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Tripeptides acting on opioid receptors in rat colon
European Journal of Pharmacology, 100 (1984) 29 39
29
Elsevier
T R I P E P T I D E S A C T I N G O N O P I O I D R E C E P T O R S IN RAT C O L O N HIDEKI MORITOKI 5, MASAO TAKEI *, MASAH1RO KOTANI, YOSHIAKI KISO **, YUKIO ISHIDA and KOUICHI ENDOH * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1, Tokushima 770, Japan, and * Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Arts and Science University, Yamashiro, Tokushima 770, Japan
Received 4 October 1983, revised MS received 2 January 1984, accepted 17 January 1984
H. MORITOKI, M. TAKEI, M. KOTANI, Y. KISO, Y. ISHIDA and K. ENDOH, Tripeptides acting on opioid receptors in rat colon, European J. Pharmacol. 100 (1984) 29-39.
The tripeptides SD-34 and SD-25 induced atropine-, guanethidine-, antihistaminics-resistant but naloxone-sensitive contractions of isolated rat distal colon. They appeared to act on an opioid receptor, probably of the ~ subtype, distinct from those for methionine enkephalin and morphine, because the pA 2 values of naloxone for the peptides were similar to those for ~-agonists but different from those for methionine enkephalin and morphine, and because the peptides caused contractions of colon that had been desensitized to morphine. Mr 2266, a supposed r-antagonist, inhibited the actions of the peptides, ethylketocyclazocine and dynorphin at concentrations much lower than those inhibiting the actions of methionine enkephalin and morphine. Thus these peptides seem to act on the bt- and/or r-receptors. The actions of the tripeptides were inhibited by methysergide and methylergometrine, but not by the 5-HT 2 antagonist ketanserin, and were not affected by 5-HT or substance P autodesensitization. Thus their actions do not seem to involve 5-HT, histamine, ACh or substance P. It seems likely that the tripeptides, through opioid receptors, directly activate the muscle, or remove some inhibitory modulation of myogenic activity, thus causing contractions. Tripeptides
Rat colon
Contraction
Naloxone
I. Introduction The predominant effects of opiates are inhibitory, but there are reports demonstrating their stimulatory actions both in vivo and in vitro. The excitatory effect of morphine on rat and dog intestine has been widely studied with special reference to the possible mediation of 5-HT or A C h in the effect (Burks, 1973; H u i d o b r o - T o r o and Way, 1981; Nijkamp and Van Ree, 1980), or to stimulation of cholinergic neurons (Bueno et al., 1981; Burks, 1973; N o r t h w a y and Burks, 1979). Other studies have suggested that morphine exerts an t To whom all correspondence should be addressed at first address. ** Present address: Department of Medicinal Chemistry, Kyoto College of Pharmacy, Yamashina, Kyoto 607, Japan. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Mr 2266
Opioid-like action
excitatory effect by removing tonic inhibitory modulation of myogenic activity via a presynaptic mechanism (Gillan and Pollock, 1980) or by stimulating a nonadrenergic, noncholinergic and nonserotonergic pathway (Scheurer et al., 1981). The physiological significance of enkephalins as modulators in the gastrointestinal tract has recently been established by the finding that these c o m p o u n d s are located in or near the myenteric plexus (Hughes et al., 1977) and are released in response to nerve stimulation (Corbett et al., 1980; Schulz et al., 1977), Moreover, enkephalins as well as morphine have been shown to stimulate rat colon (Boura and Olley, 1981; Gillan and Pollock, 1980; H u i d o b r o - T o r o and Way, 1981; Nijkamp and Van Ree, 1980; Scheurer et al., 1981). It seems therefore that enkephalins, as well as having an inhibitory action, act as excitatory modulators in
30 the gastrointestinal tract in vitro in some conditions. Opioid receptors in rat colon have been proposed to have properties of the/~ and • subtypes (Scheurer et al., 1981), or the • and /~ subtypes (Boura and Olley, 1981). Thus, the receptor subtypes in rat colon are not yet well established. We have studied the effects of enkephalin analogs on several in vitro preparations and on analgesia (Kiso et al., 1981; Moritoki et al., 1981). In the present experiments we investigated the effects of the tripeptides SD-34 and SD-25 on rat colon and compared their stimulatory actions with those of opiates and opioid peptides. We also tried to characterize the opioid receptor subtypes with which the peptides interact in the colon.
2. Materials and methods
2.1. Organ bath experiments Male Wistar rats of about 8-10 weeks old (230280 g) were used. A segment of their distal colon of 4 cm length was removed. Both ends of the segment were left open to avoid accumulation of mucosal secretion during the resting period and in long experiments. The preparation was then immersed in a 10 ml organ bath filled with Tyrode solution bubbled with oxygen. The Tyrode solution had the following composition (mM): NaC1 136.9, KCI 2.7, CaC12 1.8, MgC12 1.8, N a H z P O 4 0.42, N a H C O 3 11.9 and glucose 11.2. The oral end of the segment was connected to an isotonic transducer (Nihon Kohoden T D 112S) or a lever with a 1 g load. The bath temperature was kept at 26°C to reduce spontaneous contractions as much as possible. For preparation of dose-response curves, opiates and the tripeptides were added to the organ bath cumulatively in a volume of 1-70 /~1 and the responses were plotted as percentages of the maximal response to morphine or methionine enkephalin. Apparent activities of the peptides and opioids were expressed as pD 2 values (negative logarithms of the molar concentrations producing 50% of the maximal contraction). Inhibitory activities of antagonists were expressed as pA 2 values, de-
termined by the method of Van Rossum (1963), or as pD~ values (negative logarithms of the molar concentrations causing 50% inhibition of the maximal contractions) determined by the method of Van Rossum (1963). The pA 2 values in table 2 are means of the mean values at 3-5 different concentrations of antagonists.
2.2. Morphine tolerant preparations Rats were made morphine-dependent/tolerant by injection of morphine on 6 consecutive days: on the first 3 days, 10 m g / k g was injected subcutaneously (s.c.) 4 times a day; on the following 3 days, 30 m g / k g of morphine was given 4 times a day; on day 7, 30 m g / k g of morphine was injected 2 h before killing the rats. The colon was removed and incubated in Tyrode solution containing 5 × 10 7 M morphine throughout the experiments. This concentration of morphine has been shown to be present in the plasma 3 days after implantation of a morphine pellet (Goldstein and Schulz, 1973; Schulz and Herz, 1976a) and to be sufficient to prevent withdrawal symptoms (Schulz and Herz, 1976b).
2.3. Drugs used The drugs used were SD-34 (MeTyr-D-Met(O)Gly-methylphenethylamide; Kiso et al., 1982), SD-25 (Tyr-D-Met(O)-Gly-methylphenylalaninol; Kiso et al., 1981), [D-Ala2,D-LeuS]enkephalin (DADL; Peptide Institute, Osaka), dynorphin-(117) (Peptide Institute, Osaka), methionine enkephalin (Peptide Institute, Osaka), fentanyl (Sankyo Co., Tokyo), pentazocine (Yamanouchi, Tokyo), ethylketocyclazocine (EKC; SterlingWinthrop Research Institute, Rensselaer, NY), morphine hydrochloride (Takeda, Osaka), naloxone (Endo Laboratories, Garden, City, NY), Mr 2266 ( [ - ]-2-(3-furylmethyl)-5,9-diethyl-2'-hydroxy-6,7-benzomorphan; Boehringer Ingelheim K.G., FRD), serotonin creatinine sulfate (5-HT; Sigma Chemical Co., St. Louis, Mo), ketanserin (Janssen Pharmaceutica, Beerse, Belgium), cinanserin (Squibb Japan Inc., Tokyo), methysergide maleate (Sandoz, Basel, Switzerland), spiperone (Janssen Pharmaceutica), phenylbiguanide (AI-
31 drich Chemical Co., Milwaukee, Wis), quipazine maleate (Miles Laboratories, Elkhart, Ind), phentolamine mesylate (Ciba-Geigy Japan Inc., Osaka), tolazoline hydrochloride (Sigma Chemical Co.), yohimbine hydrochloride (Nakarai Chemical, Kyoto), prazosin (Taito-Pfeizer, Tokyo), phenoxybenzamine hydrochloride (Nakarai), guanethidine sulfate (Ciba-Geigy Japan Inc.), cimetidine (Smith Kline & French Laboratories, Philadelphia, Pa), chlorpheniramine maleate (Tokyo Kasei, Tokyo), quinacrine hydrochloride (Sigma), indomethacin (Sigma), atropine sulfate (Sigma), acetylcholine chloride (ACh, Sigma), tetrodotoxin (Sigma), bacitracin (Sigma), hexamethonium bromide (Yamanouchi) and substance P (Peptide Institute). 3. Results
3.1. Effects of the peptides The tripeptides SD-34 and SD-25 induced dose-dependent and reproducible contractions of the isolated rat distal colon. The contractions
SD-M
I00
developed rapidly and in 20 s reached a maximum which was sustained for more than 30 s followed by relaxation. Therefore, when the peptides were added cumulatively, subsequent doses were applied just when the response reached a maximum. Fig. 1 shows the dose-response curves for the peptides, morphine, ethylketocyclazocine (EKC), methionine enkephalin (Met-enk), and related opioids. The concentration producing 50% contraction (EDs0) of SD-34 was 2.95 × 10 -9 M, which was about one-tenth of that of morphine (2.75 × 10 8 M), but 3 times that of Met-enk ( 8 . 1 2 x 1 0 -x° M). SD-25 ( 6 . 0 3 x 1 0 -9 M) was about half as active as SD-34. The maximal contractions induced by SD-34, SD-25 and morphine were of similar magnitude, and corresponded to 42.0 + 3.1% (n = 8), 46.2 + 3.6% (n = 8) and 42.0 + 5.4% (n = 6), respectively, of the maximal contraction produced by 10-s M ACh. The potencies of the peptides and related opioids in inducing contractions of the colon are listed as pD z values in table 1. In subsequent experiments, SD-34, a tripeptide alkylamide, was used as representative peptide unless otherwise stated.
SD-~
EKC
x
._~ 50 co
i
i0-I0
10-9
i
i0 -8
l
i0 -7
,
iO-6
Opioids (M)
Fig. 1. Dose-responsecurves for the tripeptides and related compounds in inducing contractions of isolated rat distal colon. DADL: [D-Ala2,D-LeuS]enkephalin, Met-enk: methionine-enkephalin, dyn: dynorphin, EKC: ethylketocyclazocine, fent: fentanyl, mor: morphine, pent: pentazocine. The ordinate shows the contractions of the colon as percentages of the maximal contraction induced by Met-enk or morphine. Values (means + S.E.) were obtained with colons of 6-12 animals.
32 TABLE 1
TABLE 2
Potencies of opioid peptides and opiates in inducing contractions of isolated rat distal colon. Potencies are expressed as pD 2 values. Met-enk: methionine enkephalin, DADL: [DAla2,D-Leu~]enkephalin, EKC: ethylketocyclazocine. Numbers in parentheses show numbers of observations.
Effects of naloxone and Mr 2266 in antagonizing the actions of opioid peptides and opiates on rat colon, expressed as pA 2 values. The values are means of the mean values calculated from the values at 4-5 concentrations of the antagonists (3 x 10 9-10 0 M) in 5 animals. Abbreviations are as for table 1. pA2
pD2 SD-34 SD-25 Morphine Fentanyl Met-enk DADL EKC Pentazocine Dynorphine
-- IO0
~, 50
g,
(24) (12) (23) (10) (14) (13) (9) (12) (4)
SD-34 SD-25 Morphine Fen tanyl Met-enk DADL EKC Dynorphin Pen tazocine
Naloxone
Mr 2266
8.56 + 0.07 8.48 ± 0.11 7.85 ± 0.09 8.72 ± 0.02 7.64 ± 0.| 0 7.80 ± 0.06 7.75 ± 0.06 7.97 ± 0.09 8.30 ± 0.10
8.54 + 0.08 8.46 + 0.08 6.97 + 0.07 8.19 _+0.07 7,83 ± 0.09 7.57 + 0.10 8.56 ± 0.07 8,52 ± 0.03 8.16 _+0.06
Fent
SD-34
,///~k
¢, 10-9
= 100
8.53 _+0.05 8.22±0.12 7.56 ± 0.05 7.53 ± 0.03 9.09 ± 0.14 9.58 + 0.04 7.34±0.08 6.46 ± 0.09 8.66±0.23
10-8
10-7
10-6
10-8
I0 -7
DADL
E,c
I0-6
10-5
10-8
10-7
,,~k
X
E
0
~-
"E O
50
/,/¢ 10-8
10-7
10-6
Concentration (M)
10-5
lo-lO
10-9
Concentration (M)
Fig. 2. Antagonism by naloxone of the contractile actions of SD-34 and related compounds. EKC: ethylketocyclazocine, fent: fentanyl, DADL: [D-Ala2,D-LeuS]enkephalin. Concentration of naloxone: O, 0; O, 3 x 10 -9 M; ix, 10 8 M; &, 3 × 10 s M; O, 10 v M; m, 3 )< 10 -7 M. For comparison, the dose-response curves in the presence of 3 x 10 s M naloxone are indicated by dotted lines. The ordinate shows contraction of the colon as a percentage of the maximal contraction. Values (means ± S.E.) were obtained with colons from 5 animals.
33
3.2.1. Effect of naloxone on the actions of the tripeptides Naloxone antagonized the actions of the peptides, and of morphine and related opioids. The dose-response curves obtained are shown in fig. 2. Naloxone at concentrations of 3 x 10 -9, 10 -8, 3 × 10 8, 1 0 - 7 and 3 x 10 -7 M caused a parallel shift to the right of the dose-response curve for SD-34 2.2, 24.6, 79.4 and 281.8 times, respectively. The pA 2 values obtained with all these concentrations of naloxone were almost the same, being 8.46, 8.52, 8.61 and 8.63, respectively. The mean of the mean values was 8.56 (table 2). These results indicate that naloxone caused competitive antagonism. Naloxone antagonized the action of SD-25 as readily as that of SD-34. A similar pA z value of 8.72 was obtained with the /~-agonist fentanyl. Naloxone was more inhibitory with the peptides, fentanyl and pentazocine than with morphine, the 8-agonists [D-AlaZ,D-LeuS]en-
~= 100r SD-~ ~ ,
•
~
kephalin (DADL) and Met-enk, and the x-agonists E K C and dynorphin. At a concentration of 3 x 10-8 M, naloxone displaced in parallel to the right the dose-response curves for SD-34 and fentanyl 24.6 times and 19.9 times, respectively (fig. 2). The pA 2 values of naloxone for morphine, D A D L , Met-enk and dynorphin ranged from 7.79 to 7.64, whereas those for SD-34 and fentanyl were about 8.7 to 8.5 (table 2).
3.2.2. Effect of Mr 2266 on the actions of the peptides Mr 2266, a x-antagonist, preferentially and competitively antagonized the actions of SD-34 and SD-25 at concentrations above 10 -8 M (table 2 and fig. 3). The actions of the x-agonists EKC and dynorphin, and a K- and 8-agonist pentazocine were antagonized as effectively as those of the peptides. The pA 2 values of Mr 2266 for SD-34 and SD-25 were 8.54 and 8.46, respectively, which
Mor
E
10-9
too
10-8
10-7
EKC
10-6
10-8
10-7
10-6
OADL,,,,.~
,'~" A
E I
10-8
10-7
10-6
Concentration (M)
10-5
1'0-10
i;-9
1'0-8
Concentration (M)
Fig. 3. Antagonism by Mr 2266 of the contractile actions of SD-34 and related compounds. For comparison, the dose-responsecurves in the presence of 3 x 10-8 M Mr 2266 are indicated by dotted lines. Abbreviations and symbols are as for fig. 2.
34 TABLE 3
3.3. Effects of the peptides on morphine-tolerant preparations
Comparison of sensitivities of rat colon to opioid peptides and opiates before and after chronic morphine treatment. The ratio is the ratio of ED~o values in normal and morphine-treated preparations. Values are means for those in 2 preparations. Abbreviations are as for table 1. pD2 Normal rats SD-34 SD-25 EKC Met-enk Morphine
Morphinetreated rats
8.40 8.22 7.39 9.59 7.42
7.31 7.11 6.37 8.65 4.21
The effects of the tripeptides and related comp o u n d s were c o m p a r e d on colon chronically treated with m o r p h i n e to render it d e p e n d e n t / tolerant as described in Methods. Withdrawal was prevented by adding 5 × 10 7 M m o r p h i n e to the T y r o d e solution. After this treatment, the sensitivity of the p r e p a r a t i o n to SD-34, SD-25, Met-enk or E K C was decreased a b o u t 10 fold, whereas the sensitivity to m o r p h i n e was dramatically decreased a b o u t 1600-fold. The p D 2 values for n o r m a l and t o l e r a n t / d e p e n d e n t rat colon are compared in table 3. N a l o x o n e did not induce morphinewithdrawal contracture of the colon. The effects of SD-34 and m o r p h i n e on colon rendered acutely tolerant to m o r p h i n e were exa m i n e d next. Rat colon became unresponsive to 10 6 M m o r p h i n e after successive application of the drug without washing (fig. 4). U n d e r these conditions, however, the colon showed almost the same response to SD-34 as the control p r e p a r a t i o n before desensitization by morphine. At this time, the colon r e m a i n e d unresponsive to m o r p h i n e even at a c o n c e n t r a t i o n of 3 × 10-5 M. I m m e d i a t e l y after the application of 3 x 10 5 M morphine, the colon still responded to SD-34. This result also shows that p r e t r e a t m e n t with 3 × 10 -~ M
EDso ratio
12.9 8.7 10.5 14.8 1621.8
is very close to those for E K C (8.56), d y n o r p h i n (8.52) a n d pentazocine (8.16). M r 2266 showed high a n t a g o n i s t activity against fentanyl as well as SD-34 and EKC, the pA 2 value for fentanyl being 8.19. M u c h higher c o n c e n t r a t i o n s (10 times or more) were required to p r o d u c e the same degree of a n t a g o n i s m of the actions of so-called &agonists: the pA 2 values for D A D L a n d M e t - e n k were 7.57 a n d 7.83, respectively. M o r p h i n e was highly resistant to M r 2266 as evidenced by its significantly lower pA 2 value (6.97) than those for SD-34, SD-25 and E K C (8.5-8.6).
2o/ 7
~0 xlO-9M SD-34
•
•
•
•
•
Morphine IO-6M
•
10
I0 xlO-gM SD-34
3 min
I0
A
3
ZX
Morphine 3xlO-5M xlO-9M Morphine SD-34 3xlO-5M
Fig. 4. Morphine (10 - 6 M) was added successivelyas shown by filled triangles (usually 4-6 doses) without washing and remained in the bath throughout the experiments as indicated by the dotted line. Immediately after the last application of 10 6 M morphine, SD-34 at concentrations of above 3 x 10-9 M was added cumulatively and remained in the bath. When the colon relaxed, SD-34 (10 -8 M) was applied twice for further confirmation of the response to SD-34. Morphine 3 × 10 5 M was added thereafter.
35 morphine did not interfere with the action of SD-34. Conversely, repeated application of SD-34 at 10 7 M did not desensitize the colon to morphine.
3.4. Effect of bacitracin When preparations were treated with 10 - 4 g / m l of bacitracin for 30 min, the action of SD-34 was augmented. The p D 2 value increased from the control value of 8.52 + 0.10 to 8.92 + 0.10 (n = 5) whereas that of morphine (7.75 + 0.06) was slightly decreased (7.62 + 0.15, n = 4).
3.5. Effects of 5-HT antagonists and desensitization Methylergometrine at a concentration (10-6 M), which did not affect the contraction induced by ACh, inhibited the contractions induced by SD-34 and morphine. This inhibition was noncompetitive, and the apparent p D 2 value was 5.93 _+ 0.14 (n = 4) for SD-34 (fig. 5). Besides inhibiting the action of SD-34, this concentration of methylergometrine antagonized the contractile action of 5-HT, the pA 2 value being 8.8 (n = 2). Likewise, methysergide 10 -7 and 3 × 10 -7 M shifted the dose-response curve for SD-34 downward and to the right (fig. 5). The inhibitory potency of methysergide expressed as the pD~ value was 6.82
I0O
50 C
R
(3C
3.6. Effect of substance P autodesensitization The possible contribution of substance P (SP) to the actions of the tripeptides and related opioids were examined by testing their effects on preparations desensitized to SP by its repeated application without washout. Addition of SP at a concentration of 3 × 10 -8 M produced almost maximal contraction that gradually returned to the basal
I00
E E
+ 0.04 (n = 4). However, increasing the concentration of methysergide to 10 -5 M did not cause further suppression. The pD; values for SD-25 and morphine were 6.38 and 6.35 (n = 2), respectively. Ketanserin (10-8-10 .6 M), cinanserin (10 8_ 10 .6 M), quipazine (10-6-10 5 M), spiperone (10-8-10 -6 M) and phenylbiguanide (10-7-3 × 10-7 M) did not have any inhibitory effects on the contractions induced by SD-34. Next we examined whether autodesensitization by repeated applications of 5-HT affected the action of SD-34. 5-HT at 10 .5 M was applied to the colon to produce maximal contraction. When the intense contraction had subsided to the basal level, the same concentration of 5-HT was applied to confirm the development of tachyphylaxis. The preparation usually failed to respond to a third dose of 10-5 M 5-HT. The actions of SD-34 and morphine were not affected by this treatment.
/ 10-9
M•ethysercjide 50
go 10-6M //~~xlO-7M 10-8 SD- 34
10-7 (M)
i0-9
10-8 SD- 34 (MI
10-7
Fig. 5. Effects of methysergide and methylergometrine(Me-ergo) on the contractions of rat colon induced by SD-34. Values (means + S.E.) were obtained with colons from 5 animals.
36 level. A second application of the same dose of SP produced a contraction smaller than the first one. After 4 to 5 successive applications of SP, the colon lost its responsiveness to SP but not to the peptides or morphine.
3. 7. Effects of other agents Atropine (10 -7 M) and hexamethonium (10 5 M) had no inhibitory effects on opioid-induced contractions. Tetrodotoxin at concentrations of up to 3 × 10 7 M did not affect the contractile actions of the peptides but concentrations as high as 10 6 M or more produced dose-dependent contractions just as did the peptides, the pD 2 value being 6.64 + 0.05 (n = 3). The a-blockers phentolamine and tolazoline (3 × 10 -6 M, each) enhanced the contraction of the colon induced by SD-34. In the presence of 3 × 10 6 M tolazoline, the dose-response curve for SD-34 (pD 2 = 8.84, n = 2) was shifted slightly upward (115.4%) and 7.6 times to the left (pD 2 = 9.34, n = 2). Similarly, 3 x 10 6 M phentolamine increased the pD 2 value of SD-34 from 8.34 to 8.90 (n = 3)~ and the maximal contraction height to 122.0%. However, other a-blockers, phenoxybenzamine, prazosin and yohimbine (10 6 M each), did not show the same effect as tolazoline and phentolamine. Guanethidine (3 × 10 6 M) had no effect on the action of SD-34. The histamine H2-antagonist cimetidine (10 5_ 10 4 M), the histamine H~-antagonist chlorpheniramine (10 7 M) and the inhibitors of prostaglandin synthesis quinacrine (10 7-10 6 M) and indomethacin (10 -4 M) did not affect the contractile actions of SD-34, Met-enk and morphine.
4. Discussion The present study demonstrated that the tripeptides SD-34 and SD-25 induced contractions of isolated rat colon, just as did Met-enk and morphine. Naloxone selectively and competitively antagonized the contractile actions of the peptides, indicating that the peptides act on an excitatory opioid receptor. The order of potency of the peptides and related opioids in inducing contrac-
tions of the colon was D A D L and Met-enk (socalled 3-agonists)> SD-34, SD-25, and dynorphin (so-called ~-agonists)> fentanyl and morphine (so-called /~-agonists). Thus, judging from the affinities of the agonists, the receptors in the colon seem to consist of 3-, ~- and/~-subtypes. The pA 2 values of naloxone for the peptides were not comparable with its pA 2 values for the 3-agonists D A D L and Met-enk in the present experiments (table 2), or with its pA 2 values for 3-agonists calculated by Boura and Olley (1981), Huidobro-Toro and Way (1981), Lord et al. (1977) and Nijkamp and Van Ree (1980); they were however comparable with its pA 2 value for the /x-agonist fentanyl. In contract to the actions of 6-agonists, those of SD-34 and SD-25 were antagonized by naloxone as readily as that of the #-agonist fentanyl. About 5 to 10 times less naloxone was required to antagonize the actions of the peptides than to antagonize the actions of the 3-agonists. The pA 2 values of naloxone for SD-34 and SD-25 were 8.56 and 8.48, respectively, being similar to that of 8.7 for fentanyl and that of 8.6 reported by Boura and Olley (1981) for the /~agonist Rx 3030 on rat colon. Thus, judging from the pA 2 values of naloxone, the peptides seemed to act mainly on the ~L-receptor rather than the &receptor in the rat distal colon in the present experiments. On the other hand, opioid receptors in isolated rat colon have been suggested to have properties of the /x and K subtypes (Scheurer et al., 1981). Thus SD-34 and SD-25 could also have the characteristics of ~-agonists on the colon. In support of this possibility, Mr 2266, suggested to be a ~antagonist (Kosterlitz et al., 1981; Lord et al., 1977; Oka et al., 1982), showed a greater antagonism of the peptides as well as of EKC, dynorphin and pentazocine than of the 3-agonists Met-enk and DADL. However, the action of the ~-agonist was antagonized by Mr 2266 as effectively as the actions of EKC and dynorphin. It is noteworthy in this respect that Mr 2266 has been reported to act well on both the /~-receptor and K-receptor in guinea-pig ileum (Lord et al., 1977; Yoshimura et al., 1982). In addition, the pA 2 values of naloxone for the peptides were not comparable with those for EKC, dynorphin and penta-
37 zocine. These results argue against a major contribution of the x-receptor to the actions of the peptides. In contrast, the relative effects of Mr 2266 and naloxone, expressed as Ke n a l o x o n e / K e Mr 2266, for the peptides (0.91-0.95) were different from those for the bt-agonist fentanyl (0.30), even though the values for the peptides were not comparable with those for EKC and dynorphin (3.38-3.54). Furthermore, Mr 2266 antagonized the actions of the peptides more readily than that of fentanyl as evidenced by the pA 2 values of Mr 2266 for the peptides (8.5) and fentanyl (8.2). Thus, it is not clear whether SD-34 and SD-25 act as /~-agonists or x-agonists, but it is evident that these peptides do not have 8-agonist characteristics. Pentazocine, which is thought to be a ~- and 6-agonist (Snyder and Goodman, 1980), may also act on the #-receptor, because the pA 2 value of naloxone for pentazocine was similar to that for a /~-agonist. Morphine has been reported to be a bt-agonist in guinea-pig ileum but in rat colon it may interact with a different subtype of receptor. The pA 2 value of naloxone for morphine was found to be 7.85 in the present experiments and has been reported to be 7.67 (Huidobro-Toro and Way, 1981), these pA 2 values being similar to those for 6-agonists. However, the pA 2 value of Mr 2266 for morphine was not comparable with its value for either/~-, 8- or K-agonists, suggesting that morphine also acts on another subtype of receptor. If SD-34 produces contractions by releasing endogenous enkephalins the pA 2 values of naloxone and Mr 2266 for SD-34 should be the same as those for Met-enk. Thus SD-34 does not seem to release endogenous enkephalins. If the peptides act on the morphine receptor, selective tolerance could be produced by chronic treatment with morphine in vivo or by repeated application of morphine in vitro. In fact, after chronic morphine treatment, tolerance to morphine did develop as reported by Huidobro-Toro and Way (1981) and Whster et al. (1981), but the resulting tolerance to the peptides was less (table 3). Similarly, when acute desensitization developed after repeated application of morphine without
washout tolerance to morphine developed but cross tolerance to SD-34 did not (fig. 4). These findings also suggest that the receptor for SD-34 is different from that for morphine. The contractile actions of morphine and related opioids have been studied by many workers. The idea that these actions are mediated by 5-HT is based on the finding that they were attenuated by the 5-HT antagonist methysergide (Burks, 1976; Huidobro-Toro and Way, 1981; Nijkamp and Van Ree, 1980) or cyproheptadine (Burks, 1973; Nijkamp and Van Ree, 1980), and that morphine did not cause contraction of a preparation rendered tachyphylactic to 5-HT (Burks and Long, 1967). In fact, the present experiments demonstrated that the action of SD-34 was antagonized noncompetitively by the lysergic acid derivatives methysergide and methylergometrine at concentrations sufficient to cause competitive antagonism of the action of 5-HT. However, we also observed that SD-34 still caused contractions of rat colon desensitized to 5-HT by prolonged exposure to a high concentration of 5-HT (Gillan and Pollock, 1980). Moreover, the 5-HT2 antagonists ketanserin (Van Nueten et al., 1981), cinanserin (Rubin et al., 1964), quipazine (Lansdown et al., 1980), spiperone (Peroutka and Snyder, 1979) and phenylbiguanide (Gintzler, 1979) did not block the actions of SD-34 and morphine. The present results and the above considerations are not consistent with the idea that a serotonergic mechanism is in some way associated with the actions of SD-34 and morphine. There is evidence for a mediatory role of SP in the actions of opiates. It has been reported that SP is released from guinea-pig ileum by electrical stimulation or treatment with dimethylphenylpiperadinium (Franco et al., 1979b) and SP-like immunoreactivity has been detected in guinea-pig ileum (Costa et al., 1980; Franco et al., 1979a) and rat colon (Holzer et al., 1980). Thus SP is thought to be a putative transmitter or a modulator in the noncholinergic excitatory response of the gut (Gintzler, 1979; 1980; Gintzler and Scalisi, 1982; Tsou et al., 1982). To determine whether SP is involved in mediating the contractile action of SD-34, we used a procedure for autodesensitization to SP. As exposure of the ileum to a relatively high concentration of SP is known to render it
38 insensitive to further application of SP ( F r a n c o et al., 1979a; 1979b; M o n i e r a n d Kitabgi, 1980; Tsou et al., 1982), the same or an analogous p h e n o m e n o n might be observed in the colon if SP is involved. A l t h o u g h we d e m o n s t r a t e d that repeated application of SP to the colon made it insensitive to SP, the colon r e s p o n d e d to SD-34 a n d m o r p h i n e as well after this t r e a t m e n t as before desensitization. It thus seems unlikely that the actions of SD-34 and m o r p h i n e on the colon are mediated by SP. It has been suggested that n o r e p i n e p h r i n e is an i n h i b i t o r y m o d u l a t o r in the gut a n d that removal of this i n h i b i t o r y influence by the a-blocker phent o l a m i n e resulted in an increase in the stimulatory effect of Met-enk, which seems to act directly on the muscle ( N i j k a m p a n d Van Ree, 1980). In fact, in the present experiments, tolazoline and phent o l a m i n e slightly p o t e n t i a t e d the contractions induced by SD-34 a n d morphine. However, a n o t h e r a - b l o c k e r p h e n o x y b e n z a m i n e a n d the selective a~blocker prazosin a n d a2-blocker y o h i m b i n e did not potentiate the actions of the peptides or morphine. Thus the question arises of why selective a-blockers did not attenuate the supposed a - i n h i b i t o r y m o d u l a t i o n . A n o t h e r mechanism(s) unrelated to an a - b l o c k i n g action may be responsible for the observed p o t e n t i a t i o n b u t at present there is n o evidence for this. The failure of inhibitors of p r o s t a g l a n d i n synthesis ( q u i n a c r i n e a n d i n d o m e t h a c i n ) a n d h i s t a m i n e H 1- a n d Hz-antagonists (chlorpheniram i n e and cimetidine) to a t t e n u a t e the contractile actions of SD-34 and m o r p h i n e implies that the actions of the c o m p o u n d s are not mediated by p r o s t a g l a n d i n s or histamine. We confirmed that tetrodotoxin, like SD-34, p r o d u c e d c o n t r a c t i o n s of rat colon. Thus SD-34 m a y act by blocking tonic i n h i b i t o r y m o d u l a t i o n as has been reported by G i l l a n a n d Pollock (1980) for the stimulatory action of tetrodotoxin. We suggest, based on the results of this study, that the tripeptides SD-34 a n d SD-25 produce their contractile effects on rat colon through the opioid/~- or K-receptor or both but not through the 6-receptor. The stimulatory effects of the tripeptides a n d related c o m p o u n d s tested do not seem to be mediated by 5-HT, A C h or SP, or due
to blockade of the adrenergic inhibitory pathway. These c o m p o u n d s seem either to act by blockade through opioid receptors of the nonadrenergic, noncholinergic inhibitory pathway m o d u l a t i n g s p o n t a n e o u s myogenic activity, or to cause direct s t i m u l a t i o n of opioid receptors in the muscle.
Acknowledgements We are grateful to Dr. J.M. Van Nueten, Janssen Pharmaceutica, Beerse, Belgium, for gifts of ketanserin and spiperone, Dr. H. Merz, Boehringer Ingelheim K.G., West Germany, for a gift of Mr 2266, Smith Kline & French Laboratories, Philadelphia, Pa for supplying cimetidine, Endo Laboratories, Garden City, NY~ for supplying naloxone, Sterling-Winthrop Research Institute, Rensselaer, NY, for supplying ethylketocyclazocine,Squibb Japan Inc., Tokyo, for supplying cinanserin, Sankyo Co., Tokyo, for supplying fentanyl and Taito-Pfizer, Tokyo, for supplying prazosin.
References Boura, A.L.A. and J.E. Olley, 1981, Characterization of the excitatory opiate receptors in the rat large intestine, Br. J. Pharmacol. 74, 246p. Bueno, L., J. Floramonti and M. Ruckebusch, 1981, Comparative effects of morphine and nalorphine on colonic motility in the conscious dog, European J. Pharmacol. 75, 239. Burks, T.F., 1973, Mediation by 5-hydroxytryptamine of morphine stimulant actions in dog intestine, J. Pharmacol. Exp. Ther. 185, 530. Burks, T.F., 1976, Acute effects of morphine on rat intestinal motility, European J. Pharmacol. 40, 279. Burks~ T.F. and J.P. Long, 1967, Release of intestinal 5-hydroxytryptamine by morphine and related agents, J. Pharmacol. Exp. Ther. 156, 267. Corbett, A.D., R.P. Sosa, A.T. McKnight and H.W. Kosterlitz, 1980, Effects of electrical stimulation on the enkephalins in guinea-pig small intestine, European J. Pharmacol. 65, I 13. Costa, M., A.C. Cuello, J.B. Furness and R. Franco, 1980, Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum, Neuroscience 5, 323. Franco, R,, M. Costa and J.B. Furness, 1979a, Evidence for the release of endogenous substance P from intestinal nerves, Naunyn-Schmiedeb. Arch. Pharmacol. 306, 159. Franco, R., M. Costa and J.B. Furness, 1979b, Evidence that axons containing substance P in the guinea-pig ileum are of intrinsic origin, Naunyn-Schmiedeb. Arch. Pharmacol. 307, 57. Gillan, M.G.C. and D. Pollock, 1980, Acute effects of morphine and opioid peptides on the motility and responses of rat colon to electrical stimulation, Br. J. Pharmacol. 68, 381.
39 Gintzler, A.R., 1979, Serotonin participation in gut withdrawal from opiates, J. Pharmacol. Exp. Ther. 211, 7. Gintzler, A.R., 1980, Substance P involvement in the expression of gut dependence on opiates, Brain Res. 182, 224. Gintzler, A.R. and J.A. Scalisi, 1982, Effects of opioids on noncholinergic excitatory responses of guinea-pig ileum: inhibition of release of enteric substance P, Br. J. Pharmacol. 75, 199. Goldstein, A. and R. Schulz, 1973, Morphine-tolerant longitudinal muscle strip from guinea-pig ileum, Br. J. Pharmacol. 48, 655. Holzer, P., R. Gamse and F. Lembeck, 1980, Distribution of substance P in the rat gastrointestinal tract - Lack of effect of capsaicin pretreatment, European J. Pharmacol. 61,303. Hughes, J., H.W. Kosterlitz and T.W. Smith, 1977, The distribution of methionine enkephalin and leucine enkephalin in the brain and peripheral tissues, Br. J. Pharmacol. 61, 639. Huidobro-Toro, J.P. and E.L. Way, 1981, Contractile effect of morphine and related opioid alkaloids, fl-endorphin and methionine enkephalin on the isolated colon from Long Evans rats, Br. J. Pharmacol. 74, 681. Kiso, Y., T. Miyazaki, M. Satomi, M. Imai, T. Akita, H. Moritoki, M. Takei and H. Nakamura, 1982, Synthesis and activity of short-chain enkephalin-like peptides, Peptide Chemistry 1981, p. 65. Kiso, Y., Y. Yamaguchi, T. Akita, H. Moritoki and M. Takei, 1981, Super-active enkephalin analogues, Naturwissenschaften 68, S 210. Kosterlitz, H.W., S.J. Paterson and L.E. Robson, 1981, Characterization of the x-subtype of the opiate receptor in the guinea-pig brain, Br. J. Pharmacol. 73, 933. Lansdown, M.J.R., H.L. Nash, P.R. Preston, D.I. Wallis and R.G. Williams, 1980, Antagonism of 5-hydroxytryptamine receptors by quipazine, Br. J. Pharmacol. 68, 525. Lord, J.A.H., A.A. Waterfield, J. Hughes and H.W. Kosterlitz, 1977, Endogenous opioid peptides: multiple agonists and receptors, Nature 267, 495. Monier, S. and P. Kitabgi, 1980, Substance P-induced autodesensitization inhibits atropine-resistant, neurotensin-stimulated contractions in the guinea-pig ileum, European J. Pharmacol. 65,461. Moritoki, H., Y. Kiso, T. Kageyama and K. Matsumoto, 1981, Morphine-like activities of synthetic enkephalin analogues, J. Pharm. Pharmacol. 33, 54. Nijkamp, F.P. and J.M. Van Ree, 1980, Effects of endorphins on different parts of the gastrointestinal tract of rat and guinea-pig in vitro, Br. J. Pharmacol. 68, 599. Northway, M.G. and T.F. Burks, 1979, Indirect intestinal stimulatory effects of heroin: direct action on opiate receptors, European J. Pbarmacol. 59, 237.
Oka, T., K. Negishi, M. Suda, A. Sawa, M. Fujino and M. Wakimasu, 1982, Evidence that dynorphin (1-13) acts as an agonist on x-opioid receptor, European J. Pharmacol. 77, 137. Peroutka, S.J. and S.H. Snyder, 1979, Multiple serotonin receptors: Differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol, Mol. Pharmacol. 16, 687. Rubin, B., J.J. Piala, J.C. Burke and B.N. Craver, 1964, A new potent and specific serotonin inhibitor (SQ 10,643) 2-(3-dimethylaminopropylthio)-cinnamanilidehydrochloride: antiserotonin activity on uterus and gastrointestinal, vascular and respiratory systems of animals, Arch. Int. Pharmacodyn. 152, 132. Scheurer, U., E. Drack, L. Vara and F. Halter, 1981, Morphine-like action of enkephalin analog FK 33-539 on motility of the isolated rat colon, J. Pharmacol. Exp. Ther. 219, 534. Schulz, R. and A. Herz, 1976a, Aspects of opiate dependence in the myenteric plexus of the guinea-pig, Life Sci. 19, 1117. Schulz, R. and A. Herz, 1976b, The guinea-pig ileum as an in vitro model to analyse dependence liability of narcotic drugs, in: Opiates and Endogenous Opioid Peptides, ed. H.W. Kosterlitz (Elsevier Biomedical Press, North-Holland) p. 319. Schulz, R., M. Wi~ster, R. Simantov, S.H. Snyder and A. Herz, 1977, Electrically stimulated release of opiate-like material from the myenteric plexus of the guinea-pig ileum, European J. Pharmacol. 41,347. Snyder, S.H. and R.R. Goodman, 1980, Multiple neurotransmitter receptors, J. Neurochem. 35, 5. Tsou, K., G. Louie and E.L. Way, 1982, Manifestations of gut withdrawal contracture and its blockade by capsaicin, European J. Pharmacol. 81,377. Van Nueten, J.M., P.A.J. Janssen, J. VanBeek, R. Xhonneux, T.J. Verbeuren and P.M. Vanhoutte, 1981, Vascular effects of ketanserin (R 41 468), A novel antagonist of 5-HT2 serotoninergic receptors, J. Pharmacol. Exp. Ther. 218, 217. Van Rossum, J.M., 1963, Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int. Pharmacodyn. 143, 299. Wi~ster, M., R. Schulz and A. Herz, 1981, Multiple opiate receptors in peripheral tissue preparations, Biochem. Pharmacol. 30, 1883. Yoshimura, K., J.P. Huidobro-Toro and E.L. Way, 1982, Potency of three opiate antagonists to reverse the inhibitory activity of dynorphin, enkephalin and opioid-like alkaloids on the guinea-pig ileum, European J. Pharmacol. 84, 17.
29
Elsevier
T R I P E P T I D E S A C T I N G O N O P I O I D R E C E P T O R S IN RAT C O L O N HIDEKI MORITOKI 5, MASAO TAKEI *, MASAH1RO KOTANI, YOSHIAKI KISO **, YUKIO ISHIDA and KOUICHI ENDOH * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1, Tokushima 770, Japan, and * Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Arts and Science University, Yamashiro, Tokushima 770, Japan
Received 4 October 1983, revised MS received 2 January 1984, accepted 17 January 1984
H. MORITOKI, M. TAKEI, M. KOTANI, Y. KISO, Y. ISHIDA and K. ENDOH, Tripeptides acting on opioid receptors in rat colon, European J. Pharmacol. 100 (1984) 29-39.
The tripeptides SD-34 and SD-25 induced atropine-, guanethidine-, antihistaminics-resistant but naloxone-sensitive contractions of isolated rat distal colon. They appeared to act on an opioid receptor, probably of the ~ subtype, distinct from those for methionine enkephalin and morphine, because the pA 2 values of naloxone for the peptides were similar to those for ~-agonists but different from those for methionine enkephalin and morphine, and because the peptides caused contractions of colon that had been desensitized to morphine. Mr 2266, a supposed r-antagonist, inhibited the actions of the peptides, ethylketocyclazocine and dynorphin at concentrations much lower than those inhibiting the actions of methionine enkephalin and morphine. Thus these peptides seem to act on the bt- and/or r-receptors. The actions of the tripeptides were inhibited by methysergide and methylergometrine, but not by the 5-HT 2 antagonist ketanserin, and were not affected by 5-HT or substance P autodesensitization. Thus their actions do not seem to involve 5-HT, histamine, ACh or substance P. It seems likely that the tripeptides, through opioid receptors, directly activate the muscle, or remove some inhibitory modulation of myogenic activity, thus causing contractions. Tripeptides
Rat colon
Contraction
Naloxone
I. Introduction The predominant effects of opiates are inhibitory, but there are reports demonstrating their stimulatory actions both in vivo and in vitro. The excitatory effect of morphine on rat and dog intestine has been widely studied with special reference to the possible mediation of 5-HT or A C h in the effect (Burks, 1973; H u i d o b r o - T o r o and Way, 1981; Nijkamp and Van Ree, 1980), or to stimulation of cholinergic neurons (Bueno et al., 1981; Burks, 1973; N o r t h w a y and Burks, 1979). Other studies have suggested that morphine exerts an t To whom all correspondence should be addressed at first address. ** Present address: Department of Medicinal Chemistry, Kyoto College of Pharmacy, Yamashina, Kyoto 607, Japan. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Mr 2266
Opioid-like action
excitatory effect by removing tonic inhibitory modulation of myogenic activity via a presynaptic mechanism (Gillan and Pollock, 1980) or by stimulating a nonadrenergic, noncholinergic and nonserotonergic pathway (Scheurer et al., 1981). The physiological significance of enkephalins as modulators in the gastrointestinal tract has recently been established by the finding that these c o m p o u n d s are located in or near the myenteric plexus (Hughes et al., 1977) and are released in response to nerve stimulation (Corbett et al., 1980; Schulz et al., 1977), Moreover, enkephalins as well as morphine have been shown to stimulate rat colon (Boura and Olley, 1981; Gillan and Pollock, 1980; H u i d o b r o - T o r o and Way, 1981; Nijkamp and Van Ree, 1980; Scheurer et al., 1981). It seems therefore that enkephalins, as well as having an inhibitory action, act as excitatory modulators in
30 the gastrointestinal tract in vitro in some conditions. Opioid receptors in rat colon have been proposed to have properties of the/~ and • subtypes (Scheurer et al., 1981), or the • and /~ subtypes (Boura and Olley, 1981). Thus, the receptor subtypes in rat colon are not yet well established. We have studied the effects of enkephalin analogs on several in vitro preparations and on analgesia (Kiso et al., 1981; Moritoki et al., 1981). In the present experiments we investigated the effects of the tripeptides SD-34 and SD-25 on rat colon and compared their stimulatory actions with those of opiates and opioid peptides. We also tried to characterize the opioid receptor subtypes with which the peptides interact in the colon.
2. Materials and methods
2.1. Organ bath experiments Male Wistar rats of about 8-10 weeks old (230280 g) were used. A segment of their distal colon of 4 cm length was removed. Both ends of the segment were left open to avoid accumulation of mucosal secretion during the resting period and in long experiments. The preparation was then immersed in a 10 ml organ bath filled with Tyrode solution bubbled with oxygen. The Tyrode solution had the following composition (mM): NaC1 136.9, KCI 2.7, CaC12 1.8, MgC12 1.8, N a H z P O 4 0.42, N a H C O 3 11.9 and glucose 11.2. The oral end of the segment was connected to an isotonic transducer (Nihon Kohoden T D 112S) or a lever with a 1 g load. The bath temperature was kept at 26°C to reduce spontaneous contractions as much as possible. For preparation of dose-response curves, opiates and the tripeptides were added to the organ bath cumulatively in a volume of 1-70 /~1 and the responses were plotted as percentages of the maximal response to morphine or methionine enkephalin. Apparent activities of the peptides and opioids were expressed as pD 2 values (negative logarithms of the molar concentrations producing 50% of the maximal contraction). Inhibitory activities of antagonists were expressed as pA 2 values, de-
termined by the method of Van Rossum (1963), or as pD~ values (negative logarithms of the molar concentrations causing 50% inhibition of the maximal contractions) determined by the method of Van Rossum (1963). The pA 2 values in table 2 are means of the mean values at 3-5 different concentrations of antagonists.
2.2. Morphine tolerant preparations Rats were made morphine-dependent/tolerant by injection of morphine on 6 consecutive days: on the first 3 days, 10 m g / k g was injected subcutaneously (s.c.) 4 times a day; on the following 3 days, 30 m g / k g of morphine was given 4 times a day; on day 7, 30 m g / k g of morphine was injected 2 h before killing the rats. The colon was removed and incubated in Tyrode solution containing 5 × 10 7 M morphine throughout the experiments. This concentration of morphine has been shown to be present in the plasma 3 days after implantation of a morphine pellet (Goldstein and Schulz, 1973; Schulz and Herz, 1976a) and to be sufficient to prevent withdrawal symptoms (Schulz and Herz, 1976b).
2.3. Drugs used The drugs used were SD-34 (MeTyr-D-Met(O)Gly-methylphenethylamide; Kiso et al., 1982), SD-25 (Tyr-D-Met(O)-Gly-methylphenylalaninol; Kiso et al., 1981), [D-Ala2,D-LeuS]enkephalin (DADL; Peptide Institute, Osaka), dynorphin-(117) (Peptide Institute, Osaka), methionine enkephalin (Peptide Institute, Osaka), fentanyl (Sankyo Co., Tokyo), pentazocine (Yamanouchi, Tokyo), ethylketocyclazocine (EKC; SterlingWinthrop Research Institute, Rensselaer, NY), morphine hydrochloride (Takeda, Osaka), naloxone (Endo Laboratories, Garden, City, NY), Mr 2266 ( [ - ]-2-(3-furylmethyl)-5,9-diethyl-2'-hydroxy-6,7-benzomorphan; Boehringer Ingelheim K.G., FRD), serotonin creatinine sulfate (5-HT; Sigma Chemical Co., St. Louis, Mo), ketanserin (Janssen Pharmaceutica, Beerse, Belgium), cinanserin (Squibb Japan Inc., Tokyo), methysergide maleate (Sandoz, Basel, Switzerland), spiperone (Janssen Pharmaceutica), phenylbiguanide (AI-
31 drich Chemical Co., Milwaukee, Wis), quipazine maleate (Miles Laboratories, Elkhart, Ind), phentolamine mesylate (Ciba-Geigy Japan Inc., Osaka), tolazoline hydrochloride (Sigma Chemical Co.), yohimbine hydrochloride (Nakarai Chemical, Kyoto), prazosin (Taito-Pfeizer, Tokyo), phenoxybenzamine hydrochloride (Nakarai), guanethidine sulfate (Ciba-Geigy Japan Inc.), cimetidine (Smith Kline & French Laboratories, Philadelphia, Pa), chlorpheniramine maleate (Tokyo Kasei, Tokyo), quinacrine hydrochloride (Sigma), indomethacin (Sigma), atropine sulfate (Sigma), acetylcholine chloride (ACh, Sigma), tetrodotoxin (Sigma), bacitracin (Sigma), hexamethonium bromide (Yamanouchi) and substance P (Peptide Institute). 3. Results
3.1. Effects of the peptides The tripeptides SD-34 and SD-25 induced dose-dependent and reproducible contractions of the isolated rat distal colon. The contractions
SD-M
I00
developed rapidly and in 20 s reached a maximum which was sustained for more than 30 s followed by relaxation. Therefore, when the peptides were added cumulatively, subsequent doses were applied just when the response reached a maximum. Fig. 1 shows the dose-response curves for the peptides, morphine, ethylketocyclazocine (EKC), methionine enkephalin (Met-enk), and related opioids. The concentration producing 50% contraction (EDs0) of SD-34 was 2.95 × 10 -9 M, which was about one-tenth of that of morphine (2.75 × 10 8 M), but 3 times that of Met-enk ( 8 . 1 2 x 1 0 -x° M). SD-25 ( 6 . 0 3 x 1 0 -9 M) was about half as active as SD-34. The maximal contractions induced by SD-34, SD-25 and morphine were of similar magnitude, and corresponded to 42.0 + 3.1% (n = 8), 46.2 + 3.6% (n = 8) and 42.0 + 5.4% (n = 6), respectively, of the maximal contraction produced by 10-s M ACh. The potencies of the peptides and related opioids in inducing contractions of the colon are listed as pD z values in table 1. In subsequent experiments, SD-34, a tripeptide alkylamide, was used as representative peptide unless otherwise stated.
SD-~
EKC
x
._~ 50 co
i
i0-I0
10-9
i
i0 -8
l
i0 -7
,
iO-6
Opioids (M)
Fig. 1. Dose-responsecurves for the tripeptides and related compounds in inducing contractions of isolated rat distal colon. DADL: [D-Ala2,D-LeuS]enkephalin, Met-enk: methionine-enkephalin, dyn: dynorphin, EKC: ethylketocyclazocine, fent: fentanyl, mor: morphine, pent: pentazocine. The ordinate shows the contractions of the colon as percentages of the maximal contraction induced by Met-enk or morphine. Values (means + S.E.) were obtained with colons of 6-12 animals.
32 TABLE 1
TABLE 2
Potencies of opioid peptides and opiates in inducing contractions of isolated rat distal colon. Potencies are expressed as pD 2 values. Met-enk: methionine enkephalin, DADL: [DAla2,D-Leu~]enkephalin, EKC: ethylketocyclazocine. Numbers in parentheses show numbers of observations.
Effects of naloxone and Mr 2266 in antagonizing the actions of opioid peptides and opiates on rat colon, expressed as pA 2 values. The values are means of the mean values calculated from the values at 4-5 concentrations of the antagonists (3 x 10 9-10 0 M) in 5 animals. Abbreviations are as for table 1. pA2
pD2 SD-34 SD-25 Morphine Fentanyl Met-enk DADL EKC Pentazocine Dynorphine
-- IO0
~, 50
g,
(24) (12) (23) (10) (14) (13) (9) (12) (4)
SD-34 SD-25 Morphine Fen tanyl Met-enk DADL EKC Dynorphin Pen tazocine
Naloxone
Mr 2266
8.56 + 0.07 8.48 ± 0.11 7.85 ± 0.09 8.72 ± 0.02 7.64 ± 0.| 0 7.80 ± 0.06 7.75 ± 0.06 7.97 ± 0.09 8.30 ± 0.10
8.54 + 0.08 8.46 + 0.08 6.97 + 0.07 8.19 _+0.07 7,83 ± 0.09 7.57 + 0.10 8.56 ± 0.07 8,52 ± 0.03 8.16 _+0.06
Fent
SD-34
,///~k
¢, 10-9
= 100
8.53 _+0.05 8.22±0.12 7.56 ± 0.05 7.53 ± 0.03 9.09 ± 0.14 9.58 + 0.04 7.34±0.08 6.46 ± 0.09 8.66±0.23
10-8
10-7
10-6
10-8
I0 -7
DADL
E,c
I0-6
10-5
10-8
10-7
,,~k
X
E
0
~-
"E O
50
/,/¢ 10-8
10-7
10-6
Concentration (M)
10-5
lo-lO
10-9
Concentration (M)
Fig. 2. Antagonism by naloxone of the contractile actions of SD-34 and related compounds. EKC: ethylketocyclazocine, fent: fentanyl, DADL: [D-Ala2,D-LeuS]enkephalin. Concentration of naloxone: O, 0; O, 3 x 10 -9 M; ix, 10 8 M; &, 3 × 10 s M; O, 10 v M; m, 3 )< 10 -7 M. For comparison, the dose-response curves in the presence of 3 x 10 s M naloxone are indicated by dotted lines. The ordinate shows contraction of the colon as a percentage of the maximal contraction. Values (means ± S.E.) were obtained with colons from 5 animals.
33
3.2.1. Effect of naloxone on the actions of the tripeptides Naloxone antagonized the actions of the peptides, and of morphine and related opioids. The dose-response curves obtained are shown in fig. 2. Naloxone at concentrations of 3 x 10 -9, 10 -8, 3 × 10 8, 1 0 - 7 and 3 x 10 -7 M caused a parallel shift to the right of the dose-response curve for SD-34 2.2, 24.6, 79.4 and 281.8 times, respectively. The pA 2 values obtained with all these concentrations of naloxone were almost the same, being 8.46, 8.52, 8.61 and 8.63, respectively. The mean of the mean values was 8.56 (table 2). These results indicate that naloxone caused competitive antagonism. Naloxone antagonized the action of SD-25 as readily as that of SD-34. A similar pA z value of 8.72 was obtained with the /~-agonist fentanyl. Naloxone was more inhibitory with the peptides, fentanyl and pentazocine than with morphine, the 8-agonists [D-AlaZ,D-LeuS]en-
~= 100r SD-~ ~ ,
•
~
kephalin (DADL) and Met-enk, and the x-agonists E K C and dynorphin. At a concentration of 3 x 10-8 M, naloxone displaced in parallel to the right the dose-response curves for SD-34 and fentanyl 24.6 times and 19.9 times, respectively (fig. 2). The pA 2 values of naloxone for morphine, D A D L , Met-enk and dynorphin ranged from 7.79 to 7.64, whereas those for SD-34 and fentanyl were about 8.7 to 8.5 (table 2).
3.2.2. Effect of Mr 2266 on the actions of the peptides Mr 2266, a x-antagonist, preferentially and competitively antagonized the actions of SD-34 and SD-25 at concentrations above 10 -8 M (table 2 and fig. 3). The actions of the x-agonists EKC and dynorphin, and a K- and 8-agonist pentazocine were antagonized as effectively as those of the peptides. The pA 2 values of Mr 2266 for SD-34 and SD-25 were 8.54 and 8.46, respectively, which
Mor
E
10-9
too
10-8
10-7
EKC
10-6
10-8
10-7
10-6
OADL,,,,.~
,'~" A
E I
10-8
10-7
10-6
Concentration (M)
10-5
1'0-10
i;-9
1'0-8
Concentration (M)
Fig. 3. Antagonism by Mr 2266 of the contractile actions of SD-34 and related compounds. For comparison, the dose-responsecurves in the presence of 3 x 10-8 M Mr 2266 are indicated by dotted lines. Abbreviations and symbols are as for fig. 2.
34 TABLE 3
3.3. Effects of the peptides on morphine-tolerant preparations
Comparison of sensitivities of rat colon to opioid peptides and opiates before and after chronic morphine treatment. The ratio is the ratio of ED~o values in normal and morphine-treated preparations. Values are means for those in 2 preparations. Abbreviations are as for table 1. pD2 Normal rats SD-34 SD-25 EKC Met-enk Morphine
Morphinetreated rats
8.40 8.22 7.39 9.59 7.42
7.31 7.11 6.37 8.65 4.21
The effects of the tripeptides and related comp o u n d s were c o m p a r e d on colon chronically treated with m o r p h i n e to render it d e p e n d e n t / tolerant as described in Methods. Withdrawal was prevented by adding 5 × 10 7 M m o r p h i n e to the T y r o d e solution. After this treatment, the sensitivity of the p r e p a r a t i o n to SD-34, SD-25, Met-enk or E K C was decreased a b o u t 10 fold, whereas the sensitivity to m o r p h i n e was dramatically decreased a b o u t 1600-fold. The p D 2 values for n o r m a l and t o l e r a n t / d e p e n d e n t rat colon are compared in table 3. N a l o x o n e did not induce morphinewithdrawal contracture of the colon. The effects of SD-34 and m o r p h i n e on colon rendered acutely tolerant to m o r p h i n e were exa m i n e d next. Rat colon became unresponsive to 10 6 M m o r p h i n e after successive application of the drug without washing (fig. 4). U n d e r these conditions, however, the colon showed almost the same response to SD-34 as the control p r e p a r a t i o n before desensitization by morphine. At this time, the colon r e m a i n e d unresponsive to m o r p h i n e even at a c o n c e n t r a t i o n of 3 × 10-5 M. I m m e d i a t e l y after the application of 3 x 10 5 M morphine, the colon still responded to SD-34. This result also shows that p r e t r e a t m e n t with 3 × 10 -~ M
EDso ratio
12.9 8.7 10.5 14.8 1621.8
is very close to those for E K C (8.56), d y n o r p h i n (8.52) a n d pentazocine (8.16). M r 2266 showed high a n t a g o n i s t activity against fentanyl as well as SD-34 and EKC, the pA 2 value for fentanyl being 8.19. M u c h higher c o n c e n t r a t i o n s (10 times or more) were required to p r o d u c e the same degree of a n t a g o n i s m of the actions of so-called &agonists: the pA 2 values for D A D L a n d M e t - e n k were 7.57 a n d 7.83, respectively. M o r p h i n e was highly resistant to M r 2266 as evidenced by its significantly lower pA 2 value (6.97) than those for SD-34, SD-25 and E K C (8.5-8.6).
2o/ 7
~0 xlO-9M SD-34
•
•
•
•
•
Morphine IO-6M
•
10
I0 xlO-gM SD-34
3 min
I0
A
3
ZX
Morphine 3xlO-5M xlO-9M Morphine SD-34 3xlO-5M
Fig. 4. Morphine (10 - 6 M) was added successivelyas shown by filled triangles (usually 4-6 doses) without washing and remained in the bath throughout the experiments as indicated by the dotted line. Immediately after the last application of 10 6 M morphine, SD-34 at concentrations of above 3 x 10-9 M was added cumulatively and remained in the bath. When the colon relaxed, SD-34 (10 -8 M) was applied twice for further confirmation of the response to SD-34. Morphine 3 × 10 5 M was added thereafter.
35 morphine did not interfere with the action of SD-34. Conversely, repeated application of SD-34 at 10 7 M did not desensitize the colon to morphine.
3.4. Effect of bacitracin When preparations were treated with 10 - 4 g / m l of bacitracin for 30 min, the action of SD-34 was augmented. The p D 2 value increased from the control value of 8.52 + 0.10 to 8.92 + 0.10 (n = 5) whereas that of morphine (7.75 + 0.06) was slightly decreased (7.62 + 0.15, n = 4).
3.5. Effects of 5-HT antagonists and desensitization Methylergometrine at a concentration (10-6 M), which did not affect the contraction induced by ACh, inhibited the contractions induced by SD-34 and morphine. This inhibition was noncompetitive, and the apparent p D 2 value was 5.93 _+ 0.14 (n = 4) for SD-34 (fig. 5). Besides inhibiting the action of SD-34, this concentration of methylergometrine antagonized the contractile action of 5-HT, the pA 2 value being 8.8 (n = 2). Likewise, methysergide 10 -7 and 3 × 10 -7 M shifted the dose-response curve for SD-34 downward and to the right (fig. 5). The inhibitory potency of methysergide expressed as the pD~ value was 6.82
I0O
50 C
R
(3C
3.6. Effect of substance P autodesensitization The possible contribution of substance P (SP) to the actions of the tripeptides and related opioids were examined by testing their effects on preparations desensitized to SP by its repeated application without washout. Addition of SP at a concentration of 3 × 10 -8 M produced almost maximal contraction that gradually returned to the basal
I00
E E
+ 0.04 (n = 4). However, increasing the concentration of methysergide to 10 -5 M did not cause further suppression. The pD; values for SD-25 and morphine were 6.38 and 6.35 (n = 2), respectively. Ketanserin (10-8-10 .6 M), cinanserin (10 8_ 10 .6 M), quipazine (10-6-10 5 M), spiperone (10-8-10 -6 M) and phenylbiguanide (10-7-3 × 10-7 M) did not have any inhibitory effects on the contractions induced by SD-34. Next we examined whether autodesensitization by repeated applications of 5-HT affected the action of SD-34. 5-HT at 10 .5 M was applied to the colon to produce maximal contraction. When the intense contraction had subsided to the basal level, the same concentration of 5-HT was applied to confirm the development of tachyphylaxis. The preparation usually failed to respond to a third dose of 10-5 M 5-HT. The actions of SD-34 and morphine were not affected by this treatment.
/ 10-9
M•ethysercjide 50
go 10-6M //~~xlO-7M 10-8 SD- 34
10-7 (M)
i0-9
10-8 SD- 34 (MI
10-7
Fig. 5. Effects of methysergide and methylergometrine(Me-ergo) on the contractions of rat colon induced by SD-34. Values (means + S.E.) were obtained with colons from 5 animals.
36 level. A second application of the same dose of SP produced a contraction smaller than the first one. After 4 to 5 successive applications of SP, the colon lost its responsiveness to SP but not to the peptides or morphine.
3. 7. Effects of other agents Atropine (10 -7 M) and hexamethonium (10 5 M) had no inhibitory effects on opioid-induced contractions. Tetrodotoxin at concentrations of up to 3 × 10 7 M did not affect the contractile actions of the peptides but concentrations as high as 10 6 M or more produced dose-dependent contractions just as did the peptides, the pD 2 value being 6.64 + 0.05 (n = 3). The a-blockers phentolamine and tolazoline (3 × 10 -6 M, each) enhanced the contraction of the colon induced by SD-34. In the presence of 3 × 10 6 M tolazoline, the dose-response curve for SD-34 (pD 2 = 8.84, n = 2) was shifted slightly upward (115.4%) and 7.6 times to the left (pD 2 = 9.34, n = 2). Similarly, 3 x 10 6 M phentolamine increased the pD 2 value of SD-34 from 8.34 to 8.90 (n = 3)~ and the maximal contraction height to 122.0%. However, other a-blockers, phenoxybenzamine, prazosin and yohimbine (10 6 M each), did not show the same effect as tolazoline and phentolamine. Guanethidine (3 × 10 6 M) had no effect on the action of SD-34. The histamine H2-antagonist cimetidine (10 5_ 10 4 M), the histamine H~-antagonist chlorpheniramine (10 7 M) and the inhibitors of prostaglandin synthesis quinacrine (10 7-10 6 M) and indomethacin (10 -4 M) did not affect the contractile actions of SD-34, Met-enk and morphine.
4. Discussion The present study demonstrated that the tripeptides SD-34 and SD-25 induced contractions of isolated rat colon, just as did Met-enk and morphine. Naloxone selectively and competitively antagonized the contractile actions of the peptides, indicating that the peptides act on an excitatory opioid receptor. The order of potency of the peptides and related opioids in inducing contrac-
tions of the colon was D A D L and Met-enk (socalled 3-agonists)> SD-34, SD-25, and dynorphin (so-called ~-agonists)> fentanyl and morphine (so-called /~-agonists). Thus, judging from the affinities of the agonists, the receptors in the colon seem to consist of 3-, ~- and/~-subtypes. The pA 2 values of naloxone for the peptides were not comparable with its pA 2 values for the 3-agonists D A D L and Met-enk in the present experiments (table 2), or with its pA 2 values for 3-agonists calculated by Boura and Olley (1981), Huidobro-Toro and Way (1981), Lord et al. (1977) and Nijkamp and Van Ree (1980); they were however comparable with its pA 2 value for the /x-agonist fentanyl. In contract to the actions of 6-agonists, those of SD-34 and SD-25 were antagonized by naloxone as readily as that of the #-agonist fentanyl. About 5 to 10 times less naloxone was required to antagonize the actions of the peptides than to antagonize the actions of the 3-agonists. The pA 2 values of naloxone for SD-34 and SD-25 were 8.56 and 8.48, respectively, being similar to that of 8.7 for fentanyl and that of 8.6 reported by Boura and Olley (1981) for the /~agonist Rx 3030 on rat colon. Thus, judging from the pA 2 values of naloxone, the peptides seemed to act mainly on the ~L-receptor rather than the &receptor in the rat distal colon in the present experiments. On the other hand, opioid receptors in isolated rat colon have been suggested to have properties of the /x and K subtypes (Scheurer et al., 1981). Thus SD-34 and SD-25 could also have the characteristics of ~-agonists on the colon. In support of this possibility, Mr 2266, suggested to be a ~antagonist (Kosterlitz et al., 1981; Lord et al., 1977; Oka et al., 1982), showed a greater antagonism of the peptides as well as of EKC, dynorphin and pentazocine than of the 3-agonists Met-enk and DADL. However, the action of the ~-agonist was antagonized by Mr 2266 as effectively as the actions of EKC and dynorphin. It is noteworthy in this respect that Mr 2266 has been reported to act well on both the /~-receptor and K-receptor in guinea-pig ileum (Lord et al., 1977; Yoshimura et al., 1982). In addition, the pA 2 values of naloxone for the peptides were not comparable with those for EKC, dynorphin and penta-
37 zocine. These results argue against a major contribution of the x-receptor to the actions of the peptides. In contrast, the relative effects of Mr 2266 and naloxone, expressed as Ke n a l o x o n e / K e Mr 2266, for the peptides (0.91-0.95) were different from those for the bt-agonist fentanyl (0.30), even though the values for the peptides were not comparable with those for EKC and dynorphin (3.38-3.54). Furthermore, Mr 2266 antagonized the actions of the peptides more readily than that of fentanyl as evidenced by the pA 2 values of Mr 2266 for the peptides (8.5) and fentanyl (8.2). Thus, it is not clear whether SD-34 and SD-25 act as /~-agonists or x-agonists, but it is evident that these peptides do not have 8-agonist characteristics. Pentazocine, which is thought to be a ~- and 6-agonist (Snyder and Goodman, 1980), may also act on the #-receptor, because the pA 2 value of naloxone for pentazocine was similar to that for a /~-agonist. Morphine has been reported to be a bt-agonist in guinea-pig ileum but in rat colon it may interact with a different subtype of receptor. The pA 2 value of naloxone for morphine was found to be 7.85 in the present experiments and has been reported to be 7.67 (Huidobro-Toro and Way, 1981), these pA 2 values being similar to those for 6-agonists. However, the pA 2 value of Mr 2266 for morphine was not comparable with its value for either/~-, 8- or K-agonists, suggesting that morphine also acts on another subtype of receptor. If SD-34 produces contractions by releasing endogenous enkephalins the pA 2 values of naloxone and Mr 2266 for SD-34 should be the same as those for Met-enk. Thus SD-34 does not seem to release endogenous enkephalins. If the peptides act on the morphine receptor, selective tolerance could be produced by chronic treatment with morphine in vivo or by repeated application of morphine in vitro. In fact, after chronic morphine treatment, tolerance to morphine did develop as reported by Huidobro-Toro and Way (1981) and Whster et al. (1981), but the resulting tolerance to the peptides was less (table 3). Similarly, when acute desensitization developed after repeated application of morphine without
washout tolerance to morphine developed but cross tolerance to SD-34 did not (fig. 4). These findings also suggest that the receptor for SD-34 is different from that for morphine. The contractile actions of morphine and related opioids have been studied by many workers. The idea that these actions are mediated by 5-HT is based on the finding that they were attenuated by the 5-HT antagonist methysergide (Burks, 1976; Huidobro-Toro and Way, 1981; Nijkamp and Van Ree, 1980) or cyproheptadine (Burks, 1973; Nijkamp and Van Ree, 1980), and that morphine did not cause contraction of a preparation rendered tachyphylactic to 5-HT (Burks and Long, 1967). In fact, the present experiments demonstrated that the action of SD-34 was antagonized noncompetitively by the lysergic acid derivatives methysergide and methylergometrine at concentrations sufficient to cause competitive antagonism of the action of 5-HT. However, we also observed that SD-34 still caused contractions of rat colon desensitized to 5-HT by prolonged exposure to a high concentration of 5-HT (Gillan and Pollock, 1980). Moreover, the 5-HT2 antagonists ketanserin (Van Nueten et al., 1981), cinanserin (Rubin et al., 1964), quipazine (Lansdown et al., 1980), spiperone (Peroutka and Snyder, 1979) and phenylbiguanide (Gintzler, 1979) did not block the actions of SD-34 and morphine. The present results and the above considerations are not consistent with the idea that a serotonergic mechanism is in some way associated with the actions of SD-34 and morphine. There is evidence for a mediatory role of SP in the actions of opiates. It has been reported that SP is released from guinea-pig ileum by electrical stimulation or treatment with dimethylphenylpiperadinium (Franco et al., 1979b) and SP-like immunoreactivity has been detected in guinea-pig ileum (Costa et al., 1980; Franco et al., 1979a) and rat colon (Holzer et al., 1980). Thus SP is thought to be a putative transmitter or a modulator in the noncholinergic excitatory response of the gut (Gintzler, 1979; 1980; Gintzler and Scalisi, 1982; Tsou et al., 1982). To determine whether SP is involved in mediating the contractile action of SD-34, we used a procedure for autodesensitization to SP. As exposure of the ileum to a relatively high concentration of SP is known to render it
38 insensitive to further application of SP ( F r a n c o et al., 1979a; 1979b; M o n i e r a n d Kitabgi, 1980; Tsou et al., 1982), the same or an analogous p h e n o m e n o n might be observed in the colon if SP is involved. A l t h o u g h we d e m o n s t r a t e d that repeated application of SP to the colon made it insensitive to SP, the colon r e s p o n d e d to SD-34 a n d m o r p h i n e as well after this t r e a t m e n t as before desensitization. It thus seems unlikely that the actions of SD-34 and m o r p h i n e on the colon are mediated by SP. It has been suggested that n o r e p i n e p h r i n e is an i n h i b i t o r y m o d u l a t o r in the gut a n d that removal of this i n h i b i t o r y influence by the a-blocker phent o l a m i n e resulted in an increase in the stimulatory effect of Met-enk, which seems to act directly on the muscle ( N i j k a m p a n d Van Ree, 1980). In fact, in the present experiments, tolazoline and phent o l a m i n e slightly p o t e n t i a t e d the contractions induced by SD-34 a n d morphine. However, a n o t h e r a - b l o c k e r p h e n o x y b e n z a m i n e a n d the selective a~blocker prazosin a n d a2-blocker y o h i m b i n e did not potentiate the actions of the peptides or morphine. Thus the question arises of why selective a-blockers did not attenuate the supposed a - i n h i b i t o r y m o d u l a t i o n . A n o t h e r mechanism(s) unrelated to an a - b l o c k i n g action may be responsible for the observed p o t e n t i a t i o n b u t at present there is n o evidence for this. The failure of inhibitors of p r o s t a g l a n d i n synthesis ( q u i n a c r i n e a n d i n d o m e t h a c i n ) a n d h i s t a m i n e H 1- a n d Hz-antagonists (chlorpheniram i n e and cimetidine) to a t t e n u a t e the contractile actions of SD-34 and m o r p h i n e implies that the actions of the c o m p o u n d s are not mediated by p r o s t a g l a n d i n s or histamine. We confirmed that tetrodotoxin, like SD-34, p r o d u c e d c o n t r a c t i o n s of rat colon. Thus SD-34 m a y act by blocking tonic i n h i b i t o r y m o d u l a t i o n as has been reported by G i l l a n a n d Pollock (1980) for the stimulatory action of tetrodotoxin. We suggest, based on the results of this study, that the tripeptides SD-34 a n d SD-25 produce their contractile effects on rat colon through the opioid/~- or K-receptor or both but not through the 6-receptor. The stimulatory effects of the tripeptides a n d related c o m p o u n d s tested do not seem to be mediated by 5-HT, A C h or SP, or due
to blockade of the adrenergic inhibitory pathway. These c o m p o u n d s seem either to act by blockade through opioid receptors of the nonadrenergic, noncholinergic inhibitory pathway m o d u l a t i n g s p o n t a n e o u s myogenic activity, or to cause direct s t i m u l a t i o n of opioid receptors in the muscle.
Acknowledgements We are grateful to Dr. J.M. Van Nueten, Janssen Pharmaceutica, Beerse, Belgium, for gifts of ketanserin and spiperone, Dr. H. Merz, Boehringer Ingelheim K.G., West Germany, for a gift of Mr 2266, Smith Kline & French Laboratories, Philadelphia, Pa for supplying cimetidine, Endo Laboratories, Garden City, NY~ for supplying naloxone, Sterling-Winthrop Research Institute, Rensselaer, NY, for supplying ethylketocyclazocine,Squibb Japan Inc., Tokyo, for supplying cinanserin, Sankyo Co., Tokyo, for supplying fentanyl and Taito-Pfizer, Tokyo, for supplying prazosin.
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