senktide, respectively
(IC50(10d) = 1.1 0.082 μM; EC50(10d) = 0.37 0.13 nM;
IC50(10e) = 0.36 0.091 μM; EC50(10e) = 0.10 0.027 nM).
NK3R (EC50(senktide) = 0.012
0.011 0.004 nM).
The enzymatic stability of potent peptides 10a and 10e
against NEP 24.11-mediated digestion was also evaluated
(Figure 3). No significant cleavage of the Gly-Leu pseudopep-
tide bond in 10a was observed, while peptide 10e was
0.004 nM; EC50(10a) =
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Table 4. Biological Activities of Senktide and Peptide 10a
senktide
10a
a
NK3R activation: EC50 (nM)
human
rat
0.011 0.004
0.013 0.004
0.34 0.12
0.016 0.002
0.019 0.004
0.36 0.031
0.011 0.004
cattle
goat
0.012 0.004
b
MUA volley
c
duration of the effect (R)
60.6 9.9
3.4 1.3
92.1 18.7
c
the number of ligand-induced MUA
volleys
5.6 2.3
a
EC50 values indicate the concentration needed for 50% of the full
agonistic activity for human, rat, bovine, and goat NK3R induced by
b
100 nM senktide. MUA volley induction in OVX goats by
intravenous injection of NK3R ligands (200 nmol, n = 5). Values
c
are represented as the mean SEM. P < 0.05 (paired t-test).
Figure 3. NEP 24.11-mediated degradation of senktide and
derivatives: senktide (closed circles), 10a (open circles), and 10e
(closed triangles). Senktide, 10a and 10e were incubated in NEP 24.11
solution at 37 °C and analyzed by HPLC (detection at 220 nm). Data
represent the mean SD (n = 3).
Figure 4. Effect of senktide and peptide 10a on MUA volley in OVX
goats. Representative profiles of MUA in OVX goats that received
intravenous administration of senktide (200 nmol, A) and peptide 10a
(200 nmol, B) are shown. The arrow indicates timing of injection of
NK3R agonist. The open and closed triangles indicate spontaneous
and ligand-induced MUA volleys, respectively.
degraded. Interestingly, 10a was partially degraded in rat serum
to provide a product (t1/2 >24 h), in which the C-terminal
amide was hydrolyzed (Supporting Information). These
indicate that introduction of (E)-alkene dipeptide isostere
increased the stability of senktide against proteolytic cleavage.
As such, the senktide derivative 10a with favorable enzyme
stability may be a promising NK3R agonist for regulating
reproduction systems.
Effect of Peripheral Administration of Peptide 10a on
MUA Volley in Goats. In vivo biological activity was evaluated
by single intravenous administration of senktide and peptide
10a in ovariectomized (OVX) goats, using induction of the
MUA volley as the index of GnRH pulse generator
activation.8,12 The injection of NK3R analogues immediately
induced several MUA volleys with shorter intervolley intervals
than the predetermined average value, which were followed by
MUA volleys with the regular interval (Figure 4). It is thought
that those MUA volleys with an interval less than 80% of the
average spontaneous intervolley intervals (T) are ligand-
induced, whereas ones with an interval not less than 80% of
T were spontaneous. The duration of the analogue effect (R)
was obtained during a period from injection until the onset of
the following spontaneous MUA volley, where the number of
MUA volleys occurring during this period was counted.
Statistical analysis revealed that the duration is significantly
longer (p < 0.05) and the number of ligand-induced MUA
volleys is significantly larger (p < 0.05) in peptide 10a than with
senktide injections (Table 4). This result demonstrates that the
novel NK3R agonist 10a, with an (E)-alkene dipeptide isostere,
exhibits biological action with longer duration and more potent
activity than senktide via peripheral administration in goats.
CONCLUSIONS
■
In this study, we investigated the proteolytic degradation of
NK3R-selective agonists, [MePhe7]-NKB and senktide.
[MePhe7]-NKB was degraded in serum and hypothalamic
extracts, whereas senktide was stable under the same
conditions. Senktide was digested at the Gly-Leu peptide
bond by NEP 24.11, an inactivating enzyme of NKB. On the
basis of the proteolytic degradation profiles, we designed Gly-
Leu dipeptide isostere-containing peptides. Among these,
peptide 10a, an (E)-alkene dipeptide isostere-containing
derivative, maintained the highly potent bioactivity and
selectivity of senktide for NK3R. Peptide 10a showed
prolonged effects on the GnRH pulse generator by single
intravenous administration into OVX goats. As such,
modification at the Gly-Leu substructure of senktide resulted
in the improved metabolic stability accompanied by potent
NK3R-selective activity.
EXPERIMENTAL SECTION
■
Chemistry. The peptides were synthesized by Fmoc-based solid-
phase synthesis as described previously.28,29,32 The peptides were
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purified by HPLC on a Cosmosil 5C18-ARII preparative column
(Nacalai Tesque, 20 mm × 250 mm). All peptides were characterized
by ESI-MS, and the purity was verified as >98% by HPLC on a
Cosmosil 5C18-ARII analytical column (Nacalai Tesque, 4.6 mm ×
250 mm) (see the Supporting Information for details).
Evaluation of Peptide Stability in Serum and Pig Hypo-
thalamic Extracts. First, 4 μL of peptide solution (10 mM in DMSO
containing 0.1% of m-cresol as an internal standard) was dissolved in
rat, pig, goat, and cattle serum or pig hypothalamic extracts (196 μL).
After incubation at 37 °C, a 30 μL aliquot was sampled at intervals
(indicated time). After addition of MeCN (90 μL), the sample
solution was centrifuged (4 °C, 13000g, 10 min), and then the
supernatant was analyzed by HPLC with a linear gradient of MeCN
(5−50% over 45 min or 30−50% over 20 min; detection at 220 nm).
The compound ratio was determined by the peak areas.
In OVX goats, the intervolley interval of spontaneously occurring
MUA volleys differed slightly among individuals ranging from 20 to 35
min but was relatively constant within an individual in which the
interval variation was usually 2 min, allowing for a timed treatment
between experiments. On each experimental day, the average value of
three successive intervolley intervals (T) was calculated for each OVX
goat and sample injections made at 1/2T after the preceding MUA
volley.
NK3R agonists were initially dissolved in DMSO at 10 mM and
further diluted with saline to make a working concentration of 100
μM. All five goats received a bolus injection of senktide and peptide
10a once through a jugular catheter. MUA was recorded throughout
the experimental period. Analogue treatments were separated by at
least 1 day.
Evaluation of Peptide Stability in the Presence of NEP 24.11.
First, 8 μL of peptide solution (10 mM in DMSO containing 0.1% of
m-cresol) was dissolved in Tris·HCl (pH 7.5) containing 0.05% of
Brij-35 (WAKO) (192 μL). After addition of NEP 24.11 (R&D
Systems) (200 μL, 0.1 μg/mL in Tris·HCl (pH 7.5)), solutions were
incubated at 37 °C. After incubation, a 30 μL aliquot was sampled at
the indicated intervals and quenched by addition of MeCN (30 μL).
The sample solution was analyzed by HPLC with a linear gradient of
MeCN (5−50% over 45 min or 30−50% over 20 min; detection at
220 nm). The compound ratio was determined by the peak areas.
Evaluation of Binding Affinity of Tachykinin Peptides to
NK1R, NK2R, and NK3R. Binding inhibition assays were performed
using membranes from NK1R-, NK2R-, or NK3R-expressing CHO
cells as described previously.32 Briefly, membranes were incubated
with 50 μL of peptide, 25 μL of radioactive ligand ([125I]-BH-SP for
NK1R, [125I]-NKA for NK2R, and ([125I]His3, MePhe7)-NKB for
NK3R, respectively, 0.4 nM, PerkinElmer Life Sciences) and 25 μL of
membrane solution in assay buffer (50 mM HEPES (pH 7.4), 5 mM
MgCl2, 1 mM CaCl2, 0.1% BSA). Reaction mixtures were filtered
through GF/B filters, pretreated with 0.3% polyethylenimine. Filters
were washed with (50 mM HEPES (pH 7.4), 500 mM NaCl, 0.1%
BSA) and dried at 55 °C. Bound radioactivity was measured by
TopCount (PerkinElmer Life Sciences) in the presence of MicroScint-
O (30 μL) (PerkinElmer Life Sciences).
Evaluation of NK3R Agonistic Activity. NK3R agonistic activity
of each peptide was evaluated by [Ca2+]i flux assay as described
previously.32 NK3R expressing CHO cells (4.0 × 104 cells/50 μL/
well) were inoculated in 10% FBS/Ham’s F-12 onto a 96-well black
clear-bottom plate (Greiner), followed by incubation at 37 °C
overnight in 5% CO2. After medium removal, 100 μL of pigment
mixture (Calcium 4 assay kit, Molecular Devices) was dispensed into
each well, followed by incubation at 37 °C for 1 h. Then 10 μM
peptide in DMSO was diluted with HANKS/HEPES containing 2.5
mM probenecid, and the dilution was transferred to a 96-well sample
plate (V-bottom plate, Coster). The cell and sample plates were set in
FlexStation (Molecular Devices) and 25 μL of sample solution
automatically transferred to the cell plate.
Evaluation of Effect of NK3R Agonists on MUA Volley in
OVX Goats. All goat experiments were approved by the Committee
on the Care and Use of Experimental Animals at the National Institute
of Agrobiological Sciences (H23-002). Adult female Shiba goats
(Capra hircus, 4−8 years old, weighing 23.0−32.0 kg, n = 5) were
bilaterally ovariectomized under inhalation anesthesia. With an interval
of more than one month, they were implanted with an array of
bilateral recording electrodes consisting of six Teflon-insulated
platinum−iridium wires at the posterior region of the ARC as
described previously.8,12 After recovery, the goats were kept in a
condition-controlled room (12L/12D, 23 °C, and 50% relative
humidity) and loosely held in an individual stanchion. Animals were
maintained with a standard pellet diet and dry hay and had free access
to water and supplemental minerals. MUA was monitored in conscious
goats. Signals were passed through a buffer amplifier and integrated
circuit directly plugged into an electrode assembly. After additional
amplification and amplitude discrimination, MUA signal was stored as
counts per 20 s on a personal computer.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, characterization data, degradation
profile, and NMR spectra of peptides 10a−e. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*For S. Oishi: phone, +81-75-753-4551; fax, +81-75-753-4570;
E-mail, soishi@pharm.kyoto-u.ac.jp.
*For N. Fujii: E-mail: nfujii@pharm.kyoto-u.ac.jp.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are grateful to Dr. Akira Hirasawa (Kyoto University) for
his generous support in the experiments. We are also grateful to
■
́ ́
Dr. Nikolaus Heveker (Universite du Montreal) for helpful
discussion. This work was supported by a Grant from the
Ministry of Agriculture, Forestry and Fisheries of Japan
(Research Program on Innovative Technologies for Animal
Breeding, Reproduction, and Vaccine Development, REP-2001,
REP-2003, and REP-2005) and by Grants-in-Aid for Scientific
Research from JSPS, Japan, and by Platform for Drug
Discovery, Informatics, and Structural Life Science from
MEXT, Japan. R.M. and T.N. are grateful for JSPS Research
Fellowships for Young Scientists.
ABBREVIATIONS USED
■
ARC, arcuate nucleus; BH-SP, Bolton−Hunter labeled
substance P; GnRH, gonadotropin-releasing hormone;
MePhe, N-methylphenylalanine; MUA, multiple-unit activity;
NKA, neurokinin A; NKB, neurokinin B; NK1R, neurokinin-1
receptor; NK2R, neurokinin-2 receptor; NK3R, neurokinin-3
receptor; HPG, hypothalamo-pituitary-gonadal; OVX, ovariec-
tomized; SP, substance P
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