Major metabolites of substance P degraded by spinal synaptic membranes antagonize the behavioral response to substance P in rats

Article abstract of DOI:10.1021/js990149c

Substance P (SP) was degraded by synaptic membranes of rat spinal cord. Cleavage products were separated by reversed phase high performance liquid chromatography and identified by amino acid composition analyses. Major products of SP were phenylalanine, SP(1-4), SP(1-6), SP(1-7), SP(10-11), and SP(8-9). Both the degradation of SP and the accumulation of the major cleavage products were strongly inhibited by a metal chelator, o- phenanthroline, and also by specific inhibitors of endopeptidase-24.11, thiorphan, and phosphoramidon. Thus, endopeptidase-24.11 plays a major role in SP degradation in the rat spinal cord. N-Terminal fragments, SP(17) and SP(14), detected after incubation with spinal synaptic membranes were examined in vivo for antagonism against the scratching, biting, and licking response induced by intrathecal (IT) injection of SP (3.0 nmol) in rats. When IT coadministered with SP, SP(1-7) and SP(14) produced a significant inhibition of behavioral response to SP with ED₅₀ of 135.0 pmol and 6.2 nmol, respectively. These results suggest that the degradation of SP in the spinal cord is not only responsible for inactivation of parent peptide, but may also lead to the formation of N-terminal SP-fragments which are shown to display a novel physiological function.

Full text of DOI:10.1021/js990149c

Major Metabolites of Substance P Degraded by Spinal Synaptic
Membranes Antagonize the Behavioral Response to Substance P in
Rats
,†
†
CHIKAI SAKURADA,* CHIZUKO WATANABE,‡ SHINOBU SAKURADA,§ KOICHI TAN-NO,| AND TSUKASA SAKURADA
Contribution from Department of Biochemistry, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku,
Fukuoka 815-8511, Japan, and Center for Laboratory Animal Science, Department of Physiology and Anatomy, and
Department of Pharmacology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-0905, Japan.
Received May 12, 1999. Accepted for publication August 11, 1999.
intensely noxious chemical stimulus that directly activated
C-fibers, was significantly reduced in the knockout mice.
Abstract 0 Substance P (SP) was degraded by synaptic membranes
of rat spinal cord. Cleavage products were separated by reversed
phase high performance liquid chromatography and identified by amino
acid composition analyses. Major products of SP were phenylalanine,
SP(1−4), SP(1−6), SP(1−7), SP(10−11), and SP(8−9). Both the
degradation of SP and the accumulation of the major cleavage
products were strongly inhibited by a metal chelator, o-phenanthroline,
and also by specific inhibitors of endopeptidase-24.11, thiorphan, and
phosphoramidon. Thus, endopeptidase-24.11 plays a major role in
SP degradation in the rat spinal cord. N-Terminal fragments, SP(1−
7) and SP(1−4), detected after incubation with spinal synaptic
membranes were examined in vivo for antagonism against the
scratching, biting, and licking response induced by intrathecal (IT)
injection of SP (3.0 nmol) in rats. When IT coadministered with SP,
SP(1−7) and SP(1−4) produced a significant inhibition of behavioral
They also found decreased nociceptive response of the
knockout mice in the model of acute visceral pain by using
the procedure of the acetic acid- and MgSO4-induced
writhing assay.
The physiological action of SP is probably terminated by
a membrane-bound protease capable of degrading SP in
the synaptic region, by analogy with membrane-bound
acetylcholinesterase functioning in acetylcholine degrada-
tion in the synapse. Several membrane-bound neuropep-
tidases, endopeptidase-24.11,8-10 SP-degrading enzyme
from human brain,11 SP-degrading endopeptidase from rat
brain,12 post-proline dipeptidyl-aminopeptidase,13 and an-
giotensin-converting enzyme,10,14-16 have been reported to
be involved in the degradation of SP. SP is degraded into
N- and C-terminal fragments by endogenous neuropepti-
dase. However, the physiological function of these neu-
ropeptidases from a membrane fraction of rat spinal cord
has still remained obscure. It has been reported that some
of C-terminal SP-fragments exert actions within the central
nervous system similar to those observed with SP, whereas
N-terminal fragments have opposite or antagonizing effects
on SP action.17
response to SP with ED of 135.0 pmol and 6.2 nmol, respectively.
50
These results suggest that the degradation of SP in the spinal cord
is not only responsible for inactivation of parent peptide, but may also
lead to the formation of N-terminal SP-fragments which are shown to
display a novel physiological function.
In our previous study,18 SP(1-7) and SP(1-8) in the low
picomole range antagonize the scratching, biting, and
licking response induced by IT co-administration of SP in
mice. In this study, we examined the degradation of SP by
synaptic membranes of rat spinal cord. Additional in vivo
experiments were performed to investigate the antagoniz-
ing effect of N-terminal SP-metabolites, SP(1-7) and SP-
(1-4), on the characteristic behavioral response elicited by
IT administration of SP in the rat.
Introduction
Substance P (SP) is an undecapeptide which is widely
distributed in the central nervous system and peripheral
tissues.1 This peptide is thought to function as a neu-
rotransmitter or neuromodulator after secretion into syn-
apse.2,3 A member of tachykinins, SP is the endogenous
ligand for the tachykinin NK1 receptor, while neurokinin
A and neurokinin B are endogenous ligands for NK2 and
NK3 receptors, respectively.4,5
SP is believed to play an important role as an excitatory
peptide in the spinal cord. Recently, two groups have
reported that mice in which the preprotachykinin A gene
was disrupted showed reduced responses to pain stimuli.
Zimmer et al.6 indicated that knockout mice which cannot
produce SP and neurokinin A display no significant pain
responses following hindpaw formalin injection. In addi-
tion, the animals have an increased pain threshold in the
hot-plate test. Cao et al.7 also reported that nociceptive
response evoked by a hindpaw injection of capsaicin, an
Experimental Section
An im a lssMale Wistar rats (Shizuoka Laboratory Center,
J apan), weighing 250-300 g at the time of surgery, served as
subjects. Animals were housed in a temperature (22 ( 24 °C)- and
humidity (60-70%)-controlled room illuminated on a 12-h light
and 12-h dark cycle. Food (Clea J apan, Inc., Osaka, J apan) and
water were provided ad libitum throughout the course of the study.
P ep tid es a n d Ch em ica lssThe commercial drugs used were:
SP, phosphoramidon, chymostatin, leupeptin (Peptide Institute,
Inc., Osaka, J apan), SP (1-4), SP(1-7), SP(1-9), SP(2-11), SP-
(3-11), SP(4-11), SP(5-11), SP(8-11), SP(9-11), SP(6-11), SP-
(7-11), p-chloromercuribenzenesulfonic acid (PCMBS), thiorphan
(Sigma Chemical Co., St. Louis, MO), constant boiling hydrochloric
acid (Pierce Chemical Co., Rockford, IL), captopril and phenyl-
methyl sulfonyl fluoride (PMSF) (Nacalai tesque, INC, Kyoto,
J apan). Z-321 was obtained through courtesy of Zeria Pharma-
ceutical Co., Ltd., Saitama, J apan.
* To whom correspondence should be sent. Tel: +81-92-541-0161.
Fax: +81-92-553-5698. E-mail: chikai@daiichi-cps.ac.jp.
† Daiichi College of Pharmaceutical Sciences.
‡ Center for Laboratory Animal Science, Tohoku Pharmaceutical
University.
§ Department of Physiology and Anatomy, Tohoku Pharmaceutical
University.
P r ep a r a t ion of Syn a p t ic Mem b r a n es fr om R a t Sp in a l
Cor d sRat spinal cord was washed with 10 mM Tris-HCl (pH 7.5)
| Department of Pharmacology, Tohoku Pharmaceutical University.
© 1999, American Chemical Society and
American Pharmaceutical Association
10.1021/js990149c CCC: $18.00
Published on Web 10/05/1999
Journal of Pharmaceutical Sciences / 1127
Vol. 88, No. 11, November 1999

containing 155 mM NaCl, suspended in a 10-fold volume of 10%
(w/w) sucrose, and homogenized with a Teflon homogenizer. The
homogenate was centrifuged at 800g for 20 min and then at 9000g
for 20 min. The second pellet was suspended in about a 5-fold
volume of 5 mM Tris-HCI (pH 8. 1). Then the suspension was
incubated at 0 °C for 30 min for lysis of the pellet and subjected
to discontinuous sucrose density gradient centrifugation according
to the method of J ones and Matus.19 Particle materials present
at the interface between the layer containing 28.5% sucrose and
that containing 34% sucrose were taken, diluted with 5 mM Tris-
HCI (pH 8.1), and centrifuged at 100 000g for 1 h. The resulting
pellet was suspended in 10 mM [2-[4-(2-hydroxyethyl)1-piperazi-
nyl]ethanesulfonic acid (HEPES)-NaOH (pH 7.5) containing 155
mM NaCl and stored at -80 °C. Protein was determined by the
method of Bradford20 using bovine serum albumin (Bio-Rad) as a
standard.
Degr a d a tion of Su bsta n ce P by Syn a p tic Mem br a n es of
Ra t Sp in a l Cor d sThe degradation of SP by synaptic membranes
of rat spinal cord was carried out at 37 °C in a mixture (0.1 mL)
containing of 10 mM HEPES-NaOH (pH 7.4), 80 mM NaCl, 2.5
mM KCl, 2 mM MgCl 2, 1 mM CaCl2, 50 µM SP, and 50 µg protein
of the membrane. The reaction was terminated by heating at 100
°C for 10 min. The reaction mixture was centrifuged, filtered
through a membrane filter (Cosmonice Filter; pore size, 450 nm),
and subjected to high performance liquid chromatography (HPLC)
on a reversed phase column (4.6 × 150 mm) of SYMMETRYC18
(Waters) which had previously been equilibrated with l % (v/v)
acetonitrile in 0.05% (v/v) trifluoroacetic acid (TFA). Elution was
carried out at room temperature with a 32 min linear gradient of
1-65% acetonitrile in 0.05% TFA at a flow rate of 1 mL/min. The
peptide fragments eluted were detected by monitoring the absor-
bance at 210 nm.
Id en tifica tion of Su bsta n ce P F r a gm en tssThe cleavage
products separated by HPLC were lyophilized and hydrolyzed with
6 M hydrochloric acid containing 0.1% mercaptoethanol at 150 °C
for 1 h. Their amino acid compositions were determined as
phenylthiocarbamoyl amino acid derivatives by the reversed phase
HPLC.21
In tr a th eca l In jection sTo permit application of peptides
directly into the spinal subarachnoid space, rats were anesthetized
with pentobarbital sodium (50 mg/kg, ip) and chronically im-
planted with IT catheters.22,23 Briefly, this involved inserting a
length of polyethylene tubing (PE-10) following laminectomy
between L3 and L4 and careful placing the catheter tip in the
subarachnoid space of L5 and L6. The rats were allowed to recover
over 7 days following implantation of the catheter. The catheter
was filled with sterile artificial cerebrospinal fluid (CSF) contain-
ing (in g/L) NaCl 7.4, KCl 0.19, MgCl2 0.19, CaCl2 0.14. Peptides
used in the experiments were administered in volumes of 10 µL
followed by 15 µL of artificial CSF to ensure that peptides reached
the spinal cord. SP(1-7) or SP(1-4) was mixed and coadministered
IT with SP such that all doses were delivered in a total volume of
10 µL followed by 15 µL of artificial CSF to flush the catheter.
Beh a vior a l Obser va tion sOne hour prior to IT injection,
animals were adapted to an individual plastic cage (34 × 30 × 17
cm) which also served as the observation chamber. Immediately
following IT injection of SP, each rat was replaced into the
transparent cage, and behavioral testing was begun. The total
respose time(s) of behaviors was measured for 5 min beginning
immediately after IT injection of SP. These behaviors included
hindlimb scratching directed toward the flank, biting, or licking
of the hindpaw. All these different behaviors were pooled as a
single value for each animal. Studies on the behavioral experi-
ments were performed with the approval of the Ethics Committee
of Animal Experiment in Tohoku Pharmaceutical University.
Figure 1sDegradation of substance P by synaptic membranes of rat spinal
cord. The reaction mixture (0.5 mL, pH 7.5) containing 150 µg of the membrane
preparation was incubated 37 °C for 0, 1.5, and 3.0 h. After heating at 100
°C for 5 min, 30 µL aliquots of the mixture were analyzed by HPLC on a
reversed-phase column (4.6 × 150 mm) of SYMMETRYC18, which had been
equilibrated with 1% acetonitrile in 0.05% trifluoroacetic acid. Elution was carried
out at the room temperature with a 32-min linear gradient of 1−65% acetonitrile
in 0.05% trifluoroacetic acid at a flow rate of 1 mL/min. The absorbance at
210 nm was monitored.
14 peaks were separated and named alphabetically from
a to n according to the increasing order of their retention
times. When only the membrane was incubated in the
absence of SP or only SP was incubated, newly formed
peaks were not detected at all. The position of peak P was
identical to that of the substrate, SP, and the area of this
peak decreased as a function of time (i.e., 15% and 32% of
SP were degraded 1.5 h and 3 h after incubation, respec-
tively), whereas those of other peaks increased. The results
of analyses of amino acid compositions for the cleavage
products separated by HPLC allowed their assignment
(Table 1). Fragments b, c, g, h, l, m, and n were assignable
as SP(1-4), free phenylalanine, SP(1-7), SP(1-9), SP(8-
11), SP(3-11), and SP(5-11), respectively, by comparing
their retention times in HPLC. The yield of peaks (a to n)
was calculated on the basis of SP degraded by using
procedure of quantitative amino acid composition analysis.
Thus, N-terminal SP-fragments, SP(1-4), SP(1-6), SP(1-
7), and SP(1-9), were found as major products in addition
to free phenylalanine, SP(8-9), and SP(10-11).
Ca lcu la tion s of ED50 a n d Da ta An a lysissStatistical analy-
ses of the results were performed using the Dunnett’s test for
multiple comparisons, after analysis of variance (ANOVA). Dif-
ferences were considered to be significant if P < 0.05. All values
are expressed as means (SEM.
Results
Degr a d a tion of SP by Syn a p tic Mem br a n es of Ra t
Sp in a l Cor d sDegradation of SP by synaptic membranes
of rat spinal cord was analyzed by HPLC (Figure 1). About
1128 / Journal of Pharmaceutical Sciences
Vol. 88, No. 11, November 1999

a
Table 1−Amino Acid Composition of Fragments of SP Produced
following IT administration. In the in vivo experiments,
N-terminal SP-fragments were tested for antagonism
against the behavioral response induced by IT administra-
tion of SP. When coadministered IT with SP, SP(1-7) at
doses of 40-400 pmol reduced the SP-induced behavioral
response consisting of scratching, biting, and licking in a
dose-dependent way (Figure 3). The SP-induced response
was also dose-dependently decreased by IT coadministra-
tion of SP(1-4) (0.6-38.4 nmol) (Figure 3). The ED50 for
SP(1-7) and SP(1-4) was 135.0 pmol and 6.2 nmol against
SP, respectively. SP(1-7) and SP(1-4) in the dose-range
tested did not produce SP-like behavioral response or any
other motor disturbance.
through the Action of Synaptic Membranes of Rat Spinal Cord
amino acid (mol %)
fragment yieldb
peak Arg Pro Lys Glu Phe Gly Leu Met identified (%)
a
b
c
d
e
f
g
h
i
16 32 20 32
0
0
100
0
52 48
29
15
25 11
44 20
36
0
0
0
0
0
0
0
0
0
0
1−6
1−4
Phe
13
11
45
15
38
3
25 48 26
0
0
0
0
0
0
0
0
0
0
0
0
0
0
66 32 10−11
0
4
0
0
2
11
0
0
0
0
0
0
0
6
8
6
7
6
8−9
3−8
1−7
1−9
nd
3−10
5−9
8−11
3−11
5−11
complete
12 20 35
0
0
13 24 15 32
18
5
9
0
0
0
0
0
0
8
7
9
j
k
l
6
1
2
6
3
2
38 20
Discussion
33 27 29
27 12 11
36 16 13
22 10 13
m
n
Although a number of laboratories have demonstrated
that SP is extracellularly hydrolyzed by cell-surface en-
dopeptidase, within the central nervous system,8-16 the
concurrent monitoring of SP and its metabolic fragments
by synaptic membranes of rat spinal cord have not yet been
explored. It is extremely of importance to note metabolic
routes of SP in the spinal cord, since spinal dorsal horn is
regarded as an important site for the processing of infor-
mation related to the transmission and/or modulation of
sensory signals including pain. The present findings show
that SP was readily cleaved into SP(1-7) as a major
product when incubated with synaptic membranes of rat
spinal cord. Other N-terminal SP-fragments were also
found, including SP(1-4), SP(1-6), and SP(1-9). It is,
therefore, likely that the C-terminal SP-fragments released
through the initial cleavage of SP may be susceptible
readily to the action of neuropeptidases, since recoveries
of C-terminal fragments were low compared with those of
N-terminal fragments. These results are in accordance with
our previous data that SP(1-7) is the main metabolite after
endogenous cleavage of SP; in the dorsal spinal cord of rats
the levels of SP(1-7) were about 20% those of SP, though
in most of areas of the CNS the concentrations of SP(1-7)
were about 10% those of SP.25,26 Both the degradation of
SP and the accumulation of the major cleavage products
were strongly inhibited by a metal chelator, o-phenanthro-
line, and also by specific inhibitors of endopeptidase-24.11,
thiorphan and phosphoramidon. Formation of the fragment
SP(1-4) was also inhibited by the addition of thiorphan
and phosphoramidon to the reaction medium. Since frag-
ment SP(1-4) has not been reported to be formed directly
by the action of endopeptidase-24.11 on SP, it may arise
from the action of some other protease on the initial
cleavage products formed by endopeptidase-24.11. Other
peptide fragments were those formed by the cleavage
between the Gln6-Phe7, Phe7-Phe8, Gly9-Leu10 bonds of SP,
all of which have been reported to be the sites of cleavage
by endopeptidase-24.11 obtained from various synaptic
membranes of pig caudate,8 pig kidney,9 human kidney,10
rat substantia nigra,27 glioma C6 cells,28 and glial cells
cultured from rat fetal brain.29 In our previous report,30
we have examined the effect of protease inhibitors on the
scratching, biting, and licking response elicited by IT
injection of SP in mice. Phosphoramidon simultaneously
injected with SP remarkably enhanced and prolonged SP-
induced behavioral response in a dose-dependent manner.
SP-induced behavioral response was not enhanced by
bestatin, an aminopeptidase inhibitor, and captopril, an
angiotensin-converting enzyme inhibitor. Taking account
of these behavioral observations, the present results sug-
gest that thiorphan-sensitive metalloendopeptidse, prob-
ably endopeptidase-24.11, would play a critical role in the
initial stage of cleavage of SP by synaptic membranes of
rat spinal cord.
c
P
a The sequence, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH . b Yield
2
was determined on the basis of SP degraded. c The extent of degradation of
SP was 68%. n.d., not determined.
Table 2−Effect of Protease Inhibitors on the Degradation of SP by
Synaptic Membranes of Rat Spinal Corda
inhibitor
concentration (mM)
inhibition (%)
o-phenanthroline
phosphoramidon
thiorphan
PCMBS
PMSF
captopril
1.0
0.1
0.1
1.0
1.0
0.1
0.1
0.1
0.1
0.1
94
78
80
18
24
0
0
0
28
3
bestatin
leupeptin
chymostatin
Z-321
a The activity was measured on the basis of the disappearance of SP, as
detected by HPLC with isocratic elution.
Next, we examined the effects of various protease inhibi-
tors on the degradation of SP by synaptic membranes of
rat spinal cord. First, the inhibitory effects on the initial
cleavage rate of SP were analyzed by measuring the effects
on decrease of the HPLC peak for SP (peak P). The result
was shown in Table 2. A metal chelator, o-phenanthroline
and specific inhibitors for endopeptidase-24.11, phosphora-
midon and thiorphan, inhibited SP degradation by synaptic
membranes. Other inhibitors, including an angiotensin-
converting enzyme inhibitor (captopril), a prolylendopep-
tidase inhibitor (Z-321),24 an aminopeptidase inhibitor
(bestatin), and inhibitors (PCMBS, PMSF, leupeptin, and
chymostatin) for serine and cysteine proteases, had little
inhibitory effects on SP degradation.
Second, the inhibitory effects on the generation of
cleavage products of SP separated by HPLC were analyzed
(Figure 2). The generation of all of the peaks including
major peaks of N-terminal SP-fragments was strongly
inhibited by the addition of thiorphan. Phosphoramidon
and o-phenanthroline also showed strong inhibition, while
other inhibitors exerted little inhibitory effect on the
generation of almost all of the peaks (data not shown).
Effect of SP (1-7) a n d SP (1-4) on SP -In d u ced
Beh a vior a l Resp on sesIT injections of SP (3.0 nmol)
produced an immediate behavioral syndrome characterized
by reciprocal hindlimb scratching, caudally directed biting,
and licking. The intensity of the behavior peaked at 0-5
min, declined gradually at 5-10 min, and was no longer
apparent at 15 min postinjection. In subsequent experi-
ments, therefore, the animals were observed for 10 min
Journal of Pharmaceutical Sciences / 1129
Vol. 88, No. 11, November 1999

Figure 2sEffect of protease inhibitors on the degradation of substance P by synaptic membranes of rat spinal cord. The reaction mixture (0.5 mL, pH 7.5)
containing 50 µg of the membrane preparation was incubated 37 °C for 6 h in the absence or the presence of 0.1 mM thiorpan, 0.1 mM Z-321, or 0.1 mM
captopril. After heating at 100 °C for 5 min to stop the reaction, 20 µL aliquots of the sample were analyzed by HPLC as in Figure 1. # indicates the peak derived
from the inhibitor added.
There are some reports related to SP degradation in rat
spinal cord. Mauborgne et al.31 have previously examined
the effect of peptidase inhibitors on SP-like immunoreactive
materials released by K+-induced depolarization from slices
of the rat spinal cord. They indicated that bacitracin was
the most potent agent to protect SP-like immunoreactive
materials from degradation, and captopril and thiorphan
also protected it from degradation. Suzuki et al.32 have
reported the effects of peptidase inhibitors on the enzymatic
degradation of SP by synaptic membranes of neonatal rat
spinal cord. While they did not analyze and characterize
the cleavage products of SP, they have presented that
thiorphan, actinonin for an aminopeptidase N inhibitor,
and captopril inhibited SP degradation. The inhibition
potency of each peptidase inhibitor was lower than that
of the mixture. They have also reported in electrophysi-
ological studies that the magnitude of SP-evoked depolar-
ization of a lumbar ventral root in the isolated spinal cord
preparation was increased by a mixture of peptidase
inhibitors, consisting of actinonin for an aminopeptidase
N inhibitor, arphamenine B for an aminopeptidase B
inhibitor, bestatin for an aminopeptidase B and M inhibi-
tor, captopril, and thiorphan. These results are inconsistent
with our results. Although the reason for this discrepancy
remains unknown, in all cases, however, susceptibilities
of SP degradation to thiorphan are consistent with each
other. Recent biochemical data show that two enzymes
with SP-degrading activity from the membrane bound
fraction of the rat spinal cord are separated,33 one enzyme
exhibited similarity to endopeptidase-24.11, while the other
resembled a substance P converting endopeptidase which
has been identified and purified from human cerebro-
spinal fluid. Accordingly, SP(1-7), SP(1-8), SP(8-11), and
SP(9-11) were detected after incubation with both enzyme.
The former enzyme preparation also gave SP(1-6) as a
major product. It is still unknown whether these enzymes
play critical roles in SP degradation in rat spinal cord
synapses.
Thus, SP is enzymatically cleaved into several C-termi-
nal and N-terminal fragments, which have biological
activity in the CNS sensory system. Some of C-terminal
fragments, injected IT into mice, elicit scratching, biting,
and licking response similar to that observed with SP,
whereas N-terminal fragments have antagonizing effects
on SP-induced behavioral response.34 In addition, central
actions of N-terminal fragments have previously been
found to produce effects opposite to those of C-terminal
fragments in mice. For example, the N-terminal heptapep-
tide inhibited grooming and increased rearing, whereas the
C-terminal hexapeptide had the opposite effect.17,35 Studies
with microinjection into the substantia nigra demonstrate
that SP induces dopamine release in the ipsilateral stria-
tum and produces contralateral rotation in a dose-depend-
ent manner.36 In addition, intranigrally injected SP(1-7)
produces an antagonistic action on the SP-induced re-
sponse. In agreement with these previous findings, SP(1-
7), injected into conscious rats produced a dose-dependent
antagonizing effect on SP-induced behavioral response.
Similar effects were obtained by coadministration of SP-
(1-4), though SP(1-4) is approximately 45 times less
potent than SP(1-7) in antagonizing the response to SP.
These results are in line with our recent data in mice
(unpublished data) that SP(1-7) is much more potent
than SP(1-4) as assayed by the spinally mediated SP
response. Taken together, our biochemical and pharmaco-
logical results suggest that endogenously formed SP(1-7)
may be a main modulator of SP actions in the rat spinal
cord.
1130 / Journal of Pharmaceutical Sciences
Vol. 88, No. 11, November 1999

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Figure 3sThe effectiveness of substance P(1−7) (upper panel) and substance
P(1−4) (lower panel) in antagonizing the scratching, biting, and licking behavior
induced by substance P in rats. Substance P (3.0 nmol) was coadministered
intrathecally with substance P(1−7) or substance P(1−4). The duration of the
behavioral response induced by substance P was determined over a 10 min
period starting immediately after intrathecal injection. The data are given as
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Mechanism of spinal actions of N-terminal SP-fragments
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cant difference of potency in the antagonizing effect of
SP(1-7) on SP-induced behavioral response between
rats and mice, when comparing the ED50 value of SP(1-7)
(135.0 pmol) against SP (3.0 nmol) in rats with that of
SP(1-7) (2.9 pmol) against SP (100 pmol) in mice.34 These
data suggest that SP(1-7) may inhibit binding of [3H]-
SP(1-7) to rat and mouse spinal cord membranes with
similar potency.37 It is noteworthy that similar phenomena
are seen in affinities of CP-96,345 for tachykinin NK1
receptor; the nonpeptide tachykinin NK1 receptor antago-
nist, CP-96,345, was much less active at NK1 receptors
in rat and mouse than the other mammalian species
including human, whereas there was no significant differ-
ence in affinity of CP-96,345 for NK1 receptors between rat
and mouse.38,39
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κ-opioid receptors.40 One could speculate that a physiologi-
cal action of SP(1-7) in the spinal cord may not be
mediated by an action at a typical opioid receptor but by
an action at SP(1-7) binding site or its own receptor.
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24.11 in the spinal cord is not only responsible for inactiva-
tion of SP, but may also lead to the formation of N-terminal
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Acknowledgments
This work was partly supported by a research grant, No.
08772151, from the Ministry of Education, Science, and Culture
of J apan. The authors thank Yoshiko Inoue, Toshiko Kikuta,
Hiromi Murakami, and Kaori Sueyoshi for technical assistance.
J S990149C
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