Peptides, Vol. 17, No. 4, pp. 635-639, 1996
Copyright 0 1996 Elsevier Science Inc.
Printed in the USA. All rights reserved
0196-9781/96 $15.00 + .OO
ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
PI1 SO196-9781(96)00067-S
Vasorela.xation by Casomokinin L, a Derivative
of ,O-Casobmorphin and Casoxin D, is Mediated by
NK1 Receptor
HIROYUKI
FUJITA, * HIROYUKI
RYUSUKE
NAKAGIRI,?
SUGANUMA, t’ HACHIRO
USUI, $ KAZUYOSHI
KURAHASHI, 3
RYUZO SASAKIT
AND MASAAKI
YOSHIKAWAt** zyxwvutsrqponmlkjihgfedcbaZYXW
*Nippon Synthetic Chemical Industry Co., Ltd., Ibaraki-shi, Osaka 567, Japan
?Department of Food Science and Technology, Faculty of Agriculture and $Pharmacology
Division,
Radioisotope Research Center, Kyoto University, Sakyo-ku, Kyoto 60601, Japan
Received
7 September
1995
R. SASAKI AND M. YOSHIKAWA. zyxwvutsrqponmlkjihgfedcbaZYX
Vasorelaxarion zyxwvutsrqponmlk
PEPIIDES
17(4) 635-639,
1996.-Casomokinin
L (Tyr-Pro-Phe-Pro-Pro-Leu),
a derivative of P-casomorphin
and casoxin D, showed endothelium-dependent vasorelaxing
a.ctivity on the canine mesenteric artery (EC& = 7 X lo-* M). This relaxing activity was partly blocked by
lo-’ M NAME (nitric oxide synthase inhibitor),
and the inhibition by NAME was restored by addition of 10m4 M arginine,
suggesting the involvement of nitric oxide as an endothelium-dependent
relaxing factor. The relaxing activity was blocked by
lo-* M CP-99994 and 1O-7 M FK888 (NK, antagonists),
but not by 1O-6 M SR-48968 (NK2 antagonist).
Casomokinin L binds
to neurokinin NK, receptors (K, = 5.8 X 10-s M ), and lowered blood pressure in SHR (0.1 mglkg, IV and 3 mg/kg, PO). Thus,
despite its only three residues in common with substance P, casomokinin L binds to NK, receptors, relaxes the artery, and exerts
an antihypertensive
effect.
FUIITA, H., H. SUGANUMA,
H. USUI, K. KURAHASHI,
by casomokinin L, a derivative of P-casomorphin
Casomokinin
L
Mesenteric artery
Neurokinin NKI receptor
Vasorelaxing
peptide
R. NAKAGIRI,
and casoxin D, is mediated by NK, receptor.
Nitric oxide
Blood pressure
Endothelium-dependent
relaxing
Substance P
Casoxin D
peptide casomokinin
laxation mechanism
nin L.
MANY
kinds of biologically active peptides are released from
food proteins by enzymatic digestion ( l,lO, 14). Casoxin D (TyrVal-Pro-Phe-Pro-Pro-Phe
I), which was isolated as an antiopioid
peptide from pepsin-chymotrypsin
digest of human casein, shows
vasorelaxing activity that i.s mediated by bradykinin Br receptors
(24,25). [ des-Val’] Casoxin D shows opioid agonist activity and
vasorelaxing
activity, which is also mediated by B, receptors
(24) ; we named this pept ide casomokinin F.
It is known that [de+Arg9]bradykinin,
an endogenous Br
agonist peptide ( 18), is converted to an antagonist by substitution of Phe at the carboxy-terminus
to Leu ([des-Arg’][ Leu*] bradykinin) ( 17). We substituted Phe at the carboxy-terminus of casomokinin F to Leu, with the aim of obtaining an
opioid agonist/B1 antagonist peptide that might have potent analgesic activity. The peptide obtained, Tyr-Pro-Phe-Pro-Pro-Leu,
showed opioid activity in the guinea pig ileum assay system with
an I& value of 10 ,uM (Z!4). However, this peptide was not an
antagonist for Br receptors. Furthermore,
this peptide showed
potent vasorelaxing activity that was not antagonized by [desArg9] [Leu8]bradykinin,
a B1 antagonist (24). We named this
factor
Opioid
P-Casomorphin
L. In this study, we examined the vasoreand antihypertensive
effect of casomoki-
METHOD
Chemicals and Reagents
Aspirin, indomethacin,
nitroarginine methyl ester (NAME),
L-arginine, prostaglandin
F*u, chymostatin,
leupeptin, bacitratin, BSA (radioimmunoassay
grade), and polyethyleneimine
were obtained from Sigma Chemical Co. Acetylcholine
was
obtained from Daiichi Seiyaku Co., Ltd. Papaverine
hydrochloride was obtained from Dainihon Seiyaku Co., Ltd. Substance P and [des-Arg’]
[ Leu’] bradykinin ( 17) were obtained
from The Peptide Institute, Inc. ( + ) - (2 S,3 S) -3- (2-methoxybenzyl)amino-2-phenylpiperidine
(CP-99994)
was a gift
from Pfizer Inc. N*- [ (4 R) -4-hydroxy- 1- ( 1-methyl-lH-indol3-yl)carbonyl-~-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)-L-alanineamide
(FK888) was a gift from Fujisawa
Pharmaceutical
Co., Ltd. (S) -N-methyl-N[
4- (4-acetylamino-
1 Present address: Kagome Co., Ltd. Nishinasuno-cho,
Nasu-gun, Tochigi 329-27, Japan.
* Requests for reprints should be addressed to Masaaki Yoshikawa, Department of Food Science
University, Sakyo-ku, Kyoto 606-01, Japan.
635
and Technology,
Faculty
of Agriculture,
Kyoto
FUJITA
636
in the presence
of inhibitors
or antagonists
applied prior to prostaglandin zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH
FzIr To determine
the complete
relaxation
point, 1 @f
papaverine
was added at the end of the examination.
Binding assay. Neurokinin
NK, receptor assay was performed
(A) Intact
Casomokinin L (nM)
10s M Prostaglandin F,,
1 min
(B) Endotheliumremoved
Casomokinin L (nM)
:
A
l@ M Prostaglandin FPa
ET AL.
H
1 min
FIG. 1. Effect of removing endothelium upon vasorelaxation. Cumulative addition of casomokinin L was performed at the times indicated by
closed circles. Relaxation of the intact (A) or the endothelium-removed
canine mesenteric artery (B) by casomokinin
L. Papav.: 10m4 M papaverine.
4-phenyl piperidino) -2- (3,4-dichlorophenyl)
butyl] benzamide
(SR-48968)
was a gift from Sanofi Recherche. D-Arg[ Hyp3,
Thi’,D-Tic’,Oic’]
bradykinin (Hoe 140) ( 11) was a gift from
Hoechist Company. [ 3H] [ Sar’-Met( 0,) “1 Substance P (38.3
Ci/mmol) was obtained from Du Pont NEN Research Products.
Experimental Procedures
Amino acid sequence analysis andpeptide sy nthesis. The peptide was synthesized by the t-Boc method with a Sam Two peptide synthesizer (Biosearch Inc.). The peptide was deprotected
using the anisolelhydrogen
fluoride method and purified by reverse-phase HPLC on an ODS column. The amino acid sequence
of the purified peptide was analyzed using a 477A protein sequencer (Applied Biosystems Inc.).
Vasorelaxation assay. Beagle dogs of either sex weighing 812 kg were exsanguinated from the common carotid artery under
sodium pentobarbital anesthesia (25 mg/kg, IV). The mesenteric
arteries were then removed and cut into ring strips. The endothelium was removed mechanically by gently rubbing the intimal
surface. The absence of functional endothelium was confirmed
by the inability of acetylcholine to induce relaxation. Intact and
endothelium-denuded
arteries were mounted between two stainless steel hooks in an organ bath containing Krebs-Henseleit
solution ( 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgS04, 1.2 mM
KH2P04, 2.5 mM NaHC03, and 10 mM glucose) at 37°C and
bubbled with a mixture of 95% 02/.5% C02. The end of the hook
was attached to the lever of a force-displacement
transducer (T78-240, Orientec Co.), which was connected to a Recti-Horiz-8K
recorder (NEC San-ei Instrument Co. Ltd.,) on which the isometric tension changes were recorded. Applied tension was
adjusted to 1.5 g. The artery was then constricted by prostaglandin Fz, at 1O-6 M. The relaxing activity of samples was assayed
by measuring the binding of [ 3H] [ Sar’-Met( 02) “1 substance P
(5) to rat submaxillary gland membranes (12). Submaxillary
glands from adult male Wister rats were homogenized in 10 volumes (w/v) of ice-cold buffer A (50 mM Tris-HCl, pH 7.4,
containing 120 mM NaCl and 5 mM KCl) with a Brinkmann
Polytron PT-10 at a setting of 7 for lo- 15 s. The homogenate
was incubated on ice in the presence of 300 mM KC1 and 10
mM EDTA for 30 min with intermittent mixing and then centrifuged at 50,000 x g for 10 min. The pellet was resuspended in
20 volumes of ice-cold buffer B (50 mM Tris-HCl) by a Polytron
at a setting of 7 for 5- 10 s and centrifuged again. This washing
procedure was repeated twice, and the final pellet was resuspended in 60 volumes of buffer B. For binding experiments, the
reaction was initiated by adding 400 ~1 of freshly prepared
membrane preparation in triplicate to 0.2-10 nM [3H] [Sar’Met( 0,) ‘I] substance P at 20°C for 90 min in a final volume of
500 ~1 containing 50 mM Tris-HCl (pH 7.4)) 0.02% BSA, 1 pg
of chymostatin, 2 pg of leupeptin, 20 pg of bacitracin, and various test drugs. At the end of the incubation period, 5 ml of icecold buffer A was added to each tube and contents were filtered
immediately
under reduced pressure through Whatman GF/F
glass-fiber filters (pretreated with 0.1% polyethyleneimine
in water for 3 h prior to use). Each of the filters was then washed twice
with 5 ml of ice-cold buffer A and radioactivity was determined
by liquid scintillation spectrometry using a Tri-carb scintillation
spectrometer
D3225 (Hewlett-Packard).
Nonspecific binding
was defined as binding in the presence of 1 ph4 substance P.
Specific binding was calculated by subtracting the nonspecific
from total binding.
M easurement of blood pressure. Male spontaneously
hypertensive rats (SHR) weighing 260-290 g were used. To estimate
antihypertensive
activity of the peptide after IV administration,
SHRs were anesthetized with a-D-chloralose at a dose of 45 mg/
kg (IV). A catheter for drug injection was inserted into the femoral vein. The carotid arterial blood pressure and heart rate were
measured with a pressure transducer (Spectramed Medical Products Pte., Ltd.). For estimation of antihypertensive
activity of the
10.8
1o-7
Casomokinin L (M)
FIG. 2. Effect of nitric oxide synthase
1o-s
10-s
inhibitor on vasorelaxation.
Concentration-response
curve for casomokinin
L-induced relaxation of canine mesenteric artery in the absence of the inhibitor (O), and in the
presence of 10m5 M NAME (A)or 10e5 M NAME and 10e4 M arginine
(0). Casomokinin
L was added 30 min after the inhibitors. Each value
is expressed as the mean k SE of four experiments. Significant differences from control: *p < 0.05, **p < 0.01.
CASOMOKININ
L IS AN NK, AGONIST
637 zyxwvutsrqp
PEPTIDE
c
c
CP99994 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
SR 48968
10-7
Casomokinin
L (M)
10-6
10-e
10-7
10-6
1
5
Casomokinin L(M)
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
FIG. 3. Effects of neurokinin antagonists
on vasorelaxation.
Neurokinin NK, antagonists
(CP-99994, FK888) and NK2
antagonist (SR 48968) were added 30 min before casomokinin
L. Concentrations
of antagonists are shown in the figure.
Values are expressed as means -C SE of four experiments. Significant differences from control: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON
*p < 0.05, **p < 0.01.
peptide
after oral administration,
by the tail cuff method
using
blood pressure
was measured
a UR-5000 (Ueda Seisakusho).
SHR were used in groups of five animals each. The peptide was
dissolved in saline, then emulsified with egg yolk. Test compounds were administered orally in SHR using a metal zonde in
a volume of 0.5 ml.
Data Analyses
All results are expressed as means and SE. The differences
between the presence and absence of inhibitors or antagonists
were analyzed statistically using Student’s t-test for paired observations (p < 0.05 and 0.01).
RESULTS
Relaxation
Mechanism
of Casomokinin
activity. Thus, the relaxation caused by casomokinin L was endothelium dependent [Fig. zyxwvutsrqponmlkjihgfedcbaZYXWVUTS
1( B
zyxwvutsrqponmlkjihgfedcbaZYXW
)] .
Effects of NO synthase and cyclooxygenase inhibitors. It is
known that vasorelaxation caused by endogenous peptides is mediated by endothelium-dependent
relaxing factors, such as prostaglandin IZ (22), or nitric oxide (NO) ( 15) released from endothelium. The vasorelaxation
was partly blocked by the NO
synthase inhibitor NAME at a 10m5 M. Moreover, inhibition by
NAME was restored by addition of 10 m4M arginine (Fig. 2).
On the other hand, the relaxation caused by casomokinin L was
not blocked by 5 X 10m5 M aspirin or 3 X lo-’ M indomethacin,
both of which are cyclooxygenase
inhibitors (data not shown).
These results suggest that the relaxation caused by casomokinin
L is mediated at least partly by endothelium-dependent
relaxing
factor, NO.
L
Endothelium dependence. As shown in Fig. 1, casomokinin
L showed vasorelaxing activity in canine mesenteric artery that
had been preconstricted
b:y prostaglandin Fti. Cumulative addition of casomokinin
L showed dose-dependent
vasorelaxation
with an ECso value of 70 nM [Fig. 1 (A)]. To determine the
mechanism of vasorelaxation
of casomokinin L, we examined
whether the relaxation was endothelium dependent. In endothelium-denuded
arteries, casomokinin L showed no vasorelaxing
Determination
of the Receptor
Neurokinins are vasodilators of vascular beds and relaxants
of many isolated arteries. Vasorelaxation by neurokinins is mediated by NK, or NK, receptors in the arteries ( 19). The effects
of NKI and NK2 antagonists on the vasorelaxation
elicited by
casomokinin L were examined. As shown in Fig. 3, the relaxation
was blocked by NK, antagonists, lo-‘M CP-99994 (3) and lo-’
M FK888 (7 ). On the other hand, the relaxation was not blocked
by NK, antagonist, 10 m6M SR-48968 (6). These results suggest
that the vasorelaxing activity of casomokinin L is mediated by
NK, receptors. Substance P showed vasorelaxing activity in the
80
Saline
6
70
E
g60
;
I
Casomokinin
L
(0.1mcv%)
50
E40
i3
$
30
p
20
10
. ...#
0
10-5
10-4
Casomokinin
10-3
J
10.2
L(M)
4. Displacement
of [3H][Sar9~Met(Oz)“]substance
P by casomokinin
L. Values are expressed
as means 2 SE percentage
inhibition
of
[3H][Sar9,Met(OZ)“]substance
P binding of three experiments (0).
FIG.
FIG. 5. Antihypertensive
activity of casomokinin
L after intravenous
injection in SHR. Casomokinin L was administered at a dose of 0.1 mgl
kg. Injection of saline or casomokinin L is indicated by arrow.
FUJITA
638
ET AL. zyxwvutsrq
10
2
5-
(A)
Eo
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$
-5
I
f
m
IT -10 iv
$
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g
-20:
ii
:
C% -25a
-30
"
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2
”
4
8' a
6
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Time after Administration (hr)
aot,6,""""'.""""""'
2
4
6
6
10
12
Time after Administration (hr)
FIG. 6. Antihypertensive activity of casomokinin L after oral administration in SHR.
SHRs with systolic blood pressure of about 220-250 mmHg were used. Casomokinin
L was administered as an emulsion in 30% egg yolk at doses of 3 mglkg (A) or 10 mg/
kg (B). Control groups (0) received the same amounts of egg yolk as experimental
groups (0). Changes in systolic blood pressure from zero time are expressed as means
s SE(n = 5).
-
-
( EC50 = 3.4 X 10e6 M) mediated by bradykinin B, receptors
canine mesenteric artery with an EC5,, value of 5.0 X lOmy M
(23,25). The vasorelaxation
was blocked by aspirin and indo(data not shown), whereas that of casomokinin L was 7.0 X
methacin, cyclooxygenase
inhibitors, and mediated by prostalo-EM.
We further tested the effects of casomokinin L on the binding
glandin I?. Casomokinin F, a derivative peptide of casoxin D,
of 1.2 x 10e9 M [3H] [Sar’-Met(02)“]substance
P, a selective
also showed vasorelaxation mediated by bradykinin B, receptors
ligand for NKI receptors, to submaxillary glands of Wister rats.
(24 ) ( ECsO = 8.6 X 10 -7 M) . On the other hand, the vasorelaxAs shown in Fig. 4, the inhibition of specific binding to the NKIing activity of casomokinin L is mediated by neither bradykinin
selective radioligand by casomokinin L occurred with a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
K, value
B, nor B2 receptors but by NK, receptors. The endothelium-deof 5.8 X 10m5 h4. The Ki value of substance P was 8.6 X lo-’
pendent relaxation of the artery was partly blocked by an inhibM under the same conditions.
itor for NO synthase, but not by inhibitors for cyclooxygenase.
Substance P binds to NK, receptors, stimulating the release of
nitric oxide from the endothelium and relaxing artery smooth
Antihypertensive Effect of Casomokinin L
muscle (4). We have shown that casomokinin L evokes vasoThe endogenous NK1 receptor-selective
peptide substance P
relaxation by stimulating NKI receptors and nitric oxide release.
has antihypertensive
activity (2,16). Thus, we examined the efCasomokinin L showed vasorelaxing activity in the canine
fects of casomokinin L after IV administration in SHR. As shown
mesenteric artery with an E& value of 7.0 x 10m8 M, whereas
in Fig. 5, casomokinin L administered IV to SHR lowered blood
that of substance P was 5.0 X 10m9 M. Casomokinin L showed
pressure by 25 mmHg at a dose of 0.1 mg/kg.
similar activity in the porcine coronary artery (data not shown).
We further tested the effects of casomokinin L after oral adMoreover, casomokinin L was not bound to bradykinin B, or B2
ministration
in SHR. Antihypertensive
activities of relatively
receptors (data not shown).
large angiotensin-converting
enzyme inhibitory peptides after
The K, value of casomokinin L in the NK, binding assay was
oral administration were reported previously to be potentiated by
5.8 X lo-’ M whereas that of substance P was 8.6 X 10m8 M.
emulsification in egg yolk (21) . We also found that the antihyThe E& value of the former in the vasorelaxation assay was 7.0
pertensive activity of ovokinin, a bradykinin B, agonist octax 10m8 M whereas that of the latter was 5. 0 X 10m9 M. The
peptide derived from ovalbumin (8)) was potentiated by emulreason for the large discrepancy between the Ki value in the bindsification
in egg yolk (9). Therefore,
we examined
the
ing assay and the EC5o value in the relaxation assay for casoantihypertensive
activity of orally administered casomokinin L
mokinin L is not clear. Casomokinin L might be resistant to pepafter emulsification in 30% egg yolk. As shown in Fig. 6, casotidases because it is rich in Pro residues. This might contribute
mokinin L dose-dependently
lowered systolic blood pressure afto its potent vasorelaxing activity.
ter administration in SHR; at a dose of 3 mg/kg, blood pressure
It is generally agreed that there are three types of neurokinin
was lowered after 2 h by 11 mmHg [Fig. 6(A)]. Furthermore,
receptors, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI
NK, , NK2, and NK3, whose preferred endogenous
at a dose of 10 mg/kg, blood pressure was significantly lowered
substrates
are
substance P, neurokinin A, and neurokinin B, re4 h after administration, and this activity was observed even after
spectively. These peptides have the characteristic arrangement of
8 h [Fig. 6(B)]. These results suggest that casomokinin L, a
their C-termini Phe-X-Gly-Leu-Met-NH,.
However, casomokivasorelaxing peptide, has potent antihypertensive
activity.
nin L, of which the C-terminal arrangement is Pro-Pro-Leu,
shows vasorelaxing activity mediated by NK, receptors, because
DISCUSSION
the vasorelaxation elicited by casomokinin L was blocked only
by NK, antagonists.
Casomokinin L, a derivative of casoxin D, showed potent
Figure 7 shows primary structures of casoxin D, casomovasorelaxing activity for canine mesenteric artery ( ECso = 7.0 X
kinin F ([des-Val’]
casoxin D), casomokinin
L, and [des10m8 M). Casoxin D, the original peptide from which casomoArg’] bradykinin. Casomokinin
F is a derivative of casoxin D.
kinin F and casomokinin L are derived, showed vasorelaxation
CASOMOKININ
L IS AN NKi AGONIST
639 zyxwvutsrqp
PEPTIDE
mokinin L will be useful for studying the mode of binding to
neurokinin receptors.
Substance P lowers blood pressure in various species (2,16),
and therefore we examined whether casomokinin L also shows
Casomokinin F :
Bl
this effect. We used SHR in this study, as it is a good animal
Casoxin D
Receptor
Ides-Argg]BK
: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
model of essential hypertension. It was reported that substance P
lowered blood pressure after IV injection at a dose of 1.25-2.5
FIG. 7. Homology among substance P, casomokinin L, casomokinin F,
,ug/kg in SHR ( 16). Casomokinin L showed antihypertensive
casoxin D, and [des-Arg’lbradykinin.
Common residues are enclosed.
activity after IV administration in SHR at a dose of 100 pg/kg.
Casoxin D is a bradykinin B1 agonist peptide derived from a pepsin/
Moreover,
Casomokinin L lowered systolic blood pressure after
chymotrypsin
digest of human cu,,-casein (23,25).
oral administration
in SHR. Casomokinin L bound to NK, receptors, stimulating the release of nitric oxide and exerting a
potent antihypertensive
effect.
The vasorelaxing
activities
of casoxin
D and casomokinin
F
Substance P also act as a neuro- and immunomodulator
(20).
Casomokinins F and L also can be regarded as derivatives of pare mediated
by bradykinin
B, receptors.
It should
be noted
that the Bi agonist
casomokinin
F is converted
to the NK,
casomorphin (Tyr-Pro-Phe-Pro-Gly-Pro-Ile)
, which is an opioid
agonist
casomokinin
L by substitution
of Phe at the carboxypeptide derived from p-casein. In fact, casomokinins
F and L
terminus
to Leu. This figure also shows primary structure of
have opioid activity (24). Sizheng et al. reported that Tyr-D-Alasubstance P. It should also be noted that casomokinin
L has
Gly-Phe-Phe-Gly-Met-Leu-NH,,
a hybrid between opioid peponly three homologous
residues to substance P, even if gaps
tide and substance P, showed different neuromodulatory
propare inserted in its primary structure to fit maximally.
Casoerties from substance P ( 13).
Substance P
Casomokinin L
:
:
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