Inactivation of Dynorphin-(1-8) in Isolated Preparations by Three Peptidases

Hiroaki NUMATA, Toyokazu HIRANUMA and Tetsuo OKA*

Department of Pharmacology, School of Medicine, Tokai University, Isehara 259-11, Japan

Accepted May 6, 1988

Abstract-Inactivation of dynorphin-(1 -8) in three in vitro isolated preparations, guinea-pig ileum, mouse vas deferens and rabbit vas deferens, was estimated by employing the relatively specific inhibitors of enkephalin-hydrolyzing enzymes. All three enzyme inhibitors, amastatin, captopril and phosphoramidon, significantly enhanced the inhibitory potency of dynorphin-(1 -8) in the three isolated pre parations. The magnitude of the enhancement of the dynorphin potency by captopril was significantly higher than that by either amastatin or phosphoramidon in guinea-pig ileum; that by amastatin was significantly higher than that by either captopril or phosphoramidon in rabbit vas deferens; and that by amastatin was similar to that by captopril, but significantly higher than that by phosphoramidon in mouse vas deferens. The Ke values of three antagonists, naloxone, Mr 2266 and ICI 154129, against dynorphin-(1 -8) in the presence of the three peptidase inhibitors indicated that dynorphin-(1 -8) acted on kappa receptors in guinea-pig ileum and on both kappa and delta receptors in mouse vas deferens. Since amastatin, captopril and phosphoramidon produced the naloxone-reversible inhibition of contractions of guinea-pig ileum in the presence of dynorphin-(1 -8), all three dynorphin-inactivating enzymes were indicated to be located very close to kappa receptors.

Enkephalins have been shown to be in Additionally, enkephalin has been demon activated by three enzymes, amastatin-sensi strated to be almost exclusively hydrolyzed tive aminopeptidase, captopril-sensitive pep by these three enzymes, since enkephalin tidyl dipeptidase A (angiotensin I converting remains intact almost totally after it is in enzyme, kininase II, peptidyldipeptide hydro cubated with ileal or striatal membrane pre lase; EC 3.4.15.1) and phosphoramidon parations of guinea-pig for 60 min at 37'C sensitive endopeptidase-24.11 ("enkepha in the presence of the inhibitors against these linase", EC 3.4.24.11 ), in three in vitro isolated three enzymes, although enkephalin is com preparations, guinea-pig ileum (1), mouse pletely hydrolyzed and free tyrosine and the vas deferens (2) and rat vas deferens (3). Tyr-Gly-Gly fragment are produced when the Since these enkephalin-hydrolyzing enzymes incubation mixture does not contain the are located very close to opioid receptors peptidase inhibitor (4). (1-3), these enzymes are suggested not only Dynorphin-(1 -8), another endogenous to inactive the exogenously given enkephalin opioid peptide, has been suggested to be also in the vicinity of opioid receptors but also inactivated by peptidases in isolated pre suggested to play important roles in the termi parations such as rabbit vas deferens since its nation of the action on opioid receptors of the inhibitory action on the electrically-evoked enkephalin released from presynaptic nerve contractions of the isolated preparation has terminals. been shown to be significantly enhanced by the pretreatment of the preparation with the mixture of peptidase inhibitors (5, 6). The enzyme involved in the inactivation of dynor pig ileum were prepared and set up for elec phin-(1 -8), however, is unknown at present. trical stimulation as described previously (10, In the present investigation, therefore, it was 11 ). The % inhibition of electrically-evoked examined whether or not dynorphin-(1 -8) muscle contrations produced by an opioid was inactivated by three enkephalin-hy peptide was plotted against the log concen drolyzing enzymes in three isolated prepara tration of the peptide to estimate the IC50 tions guinea-pig ileum, mouse vas deferens (concentration of the opioid peptide to pro and rabbit vas deferens. duce 50% inhibition of contractions). Amas Additionally, the preference of the opioid tatin, captopril or phosphoramidon at the receptor type by dynorphin-(1 -8) was re final concentration of 1 pM each was em examined in isolated preparations in the ployed as an inhibitor of each peptidase in the presence of peptidase inhibitors by employing present investigation since the previous the three opioid antagonists, since dynorphin studies had shown that the enkephalin (1 -8) had been reported to act on either hydrolyzing aminopeptidase, peptidyl dipep kappa receptors in the preliminary experiments tidase A and endopeptidase-24.11 were inhi (7) or a different opioid-receptor type than the bited almost completely with 1 /tM of amas three classical opioid-receptor types, mu, tatin, 1 /iM of captopril, and 1 pM of phos kappa and delta type (8), and dynorphin phoramidon, respectively (1-4). Either a pep (1 -9) had been indicated to act on both kappa tidase inhibitor or the mixture of two or three and delta receptors (9). peptidase inhibitors was given 10 min before Moreover, it was estimated in the present the opioid administration. The ratio of the investigation whether or not dynorphin potency and the % difference, shown in the inactivating enzymes were located very close tables, were calculated from the following to kappa receptors in guinea-pig ileum, since fomulas: ratio of potency=1C50 before ad enkephalin-inactivating enzymes had been ditional treatment/IC50 after additional treat indicated to be located very close to mu re ment and % difference= [(IC50 before ad ceptors in guinea-pig ileum in the previous ditional treatment-IC50 after additional treat investigation (1). ment)/IC50 before additional treatment] x 100. The statistical significance of % dif Materials and Methods ferences between IC50 values of two adjacent Chemicals: Gifts of compounds which groups shown in the tables was determined were gratefully received were dynorphin by the paired Student's t-test. The KQ (equi (1 -8) from Prof. Y. Kiso, Kyoto Pharmaceu librium dissociation constant) values of opioid tical University (Kyoto); ICI 154129 (N,N antagonists against dynorphin-(1-8) were diallyl-Tyr-Gly-0-(CH2S)-Phe-Leu) (where determined by the 'single' dose method (12). 0-(CH2S) signifies replacement of the amide CO-NH bond by CH2S) from Dr. J.W. Results Holaday, Walter Reed Army Inst. Res. (Wash Enhancing effects of peptidase inhibitors ington, D.C., U.S.A.); Mr 2266 [(-)-2-(3 on the potency of dynorphin-(1-8): Dynor furylmethyl) 5,9 diethyl 2' hydroxy 6,7 phin-(1 -8) dose-dependently inhibited the benzomorphan)] from Nippon CH Boehringer electrically-evoked contractions of all isolated Sohn Co., Ltd. (Osaka); and captopril and preparations employed in the present in naloxone from Sankyo Company (Tokyo). vestigation. Its IC50 value in guinea-pig Amastatin and phosphoramidon were pur ileum, mouse vas deferens and rabbit vas chased from Peptide Institute, Inc. (Minoh). deferens was significantly decreased by the In vitro isolated preparations: Male ICR pretreatment of the preparation with mixture JCL mice weighing 30-50 g, male Hartley of three peptidase inhibitors, amastatin, cap guinea-pigs weighing 300-600 g, and male topril and phosphoramidon, at the final con Japanese White rabbits weighing 2.5-3.5 kg centration of 1 ,uM each at 10 min before the were used for this study. The vasa deferentia dynorphin administration (Table 1). Each from mice or rabbits, and the myenteric peptidase inhibitor was then administered plexus-longitudinal muscle strip of guinea individually in the following experiments in

In three isolated preparations, especially in guinea-pig ileum, the IC50 value of dynor phin-(1 -8) in one preparation was some times markedly different from that in another in the absence of the peptidase inhibitor. Thus, the mean of the IC50 values of dynor phin-(1-8) in one control group was some times prominently different from that in the other control group (Tables 2, 3 and 4). When

order to determine which peptidase inhibitor the effect of a particular peptidase inhibitor on had an enhancing effect on the inhibitory the inhibitory potency of dynorphin was ex potency of dynorphin-(1 -8). amined, therefore, the IC50 values of dynor phin before and after the addition of a par ticular peptidase inhibitor were estimated in the same preparation. All three peptidase inhibitors significantly enhanced the inhibitory potency of dynor phin-(1 -8) in either guinea-pig ileum (Table 2), mouse vas deferens (Table 3) or rabbit vas deferens (Table 4). In guinea-pig ileum, the magnitude of the

Table 1. Enhancing effects of peptidase inhibitors on the inhibitory potency of dynorphin-(1 -8) in isolated preparations

Table 2. The enhancing effects of peptidase inhibitors on the inhibitory potency of dynorphin-(1-8) in guinea-pig ileum

Table 3. The enhancing effects of peptidase inhibitors on the inhibitory potency of dynorphin-(1 -8) in mouse vas deferens Table 4. The enhancing effects of peptidase inhibitors on the inhibitory potency of dynorphin-(1 -8) in rabbit vas deferens

enhancement of the dynorphin potency by amastatin was significantly higher than that by phosphoramidon, but significantly lower than that by captopril (Table 2). The degree of the augmentation of the dynorphin potency by amastatin was similar to that by captopril, but significantly higher than that by phosphoramidon in mouse vas Fig. 1. Naloxone-reversible inhibitory actions of deferens (Table 3). peptidase inhibitors on electrically-evoked con In rabbit vas deferens, the magnitude of the tractions of guinea-pig ileum in the presence of enhancement of the dynorphin potency by dynorphin-(1-8). Compounds were given to the amastatin was significantly higher than that bath at the dot. DYN, 5 nM of dynorphin-(1-8). A, by captopril, and that by captopril was signifi 1 I_tMof amastatin. C, 1 fiM of captopril. P, 1 1_,Mof cantly higher than that by phosphoramidon phosphoramidon. Nal, 1 IW of naloxone. (Table 4). The inhibitory action of the peptidase inhi of relatively high doses of naloxone on some bitor on contractions of guinea-pig ileum in preparations since dynorphin-(1 -8) at doses the presence of dynorphin-(1 -8): Either below 5 x 10-9 M in the presence of the three amastatin, captopril or phosphoramidon inhi peptidase inhibitors did not inhibit the con bited the electrically-evoked contractions of tractions at all in all preparations after the ad guinea-pig ileum in the presence of dynor ministration of relatively high doses (e.g ., phin-(1-8) (Fig. 1), although these pep 10-6 M) of naloxone which produced the tidase inhibitors by themselves did not de slight inhibition of contractions by itself in press the contractions in the absence of an some preparations. opioid peptide (figures were not shown). The The Kevalues of opioid antagonists against magnitude of the inhibition by each peptidase dynorhin-(1 -8) in the presence of peptidase: inhibitor, although it was slightly altered by The Ke values against dynorphin-(1 -8) of changing the administration order of the pep naloxone, which had been shown to have a tidase inhibitor, was the highest by captopril, high affinity to mu receptors (13), were the lowest by phosphoramidon and inter relatively high, while those of Mr 2266, which mediate by amastatin (Fig. 1 ). The inhibition had been reported to have a high affinity to induced by peptidase inhibitors in the pre both mu and kappa receptors (13), were sence of dynorphin-(1 -8) was antagonized relatively low in guinea-pig ileum (Table 5). by naloxone (Fig. 1). The degree of the Additionally, the Ke values of ICI 154129 , antagonism by naloxone was not complete in which had been found to have a relatively some preparations as shown in Fig. 1, but high affinity to delta receptors, a low affinity complete in other preparations. The apparent to mu receptors, and a very low affinity to incomplete antagonism by naloxone was kappa receptors (13), were very high in 3 likely to be produced by the agonistic action among 6 preparations and they were not able Table 5. The K,, values of naloxone, Mr 2266 and ICI 154129 against dynorphin-(1-8) in isolated preparations

to be estimated in the remaining 3 prepara by phosphoramidon in mouse vas deferens. tions of guinea-pig ileum because of no In rabbit vas deferens, amastatin-sensitive antagonism by ICI 154129 (Table 5). aminopeptidase is likely to play the most im In mouse vas deferens, the Ke values portant role in the inactivation of dynorphin against dynorphin-(1 -8) of naloxone, Mr (1 -8) since the magnitude of the augmen 2266 and ICI 154129 were relatively high, tation of the dynorphin potency by amastatin relatively low and relatively low, respectively has been shown to be significantly higher (Table 5). than that by either captopril or phosphor amidon. The minor role of phosphoramidon Discussion sensitive endopeptidase-24.1 1 in the inac The present investigation indicates that tivation of dynorphin-(1 -8) in all three pre dynorphin-(1 -8) can be inactivated by three parations is indicated by the observation that enzymes, amastatin-sensitive aminopeptidase, the magnitude of the enhancement of the captopril-sensitive peptidyl dipeptidase A dynorphin potency by phosphoramidon was and phosphoramidon-sensitive endopep lower than that by either amastatin or cap tidase-24.1 1, in three in vitro isolated pre topril in the three preparations. parations, guinea-pig ileum, mouse vas de The data in the present investigation along ferens and rabbit vas deferens, since the inhi with those obtained previously (14, 15) show bitory potency of dynorphin (1 -8) has been that the relative importance of the three shown to be significantly enhanced by the enzymes in the inactivation of dynorphin pretreatment of the preparation with either (1 -8) in guinea-pig ileum is different from amastatin, captopril or phosphoramidon in all that of either [Met5] or [Leu5]-enkephalin three preparations. since the magnitude of the enhancement of The fact that the magnitude of the enhance the inhibitory potency of both [Met5] and ment of the dynorphin potency by captopril [Leu5]-enkephalin by amastatin has been was significantly higher than that by either shown to be significantly higher than that by amastatin or phosphoramidon in guinea-pig either captopril or phosphoramidon in guinea ileum suggests that captopril-sensitive pep pig ileum (14, 15). Additionally, the relative tidyl dipeptidase A plays the most important importance of the three enzymes in the in role in the inactivation of dynorphin-(1 -8) in activation of dynorphin-(1-8) in mouse vas guinea-pig ileum. However, the important deferens is different from that of [Met5] role of amastatin-sensitive aminopeptidase enkephalin but similar to that of [Leu5] as well as captopril-sensitive enzyme in the enkephalin since the magnitude of the aug inactivation of dynorphin-(1 -8) in mouse mentation of the [Met5]-enkephalin potency vas deferens is suggested by the finding that by amastatin is similar to that by either cap the degree of the enhancement of the dynor topril or phosphoramidon in mouse vas defer phin potency by amastatin was similar to that ens (14), while the magnitude of the en by captopril but significantly higher than that hancement of [Leu5]-enkephalin potency by amastatin is similar to that by captopril but enkephalin-Arg6, an initial hydrolysis product higher than that by phosphoramidon in mouse of dynorphin-(1 -8) by the captopril-sensitive vas deferens (15). enzymes, still has essentially the same potency The reports that naloxone has a high af as dynorphin-(1 -8) in mouse vas deferens finity to mu receptors (Ke values ranging from (5), the observation that captopril has signifi 1 to 5 nM) and relatively low affinity to both cantly enhanced the potency of dynorphin kappa and delta receptors (Ke values ranging (1 -8) in mouse vas deferens indicates that from 10 to 50 nM) (10, 13, 16, 17); Mr 2266 [Leu5]-enkephalin-Arg6 is further hydrolyzed has a high affinity to both mu and kappa re by captopril-sensitive peptidase to Tyr-Gly ceptors (Ke values ranging from 1 to 5 nM) Gly-Phe which is reported to have, if any, very and relatively low affinity to delta receptors weak potency on opioid receptors (20). (Ke values ranging from 10 to 50 nM) (10, 13, All three enkephalin-hydrolyzing enzymes 17); and ICI 154129 has a relatively high have been suggested to be very closely affinity to delta receptors (Ke values renging located to mu receptors in the guinea-pig from 0.1 to 0.9 /iM), the lowest affinity to ileum (1) and to delta receptors in mouse vas kappa receptors (Ke values being more than deferens (2) by employing enkephalin which 20 /W) and intermediate affinity to mu re has been indicated to act on mu receptors in receptors (Ke values ranging from 7 to 8 tiM) guinea-pig ileum (13, 16) and delta receptors (13, 18, 19) indicate that dynorphin-(1 -8) in mouse vas deferens (13, 16). In the present acts on kappa receptors in guinea-pig ileum investigation, these three enzymes were in and on both kappa and delta receptors in dicated to very closely located to kappa re mouse vas deferens. ceptors in guinea-pig ileum by employing The fact that the relative importance of dynorphin-(1 -8), which has been suggested the three enzymes in the inactivation of to act on kappa receptors in guinea-pig ileum dynorphin-(1 -8) is different from that of in the present study, since these enzymes have enkephalins may be caused by the fact that been shown to produce a significant con one opioid peptide is hydrolyzed more easily centration difference of dynorphin-(1-8) be than the other by a particular enzyme as tween the surrounding organ bath and the shown by the previous reports (14), although vicinity of kappa receptors as demonstrated the probability that the relative activity of three in Fig. 1, and such concentration difference enzymes in the vicinity of mu receptors is can be produced only when these inac different from that of kappa receptors can not tivating-enzymes are located very closely to be neglected since both [Met5] and [Leu5] kappa receptors. The previous (1-3) and enkephalin have been shown to act on mu present investigation, therefore, strongly sug receptors in guinea-pig ileum and on delta gest that there are three opioid-peptide-inac receptors in mouse vas deferens (13, 16), tivating enzymes in the vicinity of three types while dynorphin-(1 -8) has been suggested to of opioid receptors, mu, kappa and delta act on kappa receptors in guinea-pig ileum receptors, as well as an unidentified type of and on both kappa and delta receptors in opioid receptor in rat vas deferens. mouse vas deferens in the present investiga Acknowledgments:This work was supported in tion. part by both Suzuken MemorialFoundation, and a The preliminary experiment by employing Grant-in-Aid for Scientific Research from the crude enzyme preparations obtained from Ministryof Education,Science and Cultureof Japan. membrane fractions of guinea-pig ileum and The generous gifts of compounds mentioned in striatum has shown that dynorphin-(1-8) is Materialsand Methods are gratefullyacknowledged. hydrolyzed by amastatin-sensitive amino peptidase at the Tyr-Gly bond, by phosphor References amidon-sensitive endopeptidase-24.1 1 at 1 Aoki, K., Kajiwara, M. and Oka, T.: The role of the Gly-Phe bond, and captopril-sensitive bestatin-sensitive aminopeptidase, angiotensin peptidyl dipeptidase A at both Arg-Arg and converting enzyme and thiorphan-sensitive "en Phe-Leu bonds (Hiranuma, T. and Oka, T., kephalinase" in the potency of enkephalins in unpublished observation). Since [Leu5] the guinea-pig ileum. Japan. J. Pharmaco!. 36,

importance of three enzymes in the inactivation of [Met5]-enkephalin and [Met']-enkephalin Arg' in three isolated preparations by employing the inhibitor specific for each enzyme. Japan. J. Pharmacol. 44, 241-247 (1987)

in vitro potencies in pharmacological and binding assays after inhibition of peptidases reveals that dynorphin (1-9) is a potent K-agonist. Life Sci. 31, 1725-1728 (1982)

Ishizuka, Y. and Matsumiya, T.: The choice of opiate receptor subtype by neo-endorphins. Eur. J. Pharmacol. 79, 301-305 (1982)

59-65 (1984) Eur. J. Pharmacol. 73, 235-236 (1981) 2 Aoki, K., Kajiwara, M. and Oka, T.: The inacti 12 Kosterlitz, H.W. and Watt, A.J.: Kinetic para vation of [Met'] -enkephalin by bestatin-sensitive meters of narcotic agonists and antagonists, with aminopeptidase, captopril-sensitive peptidyl di particular reference to N-allylnoroxymorphone peptidase A and thiorphan-sensitive endopep (naloxone). Br. J. Pharmacol. 33, 266-276 tidase-24.11 in mouse vas deferens. Japan. J. (1968) Pharmacol. 40, 297-302 (1986) 13 Kuno, Y., Aoki, K., Kajiwara, M., Ishii, K. and 3 Cui, S., Kajiwara, M., Ishii, K., Aoki, K., Sakamoto, Oka, T.: The relative potency of enkephalins and J., Matsumiya, T. and Oka, T.: The enhancing jS-endorphin in guinea-pig ileum, mouse vas effects of amastatin, phosphoramidon and deferens and rat vas deferens after the adminis captopril on the potency of [Met']-enkephalin tration of peptidase inhibitors. Japan. J. Phar in rat vas deferens. Japan. J. Pharmacol. 42, 43 macol. 41, 273-281 (1986) 49 (1986) 14 Kuno, Y. and Oka, T.: Estimation of relative 4 Hiranuma, T. and Oka, T.: Effects of peptidase inhibitors on the [Met5]-enkephalin hydrolysis in ileal and striatal preparations of guinea-pig: Almost complete protection of degradation by the combination of amastatin, captopril and thiorphan. Japan. J. Pharmacol. 41, 437-446 15 Oka, T., Aoki, K., Kajiwara, M., Ishii, K., Kuno, (1986) Y., Hiranuma, T. and Matsumiya, T.: Inactivation 5 Corbett, A.D., Paterson, S.J., McKnight, A.T., of [Leu']-enkephalin in three isolated pre Magnan, J. and Kosterlitz, H.W.: Dynophin1-8 parations: Relative importance of aminopep and dynorphin1-9 are ligands for the K-subtype tidase, endopeptidase-24.1 1 and peptidyl di of opiate receptor. Nature 199, 79-81 (1982) peptidase A. In NIDA Research Monograph 75: 6 McKnight, A.T., Corbett, A.D., Paterson, S.J., Progress in Opioid Research, Edited by Holaday, Magnan, J. and Kosterlitz, H.W.: Comparison of J.W., Law, P.-Y. and Herz, A., p. 259-262, U.S. Government Printing Office, Washington, D.C. (1986) 16 Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.W.: Endogenous opioid peptides: 7 Oka, T., Aoki, K. and Kajiwara, M.: The choice of multiple agonists and receptors. Nature 267, opioid receptor subtype in isolated preparations 495-499 (1977) by dynorphins. Life Sci. 33, Supp. I, 311-314 17 Oka, T., Negishi, K., Kajiwara, M., Watanabe, Y., (1983) 8 Schulz, R., Wi ster, M. and Herz, A.: Receptor preference of dynorphin A fragments in the mouse vas deferens determined by different 18 Shaw, J.S., Miller, L., Turnbull, M.J., Gormley, techniques. J. Pharmacol. Exp. Ther. 230, 200 J.J. and Morley, J.S.: Selective antagonists at 204 (1984) the opiate delta-receptor. Life Sci. 31, 1259 9 Miller, L., Rance, M.J., Shaw, J.S. and Traynor, 1262 (1982) J.R.: Conversion of dynorphin-(1 -9) to [Leu5] 19 Ueki, M., Aoki, K., Kajiwara, M., Shinozaki, K., enkephalin by the mouse vas deferens in vitro. Inoue, H. and Oka, T.: Two new opioid delta Eur. J. Pharmacol. 116, 159-163 (1985) receptor ligands: a highly selective agonist and 10 Oka, T., Negishi, K., Suda, M., Sawa, A., Fujino, a potent selective antagonist in in vitro isolated M. and Wakimasu, M.: Evidence that dynorphin preparations. Japan. J. Pharmacol. 36, 485-489 (1 -13) acts as an agonist on opioid K-receptors. (1984) Eur. J. Pharmacol. 77, 137-141 (1982) 20 Morley, J.S.: Structure-activity relationships of 11 Oka, T., Negishi, K., Suda, M., Matsumiya, T., enkephalin-like peptides. Annu. Rev. Pharmacol. Inazu, T. and Ueki, M.: Rabbit vas deferens: A Toxicol. 20, 81-110 (1980) specific bioassay for opioid K-receptor agonists.