Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides

Article abstract of DOI:10.1007/PL00005168

The contents of [Met⁵]-enkephalin-Arg⁶-Phe⁷ (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37°C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enkhydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu⁵]-enkephalin (leu-enk), α- and β-neoendorphins (α- and β-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: α-neoend, β-neoend, and dyn B.

Full text of DOI:10.1007/PL00005168

Naunyn-Schmiedeberg’s Arch Pharmacol (1998) 357:276–282
ORIGINAL ARTICLE
© Springer-Verlag 1998
Toyokazu Hiranuma · Ken Kitamura · Takao Taniguchi ·
Tomomi Kobayashi · Raita Tamaki · Masayuki Kanai ·
Kazuhito Akahori · Kayoko Iwao · Tetsuo Oka
Effects of three peptidase inhibitors, amastatin, captopril
and phosphoramidon, on the hydrolysis of [Met ]-enkephalin-Arg -Phe
and other opioid peptides
5
6
7
Received: 15 July 1997 / Accepted: 20 November 1997
5
6
7
Abstract The contents of [Met ]-enkephalin-Arg -Phe phoramidon-sensitive endopeptidase-24.11, and indicate
met-enk-RF) and its six hydrolysis products: Y, YG, that an unidentified fourth enzyme(s) is involved in the
(
YGG, YGGF, YGGFM, and YGGFMR were estimated hydrolysis of another group of peptides: α-neoend, β-neo-
after incubating met-enk-RF with either a guinea-pig ileal end, and dyn B.
or striatal membrane fraction for various times at 37°C.
5
6
7
After 45 min incubation with either ileal or striatal mem- Key words [Met ]-enkephalin-Arg -Phe hydrolysis ·
branes, met-enk-RF was completely hydrolyzed, yielding Opioid peptide hydrolysis · Amastatin-sensitive
Y as the major product. Incubation with either membrane aminopeptidase · Captopril · Dipeptidyl carboxypeptidase
preparation for 60 min in the presence of the aminopepti- I · Phosphoramidon · Endopeptidase-24.11 · High-
dase inhibitor amastatin hydrolyzed 90 or 92% of met- performance liquid chromatography
enk-RF, respectively, with YGG being the major product.
If the dipeptidyl carboxypeptidase I inhibitor captopril is
also included in the incubation, met-enk-RF hydrolysis Introduction
decreases by about half for both membranes, with YGG
remaining the major product. Inclusion of three peptidase Three distinct enzymes; amastatin-sensitive aminopepti-
inhibitors, amastatin, captopril, and phosphoramidon (in- dase(s) (AsA), phosphoramidon-sensitive endopeptidase-
hibition of endopeptidase-24.11) further reduced met-enk- 24.11 (“enkephalinase”, EC 3.4.24.11) (PsE), and capto-
hydrolysis, with 87% or more remaining intact. This shows pril-sensitive dipeptidyl carboxypeptidase I (angiotensin 1
that met-enk-RF was mainly hydrolyzed by three enzymes, converting enzyme, kininase II, EC 3.4.15.1) (CsD) par-
amastatin-sensitive aminopeptidase, captopril-sensitive di- ticipate in inactivation of exogenously added opioid pep-
5
peptidyl carboxypeptidase I and phosphoramidon-sensi- tides in isolated preparations: [Met ]-enkephalin (met-
tive endopeptidase-24.11, in both ileal and striatal mem- enk) in guinea-pig ileum (Aoki et al. 1984), mouse vas
branes.
deferens (Aoki et al. 1986) and rat vas deferens (Cui et al.
5
5
Additionally, estimations of [Leu ]-enkephalin (leu-enk), 1986); [Leu ]-enkephalin (leu-enk) in guinea-pig ileum,
α- and β-neoendorphins (α- and β-neoends), and dynor- mouse vas deferens and rat vas deferens (Oka et al. 1986);
phin B (dyn B) contents after incubating the individual dynorphin A-(1-8) [dyn A-(1-8)] in guinea-pig ileum,
peptides with striatal membrane for 60 min in the pres- mouse vas deferens and rabbit vas deferens (Numata et al.
5
6
7
8
ence of the three peptidase inhibitors showed that 98, 32, 1988); and [Met ]-enkephalin-Arg -Gly -Leu (met-enk-
, and 23%, respectively, remained intact.
RGL) in guinea-pig ileum (Hiranuma et al. 1997). The
Our previous studies together with the data obtained close proximities of these enzymes to the opioid receptors
5
here show that one group of endogenous opioid peptides: in guinea-pig ileum (Aoki et al. 1984), mouse vas defer-
met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A- ens (Aoki et al. 1986), and rat vas deferens (Cui et al.
(
1-8) are largely or almost exclusively hydrolyzed by the 1986) suggest important roles in terminating physiologi-
three enzymes, amastatin-sensitive aminopeptidase, capto
-
cal actions of endogenous opioid peptides as well.
pril-sensitive dipeptidyl carboxypeptidase I, and phos-
Additionally, when ileal or striatal membranes are in-
cubated with met-enk (Hiranuma and Oka 1986) or met-
enk-RGL (Hiranuma et al. 1997) in the presence of three
peptidase inhibitors (PIs), amastatin, captopril and phos-
phoramidon, approximately 95% of both peptides remain
intact. This shows that both met-enk and met-enk-RGL
are almost exclusively hydrolyzed by the three enzymes,
T. Hiranuma · K. Kitamura · T. Taniguchi · T. Kobayashi ·
R. Tamaki · M. Kanai · K. Akahori · K. Iwao · T. Oka (౧)
Department of Pharmacology, School of Medicine,
Tokai University, Isehara 259-11, Japan

2
77
AsA, PsE and CsD, at least in these membrane prepara- Hydrolysis of met-enk-RF and four other opioid peptides. The
membrane fraction (0.5 mg of protein in 50 mM Tris-HCl buffer,
tions. Moreover, dyn A-(1-8) hydrolysis in the ileal or
pH 7.4) was incubated with either met-enk-RF, leu-enk, α-neoend,
striatal membrane preparation was essentially abolished
β-neoend, or dyn B (5 nmol) in the absence or presence of PIs for
in the presence of the three PIs (Hiranuma et al., manu-
1
5, 30, 45 and 60 min (met-enk-RF) or for 60 min (other four pep-
script submitted). Therefore, it would be interesting to de- tides) at 37°C in a total volume of 0.5 ml. The reaction was
stopped by adding 0.5 ml of 10% trichloroacetic acid, followed by
termine if hydrolyses of other endogenous opioid peptides
centrifugation at 3,000 rpm for 15 min. To assess product forma-
tion, the supernatant was analyzed by high-performance liquid
chromatography combined with electrochemical detection (HPLC-
can be prevented by the presence of the three PIs.
Numerous reports have shown that several PIs can in-
crease the potencies of various opioid peptides (e.g., Cor- ECD) in the met-enk-RF experiment or high performance capillary
electrophoresis combined with UV detection (HPCE-UVD) in ex-
bett et al. 1982; McKnight et al. 1983) or that purified en-
zymes can hydrolyze such peptides (e.g., Matsas et al.
periments on the four other peptides.
1
983; Matsas et al. 1985). However, the extents to which
Separation and detection of met-enk-RF and its hydrolysis prod-
PIs afforded protection against hydrolysis of each peptide ucts by HPLC-ECD. The apparatus consisted of a Yanagimoto
and how many peptidases are involved in the hydrolysis L-2,000 pump, a Yanapak ODS-T reverse-phase column (4 × 250
mm) and an ECD (VMD-101) (all from Yanagimoto MFG. Co.
of a particular peptide were not determined in these re-
Ltd., Kyoto, Japan). Typical chromatograms of met-enk-RF and its
ports, making it impossible to accurately assess peptide
six hydrolysis products were shown in Fig.1. Since recoveries of
met-enk-RF and its hydrolysis products added to the heat treated
potencies.
5
Here we determined the contents of [Met ]-enk- membrane fraction (100°C for 5 min) were each above 95%, the
6
7
content of each compound was estimated without correcting for %
recovery.
ephalin-Arg -Phe (met-enk-RF) and its six hydrolysis
products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR
after incubating met-enk-RF with membrane preparations
in either the absence or the presence of three PIs, amas-
tatin, captopril and phosphoramidon, to compare the hy-
drolysis pattern of the heptapeptide met-enk-RF with that
of a pentapeptide, met-enk (Hiranuma and Oka 1986) and
octapeptide, met-enk-RGL (Hiranuma et al. 1997) and
dyn A-(1-8) (Hiranuma et al., manuscript submitted). To
determine the abilities of PIs to prevent hydrolysis, we es-
timated the amounts of intact leu-enk, α- and β-neoendor-
phins (α-.and β-neoends), and dynorphin B (rimorphin)
(dyn B) remaining after their incubation with membrane
preparations in the presence or absence of PIs. Moreover,
the effects of the three PIs on the potencies of met-enk-
RF, leu-enk, α-neoend, β-neoend and dyn B in guinea-pig
ileum were examined.
Methods
Chemicals. Captopril was kindly provided by Sankyo Company
(
Tokyo, Japan). Other reagents were purchased from the following
sources: leu-enk, YGGFMR (The standard one-letter, instead of
three-letter, codes for amino acids were employed for the hydroly-
sis products of met-enk-RF), met-enk-RF, α- and β-neoend, dyn
B, [D-Ala , N-Me-Phe , Gly-ol]-enkephalin (DAMGO), and D-
Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (Peninsula
Laboratories, Inc., Belmont, Calif., USA); Y, YGGFM (met-enk),
amastatin, and phosphoramidon (Peptide Institute, Inc., Minoh,
Japan); and YG YGG, and YGGF (BACHEM Feinchemikalien
AG, Bubendorf, Switzerland).
2
4
Preparation of membrane fractions. Male Hartley guinea-pigs
weighing 400–600 g were used. The myenteric plexus-longitudinal
muscle strip of guinea-pig ileum was prepared as previously de-
scribed (Oka et al. 1982). The striatum of the brain was dissected
by the method of Glowinski and Iversen (1966). Membrane frac-
tions of the strips or striata were prepared as previously described
Fig.1 Typical chromatograms of met-enk-RF and its six hydroly-
sis products. The chromatograms were produced by applying 10 µl
of either the standard solution containing 10 pmol of each com-
pound (panel A, B and C) or the supernatant obtained after incu-
bating with a membrane fraction in the absence of met-enk-RF for
(
Hiranuma and Oka 1986; Hiranuma et al. 1997). They were sus-
6
0 min (panel D, E, and F). The chromatographic mobile phase
pended and adjusted with 50 mM Tris-HCl buffer, pH 7.4 to a con-
centration of 2 mg/ml of protein and used as the membrane frac-
tion in the following experiments. Protein concentrations were
measured according to Lowry et al. (1951), with bovine serum al-
bumin as the standard.
was 0.1 M phosphate buffer (pH 3.0) containing 5% acetonitrile
for the Y (1), YG (3) and YGG (2) assays (panel A); that contain-
ing 20% acetonitrile for the YGGF (4), YGGFM (6) and YG-
GFMR (5) assays (panel B); and that containing 30% acetonitrile
for met-enk-RF (7) assay (panel C), at the flow rate of 0.8 ml/min

278
Separation and detection of four other opioid peptides by HPCE-
UVD. HPCE separations were performed with a Model P/ACETM
System 2000 (Beckman Instruments, Inc., Palo Alto, Calif., USA).
An uncoated fused-silica capillary (75 µm i.d. × 50 cm) and on-
column UV detection at 200 nm was used. Injections were made
by nitrogen pressure (0.5 psi) for 10 s. The electrolyte used for
capillary zone electrophoresis was 20 mM phosphate buffer at pH
2.7 for leu-enk, α-neoend and β-neoend and pH 3.0 for dyn B. The
capillary was maintained at 25°C and the running voltage was 15
kV. Under these conditions, leu-enk, α-neoend and β-neoend, and
dyn B had a migration time of approximately 21.8, 11.7, 13.4 and
1
4.1 min, respectively. In these experiments only the amount of
the parent peptide was estimated.
Calculations. The content of each compound was determined by
comparing the peak heights in the sample and the standard. Y was
always detected with HPLC-ECD even when the membrane frac-
tion was incubated without met-enk-RF for 60 min (Fig.1D).
Therefore, the amount of Y in the absence of met-enk-RF was al-
ways subtracted from that in the presence of met-enk-RF. In con-
trast, met-enk-RF and its hydrolysis products other than Y were
undetectable with HPLC-ECD in any of the samples incubated in
the absence of met-enk-RF (Figs.1D–F). Additionally, four opioid
peptides, leu-enk, α-neoend, β-neoend and dyn B, were unde-
tectable with HPCE-UVD in any of the samples incubated in the
absence of each peptide.
In vitro isolated preparation. The myenteric plexus-longitudinal
muscle strip of guinea-pig ileum was set up for electrical stimula-
tion as described previously (Oka et al. 1982). The % inhibition of
the stimulated muscle twitch produced by an opioid was plotted
against the log concentration of the opioid to estimate the IC50
(opioid concentration producing 50% inhibition of the twitch). The
Ke (equilibrium dissociation constant) values of opioid antagonists
against the opioid agonists were determined by the ‘single dose’
method of Kosterlitz and Watt (1968). Because the potency of an
opioid in one preparation was sometimes significantly different
from those in the others, the same preparation was employed to de-
termine the relative potencies of opioids. When the effect of PI on
the IC50 value of an opioid peptide was studied, they were given 10
min before the administration of the opioid peptide. The signifi-
cance of % differences between IC50 in the presence of two PIs and
that of three PIs was determined by the paired Student’s t-test and
shown in Tables 4 and 5.
Fig.2 The time course of the change in the contents of met-enk-
RF (˾) and its six hydrolysis products; Y (̆), YG (̅), YGG (b),
YGGF (ć), YGGFM (२), and YGGFMR (̄) in the reaction mix-
ture during the incubation of met-enk-RF (5 nmol) with either the
ileal (panel A) or the striatal (panel B) membrane fraction (0.5 mg
of protein each). Each point represents the mean of three determi-
nations. Each standard error was within 10% of the mean for both
panels A and B
Results
Time course of met-enk-RF hydrolysis and generation
of hydrolysis products during the incubation
with membrane fractions
RGL (Hiranuma et al. 1997), or dyn A-(1-8) (Hiranuma et
al., manuscript submitted).
During the initial 15-min incubation, about 74 and 71% of
met-enk-RF was hydrolyzed in the ileal (Fig.2A) and stri- Effects of the PIs on met-enk-RF hydrolysis
atal (Fig.2B) membrane fraction, respectively, with al- by the ileal membrane fraction.
most complete hydrolysis during the subsequent 30-min
incubation in both preparations (Figs.2A and 2B). Incubation of met-enk-RF (5 nmol) with ileal membrane
Throughout the 15 to 60 min after initiation of incubation, in the absence of PI for 60 min at 37°C produced only
Y was the major hydrolysis product for either preparation free Y among the seven monitored compounds (Table 1).
(
Figs.2A and 2B).
In contrast to Y formation, production of other frag- ysis products in the presence of either captopril (1 µM) or
ments was low in both preparations at any time between phosphoramidon (1 µM) was quite similar to that in the
5 and 60 min (Figs.2A and 2B). Among these frag- absence of the PIs (Table 1). In contrast, the presence of
Additionally, the % generation of the met-enk-RF hydrol-
1
ments, the generation of the YGG fragment in the striatal 1 µM amastatin decreased the % production of free Y to
preparation was the highest (Fig.2B), although its gener- 5.6%, increased both the YGG and YGGFM fragments to
ation from met-enk-RF was significantly lower than those 78.9 and 3.71%, respectively, and left 10.2% of the met-
from either met-enk (Hiranuma and Oka 1986), met-enk- enk-RF intact (Table 1). In the presence of two PIs, amas-

2
79
Table 1 Effects of PIs on the hydrolysis of met-enk-RF incubated with an ileal membrane fraction
Inhibitors
% of met-enk-RF and its hydrolysis products
Y
YG
YGG
YGGF
YGGFM
YGGFMR
met-enk-RF
None
Ama
Cap
Pho
95.7 ± 0.35
5.60 ± 0.40
91.5 ± 1.2
91.7 ± 0.85
6.30 ± 0.51
5.63 ± 0.23
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
78.9 ± 0.69
1.13 ± 0.18
0.0 ± 0.0
31.2 ± 0.24
52.9 ± 0.41
1.61 ± 0.23
0.0 ± 0.0
1.41 ± 0.20
0.0 ± 0.0
0.0 ± 0.0
0.810 ± 0.12
1.13 ± 0.14
0.670 ± 0.34
0.0 ± 0.0
3.71 ± 0.17
0.0 ± 0.0
0.0 ± 0.0
2.09 ± 0.29
4.22 ± 0.24
1.34 ± 0.34
0.0 ± 0.0
0.130 ± 0.13
0.0 ± 0.0
0.0 ± 0.0
0.670 ± 0.33
0.260 ± 0.26
0.330 ± 0.33
0.0 ± 0.0
10.2 ± 0.18
0.0 ± 0.0
0.0 ± 0.0
Ama + Cap
Ama + Pho
Ama + Cap + Pho 4.97 ± 0.85
58.9 ± 0.83
35.8 ± 0.34
91.1 ± 0.50
The sample of a membrane fraction (0.5 mg of protein) in 50 mM tration of 1 µM each for 60 min at 37°C. Each value represents the
Tris-HCl buffer, pH 7.4, was incubated with met-enk-RF (5 nmol) mean ± SEM of three experiments. Ama, amastatin; Cap, captopril;
in either the absence or the presence of the PI at the final concen- Pho, phosphoramidon
Table 2 Effects of PIs on the hydrolysis of met-enk-RF incubated with a striatal membrane fraction
Inhibitors
% of met-enk-RF and its hydrolysis products
YG YGG
95.6 ± 0.059 0.680 ± 0.34
Y
YGGF
YGGFM
YGGFMR
met-enk-RF
None
Ama
Cap
Pho
Ama + Cap
Ama + Pho
Ama + Cap + Pho
3.11 ± 0.28 0.260 ± 0.066 0.0 ± 0.0
1.35 ± 0.18 72.7 ± 0.35 2.90 ± 0.36 7.83 ± 0.35
0.0600 ± 0.066
0.740 ± 0.18
0.260 ± 0.13
8.04 ± 0.45
0.0 ± 0.0
6.41 ± 0.41
92.5 ± 0.69
92.0 ± 0.74
7.02 ± 0.48
6.17 ± 0.36
3.13 ± 0.18
0.0 ± 0.0
0.0 ± 0.0
4.09 ± 0.25 24.5 ± 0.34 2.59 ± 0.30
2.95 ± 0.14 49.9 ± 0.57 2.54 ± 0.18
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
3.06 ± 0.11
6.78 ± 0.29
0.0
0.0
± 0.0
± 0.0
0.0 ± 0.0
2.66 ± 0.32
2.07 ± 0.29
56.0 ± 0.064
29.5 ± 0.20
87.4 ± 0.21
3.60 ± 0.23
1.53 ± 0.24 0.930 ± 0.066 1.06 ± 0.066 2.33 ± 0.33
The sample of a membrane fraction (0.5 mg of protein) in 50 mM tration of 1 µM each for 60 min at 37°C. Each value represents the
Tris-HCl buffer, pH 7.4, was incubated with met-enk-RF (5 nmol) mean ± SEM of three experiments. Ama, amastatin; Cap, captopril;
in either the absence or the presence of the PI at the final concen- Pho, phosphoramidon
Table 3 Effects of PIs on the hydrolysis of four opioid peptides phoramidon were all present, 91.1% of the met-enk-RF
incubated with a striatal membrane fraction
remained intact (Table 1).
Opioid
peptides
% of opioid peptides remaining intact
after incubation
Effects of the PIs on met-enk-RF hydrolysis
by the striatal membrane fraction
Absence of PI
Presence of three PIs
Leu-enk
α-Neoend
β-Neoend
Dyn B
0.0 ± 0.0
24.2 ± 1.4
3.4 + 0.5
97.8 ± 0.3
32.2 ± 1.4
4.8 ± 0.8
The hydrolysis products of met-enk-RF formed during a
6
0-min incubation with the striatal membrane fraction
were qualitatively similar to, but quantitatively different
from, those yielded by the ileal membrane fraction in ei-
21.8 ± 1.3
22.7 ± 2.1
The sample of a membrane fraction (0.5 mg of protein) in 50 mM ther the absence or the presence of the PIs (Tables 1 and
Tris-HCl buffer, pH 7.4, was incubated with each opioid peptide (5
2
). The formation of YGG in the presence of both amas-
nmol) in either the absence or the presence of three PIs, amastatin,
captopril and phosphoramidon, at the final concentration of 1 µM
each for 60 min at 37°C. Each value represents the mean ± SEM
of three experiments
tatin and captopril after the incubation with the striatal
membrane fraction was lower than that observed with the
ileal membrane fraction (Tables 1 and 2). This indicates
that the PsE activity in the striatal preparation is lower
than that in the ileal preparation, being consistent with the
previous observations with both met-enk (Hiranuma and
tatin and captopril, or amastatin and phosphoramidon, Oka 1986) and met-enk-RGL (Hiranuma et al. 1997).
YGG was the main product and 58.9 or 35.8% of the met-
enk-RF remained intact, respectively (Table 1). The %
generation of YGG in the presence of both amastatin and Effects of the PIs on the hydrolysis for four endogenous
phosphoramidon was higher than that in the presence of opioid peptides by the striatal membrane fraction
both amastatin and captopril (Table 1), indicating that the
involvement of CsD was larger than that of PsE in met- The percentages of leu-enk, α-neoend, β-neoend, and dyn
enk-RF hydrolysis. When amastatin, captopril and phos- B remaining intact 60 min after the incubation with stri-

280
Table 4 Effects of PIs on potencies of met-enk-RF and leu-enk in guinea pig ileum
PIs
Met-enk-RF
Leu-enk
n
IC50 (nM)
% Difference)
Relative
potency
n
IC50 (nM)
(% Difference)
Relative
potency
(
Cap + Pho
Cap + Pho + Ama
4
4
158 ± 36
16.0 ± 2.5
1
4
4
133 ± 21
23.1 ± 2.1
1
9.41 ± 1.1
5.83 ± 0.82
(88.8 ± 1.7***)
(81.8 ± 2.5***)
Ama + Pho
Ama + Pho + Cap
5
5
121 ± 24
69.5 ± 20
1
5
5
49.7 ± 13
38.9 ± 8.9
1
1.89 ± 0.19
1.25 ± 0.030
(45.0 ± 5.5***)
(19.9 ± 1.8***)
Ama + Cap
Ama + Cap + Pho
4
4
22.2 ± 3.9
14.7 ± 2.3
1
8
8
116 ± 25
99.9 ± 21
1
1.51 ± 0.11
1.15 ± 0.032
(32.8 ± 4.8***)
(12.5 ± 2.4***)
The mixture of either two or three PIs, amastatin (Ama), captopril tion. Each value represents the mean ± SEM of n experiments.
Cap) and phosphoramidon (Pho), at the final concentration of ***P < 0.01
µM each was given 10 min before the opioid peptide administra-
(
1
Table 5 Effects of PIs on potencies α-Neoend, β-Neoend, and dyn B in guinea pig ileum
PIs
α-Neoend
β-Neoend
Dyn B
n IC50 (nM)
Relative
potency
n
IC50 (nM)
(% Difference)
Relative
potency
n IC50 (nM)
Relative
potency
(% Difference)
(% Difference)
Cap + Pho
Cap + Pho + Ama
4
4
817
77.4 ± 13
88.2 3.4***)
± 190
1
3
3
584 ± 220
53.4 ± 8.8
(88.6 ± 3.0***)
1
4
4
9.81 ± 3.7
2.93 ± 0.54
(61.8 ± 8.4***)
1
11.3 ± 3.4
10.3 ± 3.1
3.12 ± 0.79
(
±
Ama + Pho
Ama + Pho + Cap
3
3
780
170
± 410
± 88
1
4
4
75.0 ± 22
26.4 ± 3.6
(57.9 ± 7.9***)
1
6
6
1.92 ± 0.46
1.39 ± 0.26
(22.2 ± 5.9**)
1
4.09 ± 0.67
2.65 ± 0.50
1.33 ± 0.11
(74.0
±
5.0***)
Ama + Cap
Ama + Cap + Pho
8
8
66.5 ± 12
64.5 ± 10
1
4
4
33.8 ± 3.7
33.8 ± 1.8
(–2.04 ± 6.4)
1
7
7
3.00 ± 0.75
3.10 ± 0.96
(2.01 ± 6.5)
1
1.02 ± 0.036
0.992 ± 0.065
1.05 ± 0.071
(0.598 ± 4.0)
The mixture of either two or three PIs, amastatin (Ama), captopril Each value represents the mean ± SEM of n experiments. **P <
Cap) and phosphoramidon (Pho), at the final concentration of 1 µM 0.02; ***P < 0.01
(
each was given 10 min before the opioid peptide administration.
atal membrane were 0, 24.2, 3.4, and 21.8, respectively, in with three PIs, captopril, phosphoramidon and amastatin
the absence of PI, while they were 97.8, 32.2, 4.8, and (Tables 4 and 5), showing that amastatin can enhance the
2
2.7, respectively, in the presence of the three PIs, amas- inhibitory potencies of all these endogenous opioid pep-
tatin, captopril and phosphoramidon (final concentration tides in guinea-pig ileum by inhibiting AsA. Additionally,
of 1 µM each) (Table 3).
data obtained with the guinea-pig ileum pretreated with
either amastatin plus phosphoramidon or all three PIs (Ta-
bles 4 and 5) showed that captopril also augmented the
potencies of all five peptides. Moreover, the IC values of
opioid peptides in guinea-pig ileum pretreated with either
amastatin plus captopril or all three PIs (Tables 4 and 5)
showed that phosphoramidon increased the potencies of
Effects of PIs on potencies of met-enk-RF
and four other endogenous opioid peptides
in guinea-pig ileum
50
The ability of one specified enzyme among three en- met-enk-RF and leu-enk but not those of α-neoend, β-
zymes, AsA, CsD and PsE, to inactivate met-enk-RF, leu- neoend, and dyn B. The enhancement of met-enk-RF po-
enk, α- and β-neoend, and dyn B was examined in guinea- tency in guinea-pig ileum by captopril was lower than that
pig ileum in which the residual two enzymes had been in- by amastatin but higher than that by phosphoramidon
hibited in advance. The IC50 value of either met-enk-RF, (Table 4), being consistent with the magnitude of the inhi-
leu-enk, α-neoend, β-neoend, or dyn B in the guinea-pig bition of the met-enk-RF hydrolysis in the ileal membrane
ileum pretreated with two PIs, captopril and phospho- fraction by each PI (Table 1).
ramidon, was significantly higher than that pretreated

2
81
Table 6 The potencies of met-enk-RF and met-enk relative to that
of DAMGO in guinea pig ileum pretreated with the three PIs
The aminopeptidase activity in both membrane frac-
tions was identified largely as AsA because free Y gener-
ation during the 60-min incubation was largely prevented
by the presence of amastatin, a potent inhibitor of amino-
peptidase (Matsas et al. 1985). Similarly, the enzyme ac-
tivity producing the YGG fragment can be identified as
either PsE or CsD because the production YGG in the
presence of amastatin was almost completely prevented
by the presence of both captopril, a specific inhibitor of
dipeptidyl carboxypeptidase I (Rubin et al. 1978), and
phosphoramidon, a specific inhibitor of endopeptidase-
Opioid agonists
IC50 (nM)
Relative potency
DAMGO
Met-enk-RF
DAMGO
Met-enk
22.8 ± 2.7
28.9 ± 5.8
6.45 ± 0.97
12.0 ± 3.4
1
0.855 ± 0.12
1
0.588 ± 0.058
The mixture of three PIs, amastatin, captopril and phosphorami-
don, at the final concentration of 1 µM each was given 10 min be-
fore the opioid administration
Each value represents the mean ± SEM of four experiments
24.11 (Matsas et al. 1983), but it was partially blocked
by either captopril alone or phosphoramidon alone.
The time course of the met-enk-RF hydrolysis (Fig.2)
indicates that CsD and PsE contribute little to met-enk-RF
hydrolysis. In contrast, in both met-enk-RGL and dyn A-
Potencies of met-enk-RF and met-enk relative
to that of DAMGO in guinea-pig ileum
(
1-8) hydrolyses, CsD involvement is greater than that of
The Ke values of CTOP, a selective µ-opioid receptor an-
tagonist (Pelton et al. 1986; Gulya et al. 1986), and nor-
BNI, a selective κ-antagonist (Portoghese et al. 1988),
against either met-enk-RF or met-enk in guinea-pig ileum
pretreated with the three PIs were 19.3 and 25.8 nM, or
AsA, because generations of YGGFMR from met-enk-
RGL (Hiranuma et al. 1997) and YGGFLR from dyn A-
(1-8) (Hiranuma et al., manuscript submitted) were higher
than free Y generation from two these peptides during the
initial incubation time.
1
6.2 and 15.1 nM, respectively, indicating that both met-
Met-enk-RF hydrolysis products by either AsA or PsE
such as free Y and the YGG, [des-Y]-met-enk-RF and [des-
YGG]-met-enk-RF fragments are suggested to have little
agonist activity against opioid receptors (Morley 1980), so
the potency of met-enk-RF should be decreased by its hy-
drolysis by these two peptidases. In fact, we did find that
amastatin or phosphoramidon significantly increased the
potency of met-enk-RF in guinea-pig ileum. Additionally,
the present pharmacological evidence that enhancement of
met-enk-RF potency by amastatin is significantly greater
than that by phosphoramidon is consistent with the bio-
chemical data indicating that AsA involvement in met-enk-
RF hydrolysis is significantly greater than that of PsE.
In contrast to the met-enk-RF hydrolysis by AsA and
PsE, the change in the potency of met-enk-RF after the
hydrolysis by CsD depends on the balance between the
rate of the metabolic change from met-enk-RF to met-enk
and that from met-enk to the YGG fragment, because
met-enk, the initial hydrolysis product of met-enk-RF by
CsD, is slightly less potent than met-enk-RF, while the
YGG fragment, the subsequent hydrolysis product by
enk-RF and met-enk acted on µ-receptors. Therefore, we
examined the potencies of met-enk-RF and met-enk rela-
tive to that of DAMGO, a selective µ-agonist (Kosterlitz
and Paterson 1981; Handa et al. 1981) in guinea-pig ileum
pretreated with all three PIs at the final concentration of 1
µM each. Pretreatment of the preparation with the three
PIs significantly decreased the respective IC50 value of
met-enk-RF and met-enk but not DAMGO in guinea-pig
ileum (data are not shown). In the presence of the three
PIs, the potencies of met-enk-RF and met-enk were about
8
6% and 59% that of DAMGO, respectively (Table 6),
showing that met-enk-RF is more potent than met-enk at
µ-opioid receptors. The IC50 value of YGG, a major hy-
drolysis product of met-enk-RF in the presence of amas-
tatin (Tables 1 and 2), was higher than 100 µM in guinea-
pig ileum pretreated with the three PIs.
Discussion
The hydrolysis of met-enk-RF has been examined by in- CsD, is more than 1000-fold less potent than met-enk-RF
cubating with the membrane fraction prepared from either in guinea-pig ileum. Since a previous study (Aoki et al.
the myenteric plexus-longitudinal muscle strip of guinea- 1984) showed that captopril signgificantly enhances met-
pig ileum or guinea-pig striatum in either the absence or enk potency in guinea-pig ileum, met-enk must be further
the presence of PIs.
hydrolyzed to the YGG fragment by CsD. Since the en-
In the absence of PIs, more than 60% of the met-enk- hancement of met-enk-RF potency by captopril is less
RF molecules are hydrolyzed to free Y during the first 15 than twofold, it is difficult to know if the inhibition of
min of incubation in both the ileal and striatal membrane YGG fragment formation contributes more to enhance-
fractions, indicating that aminopeptidase activity is ment of met-enk-RF potency by captopril than the pre-
mainly involved in the met-enk-RF hydrolysis. When vention of met-enk generation.
compared with our previous data, free Y generation from
The electrically-evoked contractions of guinea-pig ileum
met-enk-RF was significantly higher than those from met- were shown to be inhibited by µ- and κ-agonists but not δ-
enk RGL (Hiranuma et al. 1997) and dyn A-(1-8) (Hi- agonists (Lord et al. 1977). Here, we showed that met-
ranuma et al., manuscript submitted) but was similar to enk-RF and met-enk act on the µ-receptors in guinea-pig
that from met-enk (Hiranuma and Oka 1986) in both ileum pretreated with the three PIs. Because met-enk-RF
preparations.
potency has yet to be estimated under conditions prevent-

282
ing its degradation, we determined its potency and com- Glowinski J, Iversen LL (1966) Regional studies of catecho-
3
lamines in the rat brain-1: the disposition of [ H]norepineph-
rine, [ H]dopamine and [ H]dopa in various regions of the
brain. J Neurochem 13:655–669
pared it with those of representive µ-agonists in guinea-
3
3
pig ileum pretreated with three PIs, which would largely
prevent hydrolysis. The present results coupled with pre-
vious ones (Hiranuma et al. 1997) show that the rank or-
der of potencies is as follows: YGGFMR > DAMGO >
met-enk-RF > met-enk > met-enk-RGL > morphine >
YGGF.
Our previous data on the effects of three PIs, amas-
tatin, captopril and phosphoramidon, on the hydrolysis of
either met-enk (Hiranuma and Oka 1986), met-enk-RGL
Gulya K, Pelton JT, Hruby VJ, Yamamura HI (1986) Cyclic so-
matostatin octapeptide analogues with high affinity and selec-
tivity toward mu opioid receptors. Life Sci 38:2221–2229
Handa BK, Lane AC, Lord JAH, Morgan BA, Rance MJ, Smith
CFC (1981) Analogues of β-LPH61–64 possessing selective ag-
onist activity at µ-opiate receptors. Eur J Pharmacol 70:531–
540
Hiranuma T, Oka T (1986) Effects of peptidase inhibitors on the
5
[
Met ]-enkephalin hydrolysis in ileal and striatal preparations
of guinea-pig: almost complete protection of degradation by
the combination of amastatin, captopril and thiorphan. Jpn J
Pharmacol 41:437–446
(Hiranuma et al. 1997), or dyn A-(1-8) (Hiranuma et al,
manuscript submitted) along with the present observa-
tions show that met-enk, met-enk-RGL, dyn A-(1-8), and Hiranuma T, Iwao K, Kitamura K, Matsumiya T, Oka T (1997)
5
6
Almost complete protection from [Met ]-enkephalin-Arg -
leu-enk as well as met-enk-RF are almost exclusively hy-
drolyzed by three distinct enzymes, AsA, CsD and PsE, in
membrane preparations. Because all three enzymes are in
7
8
Gly -Leu (Met-enk-RGL) hydrolysis in membrane prepara-
tions by the combination of amastatin, captopril and phospho-
ramidon. J Pharmacol Exp Ther 281:769–774
close proximity to opioid receptors (Aoki et al. 1984, Kosterlitz HW, Paterson SJ (1981) Tyr-D-Ala-Gly-MePhe-
NH(CH ) OH is a selective ligand for the µ-opiate binding site.
Br J Pharmacol 73:299P
1
986; Cui et al. 1986; Numata et al. 1988), they must play
2 2
critical roles in the physiological inactivation of several
endogenous opioid peptides. In contrast to endogenous
opioid pentapeptides (met-enk and leu-enk), heptapeptide
Kosterlitz HW, Watt AJ (1968) Kinetic parameters of narcotic ag-
onists and antagonists, with particular reference to N-allyl-
noroxymorphone (naloxone). Br J Pharmacol 33:266–276
Lord JAH, Waterfield AA, Hughes J, Kosterlitz HW (1977) En-
dogenous opioid peptides: multiple agonists and receptors. Na-
ture 267:495–499
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the Folin phenol reagent. J Biol Chem 193:
265–275
Matsas R, Fulcher IS, Kenny AJ, Turner AJ (1983) Substance P
and [Leu]enkephalin are hydrolyzed by an enzyme in pig cau-
date synaptic membranes that is identical with the endopepti-
dase of kidney microvilli. Proc Natl Acad Sci USA 80:3111–
3115
Matsas R, Stephenson SL, Hryszko J, Kenny AJ, Turner AJ (1985)
The metabolism of neuropeptides. Biochem J 231:445–449
McKnight AT, Corbett AD, Kosterlitz HW (1983) Increase in po-
tencies of opioid peptides after peptidase inhibition. Eur J Phar-
macol 86:393–402
(met-enk-RF) and octapeptides [met-enk-RGL and dyn
A-(1-8)], the three PIs only partially protected endoge-
nous opioid peptides having nine or more amino acid
residues (α-neoend, β-neoend and dyn B) against metab-
olism, suggesting the presence of a fourth hydrolytic en-
zyme(s) in addition to AsA, CsD and PsE in striatal mem-
branes. The potencies of these peptides in isolated guinea-
pig ileum preparation are significantly increased by the
presence of both amastatin and captopril, showing that
both AsA and CsD are involved in the hydrolysis of α-
neoend, β-enoend and dyn B. In contrast we obtained no
evidence that PsE is involved in the hydrolysis of these
three peptides.
Morley JS (1980) Structure-activity relationships of enkephalin-
like peptides. Annu Rev Pharmacol Toxicol 20:81–110
Numata H, Hiranuma T, Oka T (1988) Inactivation of dynorphin-
Acknowledgements This work was supported in part by a Grant-
in-Aid for Scientific Research from the Ministry of Education, Sci-
ence and Culture of Japan. The authors thank Dr. S. M. Jung, In-
stitute of Life Science, Kurume University, Kurume, Japan, for
helpful comments and thank Mrs. Kazuko Aoki for her expert
technical assistance. The generous gift of captopril described in the
Methods is gratefully acknowledged.
(
1-8) in isolated preparations by three peptidases. Jpn J Phar-
macol 47:417–423
Oka T, Negishi K, Suda M, Sawa A, Fujino M, Wakimasu M
(
1982) Evidence that dynorphin-(1-13) acts as an agonist on
opioid κ-receptors. Eur J Pharmacol 77:137–141
Oka T, Aoki K, Kajiwara M, Ishii K, Kuno Y, Hiranuma T, Mat-
5
sumiya T (1986) Inactivation of [Leu ]- enkephalin in three
isolated preparations: relative importance of aminopeptidase,
endopeptidase-24.11 and peptidyl dipeptidase A. In: Holaday
JW, Law PY, Herz A (eds) NIDA Research Monograph 75:
Progress in Opioid Research. U.S. Government Printing Office,
Washington, DC, pp 259–262
References
Aoki K, Kajiwara M, Oka T (1984) The role of bestatin-sensitive
aminopeptidase, angiotensin converting enzyme and thiorphan-
sensitive “enkephalinase” in the potency of enkephalins in the
guinea-pig ileum. Jpn J Pharmacol 36:59–65
Pelton JT, Kazmierski W, Gulya K, Yamamura HI, Hruby VJ
(
1986) Design and synthesis of conformationally constrained
5
Aoki K, Kajiwara M, Oka T (1986) The inactivation of [Met ]-
somatostatin analogues with high potency and specificity for µ
opioid receptors. J Med Chem 29:2370–2375
enkephalin by bestatin-sensitive aminopeptidase, captopril-
sensitive peptidyl dipeptidase A and thiorphan-sensitive en-
dopeptidase-24.11 in mouse vas deferens. Jpn J Pharmacol 40:
Portoghese PS, Nagase H, Lipkowski AW, Larson DL, Takemori
AE (1988) Binaltorphimine-related bivalent ligands and their κ
opioid receptor antagonist selectivity. J Med Chem 31:836–
2
97–302
Corbett AD, Paterson SJ, McKnight AT, Magnan J, Kosterlitz HW
1982) Dynorphin1–8 and Dynorphin1–9 are ligands for the κ-
8
41
(
Rubin B, Laffan RJ, Kotler DG, O’Keefe EH, Demaio DA, Gold-
berg ME (1978) SQ 14,225 (D-3-mercapto-2-methyl propa-
royl-L-proline), a novel orally active inhibitor of angiotensin
I converting enzymes. J Pharmacol Exp Ther 204:271–280
subtype of opiate receptor. Nature 299:79–81
Cui S, Kajiwara M, Ishii K, Aoki K, Sakamoto J, Matsumiya T,
Oka T (1986) The enhancing effects of amastatin, phospho-
5
ramidon and captopril on the potency of [Met ]-enkephalin in
rat vas deferens. Jpn J Pharmacol 42:43–49