Opioid peptide participates in post-tetanic twitch inhibition in guinea pig isolated ileum

Journal of the

Autonomic Nervous System

ELSEVIER

Journal of the Autonomic Nervous System 58 (1996) 147-152

Opioid peptide participates in post-tetanic twitch inhibition in guinea pig isolated ileum Masanobu Ozaki a, *, Yasusuke Masuda a, Hiroyuki Yamamoto b a Department of Toxicology, Niigata College of Pharmacy, 5-13-2 Kamishin' eicho, Niigata-City, Niigata 950-21, Japan b Department of Pharmacology, WakayamaMedical College, Wakayama 640, Japan Received 22 August 1995; revised 30 October 1995; accepted 30 October 1995

Abstract

The effects of a mixture of peptidase inhibitors, consisting of 100 nM each of amastatin, phosphoramidon, and captopril, on the twitch inhibitory response (0.1 Hz, 0.5 ms duration, maximum intensity) exerted by opioid peptides were investigated. The opioid peptides, Met-enkephalin (50-200 nM), dynorphin(l-13) (0.2-1 nM), and /3h-endorphin (20-100 nM) concentration-dependently inhibited the electrically evoked twitch response. In the presence of the mixture of peptidase inhibitors, the twitch inhibition evoked by Met-enkephalin was significantly increased; however, the twitch inhibition evoked by flh-endorphin and dynorphin(l-13) was only slightly increased. These increases were abolished by naloxone (NLX; 100 nM). Inhibition of the twitch response (0.1 Hz, 0.5 ms duration, maximum intensity) induced after high frequency stimulation (10 Hz, 0.5 ms pulse width, maximum voltage for various lengths of time) (post-tetanic twitch inhibition) was investigated in isolated guinea pig ileal longitudinal muscles. The mixture of peptidase inhibitors, which did not affect the twitch response or ACh-contraction, increased post-tetanic twitch inhibition. This increase was abolished by naloxone (100 nM). These results suggested that the potentiated post-tetanic twitch inhibition evoked by the peptidase inhibitors in the ileum was due primarily to /z-ligand(s) rather than to the K-type of endogenous opioid ligand(s) released from opioidergic neurons. Keywords: Peptidase inhibitor; Post-tetanic twitch inhibition; Methionine-enkephalin; fl-Endorphin , Human; Dynorphin(l-13); Ileum

1. Introduction The isolated guinea pig ileum is a useful model to investigate the dynamics of opiate action in vitro, since the opioid action can be detected through the inhibition of the twitch response induced by field stimulation [3]. However, the action of peptidase inhibitors on endogenous opioid peptide(s) originally present in the tissue has not been reported. Puig et al. [8,9] demonstrated a naloxone-sensitive opiate-like inhibition of the twitch responses induced by low frequency electrical stimulation after tetanic stimulation in a guinea pig ileal preparation. This post-tetanic opiate-like inhibition of the twitch response is a useful indicator for studying the role of endogenous opioids in this tissue [5,6]. However, the characteristics of this posttetanic twitch inhibition are not well understood.

* Corresponding author. Tel.: (81-25) 269-317.1 ext. 206; Fax: (81-25) 268-1230. 0165-1838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 65- 1 8 3 8 ( 9 5 ) 0 0 1 24-7

We have previously reported that post-tetanic twitch inhibition in isolated guinea pig ileal myenteric plexuslongitudinal muscles was increased by some peptidase inhibitors [5] and that both /x- and K-opioid receptor types participated in this post-tetanic twitch inhibition [6]. However, the type of endogenous opioids released which potentiate the post-tetanic twitch inhibition evoked by peptidase inhibitors has not yet been elucidated. In this study, we investigated the effects of a mixture of peptidase inhibitors (amastatin, an aminopeptidase inhibitor; phosphoramidon, an enkephalinase inhibitor; and captopril, an angiotensin I-converting enzyme inhibitor) on the twitch inhibition induced in isolated ileal muscle by the /z- and K-type of endogenous opioids (Met-enkephalin and /3h-endorphin for the /x-ligand and dynorphin(l-13) for the K-ligand). We selected this mixture of inhibitors since it is well known that the enkephalin-degrading enzymes, aminopeptidase, enkephalinase (EC 3.4.24.11) and peptidyl dipeptidase A (angiotensin I-converting enzyme; EC 3.4.15.1), play a major role in terminating opioid action [5]. The purpose of

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M. Ozaki et al./Journal of the Autonomic Nervous System 58 (1996) 147-152

this study was to clarify the type of endogenous opioid ligand that was involved in the potentiated post-tetanic twitch inhibition exerted by this mixture of peptidase inhibitors.

2. Materials and methods

Experiments were performed essentially as we have described previously [5,6].

2.1. Animals and drugs Random-bred male guinea pigs, weighing 350-450 g, were used. The animals were stunned and decapitated and the ileum was quickly isolated about 10 cm from the ileocecal junction. A myenteric plexus-longitudinal muscle (MPLM) preparation was prepared by the method of Rang [10]. A glass rod was inserted into the lumen of an intestinal segment and the MPLM was removed by rubbing the segment with a cotton swab soaked in Krebs' solution. The preparation (2-2.5 cm) was suspended, at a resting tension of 500 mg, in a 5-ml organ bath containing Krebs-bicarbonate solution (mM: NaCI, 118; KCI, 4.7; CaC12, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO 3, 25; glucose 10) at 37°C, bubbled with 95% 0 2 / 5 % CO 2. The sources of the drugs were: amastatin (Peptide Institute Inc., Minoh, Osaka); captopril (Sankyo Inc, Tokyo); dynorphin(1-13) (Peninsula Laboratories, Belmont, CA); human /3-endorphin (/3h-endorphin; Peptide Institute Inc.); Met-enkephalin (Peptide Institute Inc.); naloxone hydrochloride (Endo Laboratories Inc., Garden City, NY); phosphoramidon (Peptide Institute Inc.). Dynorphin(1-13) was dissolved in equal volumes of methanol and 0.1 N hydrochloric acid [11]. All other drugs were obtained from commercial sources and all were dissolved in distilled water.

2.2. Electrical field stimulation Rectangular electrical pulse field stimulations were applied at 0.1 Hz, 0.5 ms pulse width and maximum voltage, using a DSP-160B stimulator (NEC-Sanei Co., Japan) with a DSP-122 isolator (NEC-Sanei Co., Japan). The responses were recorded isometrically on a SP-H5P recorder (Riken Denshi Co., Japan) with a SD-IT force displacement transducer (Nihon Kohden Co., Japan).

2.3. Post-tetanic twitch response Electrical field stimulations at 0.1 Hz were applied continuously throughout the experiments. Tetanic stimulations of 10 Hz (0.5 ms pulse width, maximum voltage for 1 min), inducing an inhibition of the twitch contractions

brought about by the electrical field stimulation of 0.1 Hz (0.5 ms pulse width, maximum voltage), were repeated every 30 min. The first tetanic stimulation was applied 40-50 min after the preparation was set up. We have described the experimental protocol in a previous study [7]. The preparation was washed 6 times with 5 ml of Krebsbicarbonate solution 13-15 min after tetanic stimulation. The effects of the drugs on post-tetanic twitch inhibition were investigated after 3 tetanic stimulations, carried out at 30-min intervals in the same preparation; tetanic stimulation (10 Hz, 0.5 ms duration, maximum voltage) was applied for 1, 1.5, and 2 min. The mixture of peptidase inhibitors and 100 nM naloxone (NLX) was applied to the bath 10 min before tetanic stimulation.

2.4. Effects of the mixture of peptidase inhibitors on opioid peptide-induced twitch inhibition The peptidase inhibitors (100 nM each of amastatin, phosphoramidon, and captopril) were added singly to the bath in volumes of 5 /xl, 10 min before the addition of Met-enkephalin, flh-endorphin and dynorphin(l-13). Naloxone (100 nM) was added to the bath together with the mixture of peptidase inhibitors 10 min before the addition of the respective opioid peptide.

2.5. Effects of the mixture of peptidase inhibitors and opioids on ACh-induced basal contraction The effect of ACh on basal tension was investigated. The mixture of peptidase inhibitors (100 nM each of amastatin, phosphoramidon, and captopril) and the opioid peptides (50, 100, and 200 nM Met-enkephalin; 20, 50, and 100 nM flh-endorphin; and 0.2, 0.5, and 1 nM dynorphin(1-13)) were added to the bath in volumes of 5 ALl, 10 min before the single application of ACh (10, 30, and 100 nM).

2.6. Evaluation of effects of the mixture of peptidase inhibitors on opioid peptide-induced twitch inhibition The effects of the mixture of peptidase inhibitors on the electrically evoked opioid peptide-induced twitch inhibition were examined. The effect of twitch inhibition was expressed as the maximum inhibition induced by the respective opioid peptide. The maximum twitch response was expressed as the percent inhibition of the twitch contraction before exposure to the respective opioid peptide.

2.7. Quantitation of post-tetanic twitch inhibition The effect of tetanic stimulation (post-tetanic twitch inhibition) was quantified: the sum of the twitch amplitude

M. Ozaki et al. / Journal of the Autonomic Nervous System 58 (1996) 147-152

(mm) during the 1 min preceding and following tetanic stimulation (,~pre-T and ,~post-T, respectively) which contained the same number of twitches was calculated, and the results were expressed as a percentage of the inhibitory response.

~100

149

[]

Non-treatment

•

Peptidase inhibitors (n=12)

(n=12)

¢.,

,O= ,Q e-

Post-tetanic inhibition (%) ,,C

u 50

= 100 x ( ~ p r e - T - E p o s t - T ) / ] £ p r e - T

2.8. Evaluation of effects of the mixture of peptidase inhibitors on ACh-contraction

E

=1

E X el

ACh-induced basal contraction in the presence of the mixture of peptidase inhibitors and in the presence of the

Met-enkephalin (nM)

Non-treatment

50

A

Peptidase inhibitors

[]

v

"100I

•

100 Met-enkephalin

200 (nM)

Non-treatment (n=12) Peptidase inhibitors (n=12)

.o_

50

¢,.. L°= 100

]=

"~ 50

E [3h-endorphin (nM)

~o

~

50

m

E Non-treatment

Peptidase inhibitors

eB

•

20

.~"

[]

50 13-endorphin

Non-treatment

100 (nM)

(n=12)

C O ¢J .Q

100

Dynorphin 1-13 (riM}

0

e* ,m Non-treatment

Peptidase inhibitors .g=

o 50

0.2

E E X (g

0,5

•

0 0.2

t

2re=n,0

Fig. I. Representative dynograph tracing of concentration-dependent electrically evoked twitch inhibition (0.1 Hz, 0.5 ms pulse width, maximum voltage) induced by Met-enkephalin (upper), dynorphin(l-13) (middle), and ,Sh-endorphin (lower) in the presence and absence of the mixture of peptidase inhibitors in guinea pig ileal longitudinal muscle. Met-enkephalin, ~Sh-endorphin, and dynorphin(l-13) were added at the times indicated by the arrowheads. The mixture of peptidase inhibitors was added to the bath 10 min before Met-enkephalin, dynorphin(l-13), and /3h-endorphin.

O.5 Dynorphin 1-13

1 (nM)

Fig. 2. Concentration-dependent electrically evoked twitch inhibition (0. l Hz, 0.5 ms duration, maximum voltage) induced by Met-enkephalin (upper), dynorphin(l-13) (middle), and ~h-endorphin (lower) in the presence (closed columns) and absence (open columns) of the mixture of peptidase inhibitors in guinea pig ileal longitudinal muscle. The mixture of peptidase inhibitors was added to the bath 10 rain before Met-enkephalin, dynorphin(l-13), and /3h-endorphin. The values and vertical bars represent the means and S.E.M. of the number of experiments shown in parentheses. * *P < 0.01, different from non-treated control at the respective concentration of opioid peptide.

150

M. Ozaki et al./Journal qlthe Autonomic Nervous 5~vstem 58 (1996) 147 152 Tetanic Stimulation

Non-treatment

(sec)

Peptidase inhibitors

[

60

90

I

]~! i ,

120

.....~III~IItlIIIIIIIItlIIIIIIIIIII!IfilIWltlIIIIIIIIIilIIItl

i 2rain

lg

Fig. 3. Representative dynograpb tracing of the effect of peptidase inhibitors on post-tetanic twitch inhibition in guinea pig ileal longitudinal muscle. Tetanic stimulation (10 Hz, 0.5 ms pulse width, maximum intensity) was applied for l, 1.5, and 2 min as indicated by the underlines. Brackets in the tracing indicate post-tetanic twitch inhibition for 1 min. Each preparation was treated with a mixture of peptidase inhibitors, consisting of I00 nM each of amastatin, phosphoramidon, and captopril. This mixture was added to the bath 10 rain before the tetanic stimulation.

opioid peptide was determined as a percentage of the 200 nM ACh-contraction in the absence of the mixture of inhibitors or the opioid peptide, respectively.

Fable I Effects of the mixture of peptidase inhibitors or the opioids on ACh-induced basal contraction Drugs (nM)

ACh (nM) I0

C]

°~" 1 0 0

g

"[] • []

Non troalment (n.15]

Peptidase inhibilors (n=5) NLX 100 nM (n=5) Peplldase inblbitors + NLX 100 nM (n=5)

.=_ NS

:

51- ]

I ++

qJ

L

1 Tstanlc

1.5

stimulation

2 (min)

Fig. 4. Effect of the mixture of peptidase inhibitors on post-tetanic twitch inhibition. The mixture of peptidase inhibitors was added to the bath 10 min before tetanic stimulation. Tetanic stimulation (10 Hz, 0.5 ms pulse width, maximum voltage) was applied for 1, 1.5, and 2 min. The mixture of peptidase inhibitors increased post-tetanic twitch inhibition in a manner dependent on the duration of tetanic stimulation. These increases were antagonized by 100 nM naloxone (NLX). The values and vertical bars represent the means and S.E.M. of the number of experiments shown in parentheses. " "P < 0.01, different from non-treated control; + ' P < 0.01, different from peptidase inhibitors, at the respective durations of tetanic stimulation. N.S., not significant.

Met-enkephalin 0 40.1 + 4 . 4 ( 2 1 ) 50 37.9±2.5(9) I(X) 44.4-+4.1 (6) 200 45.2 -+ 3.7 (6) /3wEndorphin 0 34.7_+4.9(27) 20 33.5 ± 5.5 (9) 50 38.9_+8.0(9) 100 32.8+4.7(9) Dynorphin( 1 13) 0 33.6-+3.0(27) (!.2 38.6±8.9(9) 0.5 29.3±5.1 (9) I 33.8±2.3(9) Peptidase inhibitors Without 39.6±5.4(9) With 41.1±5.0(9)

I00

30 69.4± 66.7+ 71.4± 78.2 ±

7.1 (21) 3.9(9) 6.4(6) 5.3 (6)

85.3_+4.9(21) 77.9±3.3(9) 88.4-+5.1 (6) 86.1 ± 4.7 (6)

64.4± 59.9± 65.0± 59.2_+

8.2(27) 10.5 (9) 10.3 (9) 8.0(9)

73.3-+ 7.8 (27I 73.1±9.3(9) 71.1 _+7.8 (9) 73.2-+7.2(9)

73.4± 71.2-+ 72.3_+ 79.9±

5.4(27) 5.2(9) 1.6(9) 4.4(9)

89.0±6.6(9) 92.2-+5.4(3) 86.2± 1.7(3) 89.7-+2.1 (3)

69.9_+ 6.5(9) 72.1± 5.4(9)

84.6±5.1 (9) 80.0±4.7 (9)

The mixture of peptidase inhibitors (100 nM each of amastatin, phosphoram±don, and captopril) and the opioid peptides (50, 100, and 200 nM Met-enkephalin; 20, 50, and 100 nM /3h-endorphin; and 0.2, 0.5, and 1 nM dynorphin(l - 13)) were added to the bath in volumes of 5/zl, 10 min before the single application of ACh (10, 30, and 100 nM). ACh-induced basal contraction in the presence of the mixture of peptidase inhibitors and in the presence of the opioid peptides was determined as a percentage of the 200 nM ACh contraction in the absence of the opioid peptide and the mixture of peptidase inhibitors, respectively. The values represent the means±S.E.M, of the number of experiments shown in parentheses. Results were not significantly different from those in the absence of opioid peptides or without the mixture of peptidase inhibitors.

M. Ozaki et al./Journal of the Autonomic Nervous System 58 (1996) 147-152

2.9. Statistics Values are expressed as means and standard error of the mean (S.E.M.) of the number of preparations. The significance of differences in the presence or absence of an antagonist at the corresponding time or concentration of the drug was evaluated by two-way analysis of variance with Student's t-test. Differences were considered significant at P < 0.05.

3. Results

3.1. Effects of the mixture of peptidase inhibitors on electrically evoked twitch inhibition exerted by opioid peptides Typical tracings of the effect of the peptidase inhibitors (100 nM each of amastatin, phosphoramidon, and captopril) on the twitch inhibition evoked by Met-enkephalin, dynorphin(1-13), and /3h-endorphin are shown in Fig. 1. The mixture of peptidase inhibitors itself did not affect the twitch response. Met-enkephalin concentration-dependently inhibited the twitch response. The twitch inhibition evoked by Met-enkephalin was significantly potentiated in the presence of the peptidase inhibitors ( Figs. 1 and 2, upper), flh-Endorphin and dynorphin(1-13) also concentration-dependently inhibited the twitch response. However, although the twitch inhibition evoked by /3h-endorphin and dynorphin(1-13) was potentiated in the presence of the peptidase inhibitors ( Figs. 1 and 2 middle and lower panel, respectively), this effect was not significant. These increases evoked by opioid peptides in the presence and absence of peptidase inhibitors were abolished by naloxone (100 nM) (data not shown).

3.2. Effects of the mixture of peptidase inhibitors on post-tetanic twitch response Typical tracings of the effects of the peptidase inhibitors (100 nM each of amstatin, phosphoramidon, and captopril, on post-tetanic twitch inhibition are shown in Fig. 3. In the presence of the mixture of peptidase inhibitors, the post-tetanic twitch inhibition was increased in a manner that depended on the length of the tetanus (1, 1.5, and 2 min) compared with non-treatment ( Figs. 3 and 4). Naloxone (100 nM) inhibited the post-tetanic twitch inhibition both in the presence and absence of the mixture of peptidase inhibitors, and the antagonism exerted by naloxone of the increased post-tetanic twitch inhibition induced by the mixture of peptidase inhibitors was equivalent to that exerted by naloxone alone (Fig. 4).

3.3. Effects of the mixture of peptidase inhibitors and opioid peptides on ACh-contraction The mixture of peptidase inhibitors, (100 nM each of amastatin, phosphoramidon and captopril) and the opioid

151

peptides did not affect the ACh-induced basal contraction (Table 1).

4. Discussion

The post-tetanic opiate-like inhibition of twitch contractions is a useful indicator for studying the mechanism of endogenous opioid action in this preparation [5,6,8,9]. However, the characteristics of the post-tetanic twitch inhibition and the type of endogenous opioids released by tetanic stimulation are not fully understood. As we have previously reported, /x- and K-opioid ligands seem to be candidates for the endogenous opioid ligands released by tetanic stimulation [6]. Concerning the release of endogenous opioid peptides evoked by electrical stimulation, the release of Met-enkephalin [2] and pro-enkephalin-derived peptide [1], /z-ligands, from the myenteric plexus in the ileum evoked by tetanic stimulation has been demonstrated. However, the K-ligand released by tetanic stimulation has not been determined. Furthermore, whether exogenously applied endorphins and dynorphins are influenced by peptidase inhibitors is still under discussion. This experiment was performed to elucidate the opioid ligand type that potentiated the post-tetanic twitch inhibition by the peptidase inhibitors. The effect of peptidase inhibitors on the twitch inhibition evoked by /z- and K-ligands, which have enkephalin residues, was investigated. Met-enkephalin and /3h-endorphin for the /x-ligand, and dynorphin(l13) for the K-ligand, concentration-dependently inhibited the twitch response. The twitch inhibition evoked by Metenkephalin was significantly potentiated in the presence of the peptidase inhibitors. However, the twitch inhibition evoked by /3h-endorphin and dynorphin(1-13) was only slightly increased in the presence of the peptidase inhibitors. These results indicate that the Met-enkephalin molecule is a shorter endogenous opioid peptide that is more readily subjected to enzymatic degradation than the other longer peptides, i.e. dynorphins and endorphins, and that Met-enkephalin is readily degraded by the peptidases that were blocked by the mixture of inhibitors used in this study [4]. The post-tetanic twitch inhibition was potentiated by the mixture of peptidase inhibitors. Naloxone, an opioid antagonist, inhibited the post-tetanic twitch inhibition both in the presence and absence of the mixture of peptidase inhibitors, and, as stated above, the antagonism exerted by naloxone of the increased post-tetanic twitch inhibition induced by the mixture of peptidase inhibitors was equivalent to that exerted by naloxone alone. Moreover, neither the effect of peptidase inhibitors on the twitch response nor the ACh-induced basal contraction was changed in the presence of the mixture of peptidase inhibitors or the respective opioid peptide. These results suggest that these potentiations of the twitch responses by peptidase in-

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M. Ozaki et al./Journal of the Autonomic Nervous System 58 (1996) 147-152

hibitors were not due to the cholinergic-receptor blockade of ACh released from cholinergic neurons by the field stimulation, but were due to the inhibition of peptidases that degrade endogenous opioid peptides. These results also suggest that the degradation of the /x-opioid receptor ligands, Met-enkephalin and flh-endorphin, was involved in the post-tetanic twitch inhibition which was protected by peptidase inhibitors to a greater extent than the K-type of opioid receptor ligand. Furthermore, it appears that the response of the jz-opioid receptor type is concomitantly potentiated when endogenous opioids are released by tetanic stimulation from opioidergic nerve endings. In other words, the potentiation of post-tetanic twitch inhibition by the peptidase inhibitors used here appears to be due primarily to the /z-ligands, which are degraded to a greater extent than the K-ligands by the peptidases. In conclusion, our findings suggest that the potentiated post-tetanic twitch inhibition evoked by peptidase inhibitors is due primarily to the /z-ligands rather than to the K-ligands released from opioidergic neurons by tetanic stimulation. References [1] Corbett, A.D., Gillan, M.G.C. and Kosterlitz, H.W., Electrically-induced release of opioid peptides from the guinea-pig myenteric plexus preparation, J. Recept. Res., 11 (1991) 665-673. [2] Glass, J., Chan, W.C. and Gintzler, A.R., Direct analysis of the

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10] [I 1]

release of methionine-enkephalin from guinea pig myenteric plexus: modulation by endogenous opioids and exogenous morphine, J. Pharmacol. Exp. Ther., 239 (1986) 742-747. Kosterlitz, H.W. and Watertield, A.A., In vitro models in the study of structure-activity relationships of narcotic analgesics, Annu. Rev. Pharmacol., 15 (1975) 29-47. McKnight, A.T., Corbett, A.D. and Kosterlitz, H.W., Increase in potencies of opioid peptides after peptidase inhibition, Eur. J. Pharmacol., 86 (1983) 393-402. Ozaki, M., Miyamoto, Y., Kishioka, S., Masuda, Y. and Yamamoto, H., Effect of some peptidase inhibitors on exogenous and endogenous opioid action in isolated guinea-pig ileum, Biol. Pharm. Bull., 17 (1994) 62-69. Ozaki, M., Miyamoto, Y., Kishioka, S., Masuda, Y. and Yamamoto, H., Participation of opioid receptor types on post-tetanic twitch inhibition of isolated guinea-pig ileal longitudinal muscle, Neuropharmacology, 33 (1994) 671-679. Ozaki, M., Miyamoto, Y., Kishioka, S., Masuda, Y., Yamamoto, H., The dopaminergic system modulates the endogenous opioid system in guinea-pig isolated ileal longitudinal muscle, Neuropharmacology, 34 (1995) 473-480. Puig, M.M., Gascon, P., Craviso, G.L. and Musacchio, J.M., Endogenous opiate receptor ligand: electrically induced release in the guinea-pig ileum, Science, 195 (1977) 419-420. Puig, M.M., Gascon, P. and Musacchio, J.M., Electrically induced opiate-like inhibition of guinea-pig ileum: cross tolerance to morphine, J. Pharmacol. Exp. Ther., 206 (1978) 289-302. Rang, H.P., Stimulant actions of volatile anaesthetics on smooth muscle, Br. J. Pharmacol., 22 (1964) 356-365. Yoshimura, K., Huidobro-Toto, J.P., Lee, N.M., Loh, H.H. and Way, E.L., Kappa opioid properties of dynorphin and its peptide fragments on the guinea-pig ileum, J. Pharmacol. Exp. Ther., 222 (1982) 71-79.