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Home , Amastatin, Enkephalinase

The Enhancing Effects of , Phosphoramidon and Captopril on the Potency of [Met5]- in Rat Vas Deferens

Suying CUI", Midori KAJIWARA,Kaori ISHII, KazukoAOKI, Junshi SAKAMOTO,Teruhiko MATSUMIYAand Tetsuo OKA* Departmentof ,School of Medicine,Tokai University, Isehara259-11, Japan AcceptedMay 29, 1986

Abstract-The enkephalin-inactivating enzymes in rat vas deferens were studied by using the relatively specific inhibitor of each enzyme. The results showed that the rat vas deferens, like the other three preparations, guinea-pig ileum, mouse vas deferens and striatal membranes of guinea-pig brain, which had been investigated previously, contained three distinct enkephalin-hydrolyzing peptidases. Additionally, the enkephalin-hydrolyzing , -24.11 and peptidyl A in rat vas deferens were found to be inhibited maximally with 1 IN of amastatin, 1 iiM of phosphoramidon and 1 ,IM of captopril, respectively. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D--sensitive were suggested not to be involved significantly in the inactivation of exogenously given enkephalin in rat vas deferens. The characteristics of the enkephalin-degradative enzymes in rat vas deferens were discussed in terms of their similarities to and differences from those in the other preparations.

Three enzymes, both bestatin and peptidases, since enkephalin remains intact amastatin-sensitive aminopeptidase, both almost totally after it is incubated with either and phosphoramidon-sensitive ileal or striatal membrane fractions of guinea endopeptidase-24.11 ("", EC pig for 60 min at 37'C in the presence of 3.4.24.11), and captopril-sensitive peptidyl three peptidase inhibitors, amastatin, thior dipeptidase A (angiotensin I converting phan and captopril, although free tyrosine enzyme, kininase II, peptidyldipeptide hy and the Tyr-Gly-Gly fragment are produced drolase, EC 3.4.15.1), have been shown to when the incubation mixture does not play important roles in the inactivation of contain the peptidase inhibitor (3). exogenously given enkephalin in two in vitro On the other hand, the structure of phos isolated preparations, guinea-pig ileum (1) phoramidon-sensitive endopeptidase-24.1 1 and mouse vas deferens (2). Additionally, in the kidney has been reported to be slightly these three enzymes have been indicated to different from either that in the intestine (4) be located very close to receptors (1, or that in the brain (5). Additionally, the lung 2). Moreover, enkephalin has been shown to and brain peptidyl have been be exclusively inactivated by these three shown to differ markedly in their substrate preference with the lung enzyme, failing to degrade substance K, while the brain enzyme ~r Present address: Department of Physiology , Institute of Clinical Research, China-Japan displays similar activity toward substance K Friendship Hospital, Heping North Street, Beijing, and substance P (6). Moreover, the inhibitory China. potency of /9-endorphin has been shown to * To whom reprint requests should be addressed . be significantly enhanced by the pretreatment of the mouse vas deferens with three deferens in order to examine the stabi ity of peptidase inhibitors, amastatin, captopril and the response of the preparation to an opioid phosphoramidon, while it is not augmented during the usual experimental period, since in both guinea-pig ileum and rat vas deferens it has been shown that the sensitivity to an by the same pretreatment (7). These four opioid of rabbit vas deferens was significantly reports suggest that the structure or the increased with the passage of time (9), while function of a particular peptidase in one that of guinea-pig ileum was not significantly preparation or tissue may not always be the changed during the usual experimental same as that in the other. period (1). The IC50 values of [Met5] In the present investigation, therefore, the enkephalin estimated initially and secondarily effect of peptidase inhibitor on the potency were significantly higher than those estimated of [Met5]-enkephalin in rat vas deferens was secondarily and thirdly, respectively (Table examined in order to e'ucidate whether or 1). On the other hand, the IC50 values not the enkephalin-hydrolyzing peptidase in estimated thirdly and fourthly were not sig rat vas deferens had the same characteristics nificantly different from those estimated as that in guinea-pig ileum (1) or mouse vas fourthly and fifthly, respectively (Table 1). deferens (2). Since the increased sensitivity of the pre paration to an opioid during the per od from Materials and Methods the initial to the third determination of the Chemicals: Gifts of compounds which IC50 value (Table 1) might be caused by were gratefully received were amastatin and the decreased activity of the enkephalin phosphoramidon from Dr. T. Aoyagi, Inst tute hydrolyzing enzymes during the identical of Microbial Chemistry (Tokyo), and captopril period, the IC50 value of [Met5]-enkephalin from Sankyo Company (Tokyo). [Met5] was then repeatedly estimated in the presence Enkephalin and D-phenylalanine were of three peptidase inhibitors, amastatin, purchased from Institute, Inc. captopril and phosphoramidon, at the final (Minoh), and L-tyrosyl-L-tyrosine was from concentration of 1 /tM each. The IC50 values Miles-Yeda, Ltd. (Israel). of [Met5]-enkephalin in the presence of In vitro isolated preparations: Male Wistar three peptidase inhibitors were also decreased rats weighing 300-500 g were used for this during the period from their initial to third study. The vasa deferentia from rats were determinations (Table 1), indicating the prepared and set up for electrical stimulation negative involvement of the peptidases in the as described previously (8). The % inhibition increased sensitivity of the preparation to of the stimulated muscle twitch produced by an opioid during the early phase of the [Met5]-enkephalin was plotted against the experiment. Since the results obtained in log concentration of the opioid to estimate the both the absence and the presence of the IC50 (concentration of the opioid to produce peptidase inhibitors showed that the response 50% inhibition of the twitch). When the of the preparation to an opioid was not effect of peptidase inhibitors on the IC50 significantly changed during the period from value of [Met5]-enkephalin was studied, the third to the fifth estimation of the IC50 they were given at least ten minutes before value, the following experiments on the effect the enkephalin administration. The % of the peptidase inhibitor on the potency of difference shown in the table was calculated [Met5]-enkephalin in rat vas deferens were as described previously (2). The significance carried out during the equivalent period from of % differences between IC50 values of the third to the fifth estimation of the IC50 two adjacent groups shown in the table was value. determined by the paired Student's t-test. Enhanced effect of the peptidase inhibitor on the potency of [Met5]-enkephalin: Results [Met5]-Enkephalin had been shown to be The control experiment: The IC50 value of hydrolyzed by three distinct enzymes in [Met5]-enkephalin was repeatedly estimated several preparations such as guinea-pig for five times in six preparations of rat vas ileum (1), mouse vas deferens (2), and both Table 1. The repeated estimation of the IC50 values of [Met5]-enkephalin in either the presence or the absence of the mixture of peptidase inhibitors

The approximate interval of each [Met5]-enkephalin administration was 30 min. Each value represents the mean±S.E. of 6 experiments. :,The mixture of three peptidase inhibitors, amastatin, captopril and phosphoramidon, was given 10 min before the enkephalin administration at the final concentration of 1 fiM each. ***P<0.01.

Table 2. Enhancing effect of amastatin on the inhibitory potency of [Met5]-enkephalin in rat vas deferens pretreated with both phosphoramidon and captopril

The mixture of two peptidase inhibitors, phosphoramidon and captopril, was added 10 min before the enkephalin administration at the final concentration of 1 ,uM each. Amastatin was given immediately after the mixture administration. Each value represents the mean±S.E. of 4 experiments. ***P<0.01. ileal and striatal membrane fractions of of 1 ,uM of amastatin was not significantly guinea-pig (3). Therefore, the presence of different from that of 2 pM of amastatin, but the enzyme with low enkephalin-hydrolyzing significantly lower than that of 0.1 uM of activity was sometimes difficult to detect amastatin (Table 2). This implied that the when the residual enzyme with high enkephalin-hydrolyzing aminopeptidase in rat enkephalin-hydrolyzing activity was not vas deferens was inhibited maximally with inhibited (3). Then, when the enkephalin amastatin at the final concentration of 1 ,uM, hydrolyzing ability of a particular enzyme being consistent with the previous findings among three enzymes was examined, the obtained with guinea-pig ileum (1) and residual two enzymes were inhibited in mouse vas deferens (2). However, Corbett advance in the present study. et al. (10) and McKnight et al. (11) have Initially, the effect of amastatin, an amino suggested that the susceptibility to bestatin, peptidase inhibitor, on the inhibitory potency another aminopeptidase inhibitor, of the of [Met5]-enkephalin in the rat vas deferens enkephalin-hydrolyzing aminopeptidase in pretreated with two peptidase inhibitors, rat vas deferens was lower than that in phosphoramidon and captopril, at the final guinea-pig ileum or mouse vas deferens, concentration of 1 /iM each was examined. since they had used 30 pcM of bestatin in rat The IC50 value of enkephalin in the presence vas deferens but 10 uM of bestatin in both guinea-pig ileum and mouse vas deferens to by increasing the dose of amastatin to 10 iiM inhibit the enkephalin-hydrolyzing amino (Fig. 1). The inhibition produced by the peptidase. In order to confirm the present administration of both [Met5]-enkephalin and observation, therefore, the effect of the amastatin was completely reversed by 1 ,uM increasing doses of amastatin on the of , a specific (Fig . electrically-evoked contractions of the rat 1). The result suggested that the enkephalin vas deferens pretreated with both captopril hydrolyzing aminopeptidase was inhibited and phosphoramidon was examined in the maximally with amastatin at the final concen presence of [Met5]-enkephalin in the organ tration of 1 /iM, and the amastatin-induced bath. Amastatin at the final concentration of inhibition of the contractions of the rat vas 0.1 /cM significantly enhanced the enkephalin deferens in the presence of enkephalin was induced inhibition of the contractions of the caused by the increased amount of an opioid rat vas deferens pretreated with both captopril in the vicinity of receptors . and phosphoramidon (Fig. 1) . Additionally, Secondarily, the effect of phosphoramidon, the enhanced inhibition produced by 0 .1 ,cM an inhibitor of endopeptidase-24.11, on the of amastatin was further augmented by inhibitory potency of [Met5]-enkephalin in increasing the dose of amastatin to 1 /iM the rat vas deferens pretreated with two (Fig. 1). However, the inhibition induced by peptidase inhibitors, amastatin and captopril, 1 /IM of amastatin was not further enhanced at the final concentration of 1 tiM each was estimated. The IC50 value of enkephalin in the presence of 1 /-,M of phosphoramiclon was not significantly different from that of 2 uM of phosphoramidon, but significantly lower than that of 0.1 /iM of phosphoramidon (Table 3). This indicated that the enkephalin hydrolyzing endopeptidase-24.11 in rat vas deferens was inhibited maximally with phos phoramidon at the final concentration of 1 riM, being consistent with the previous Fig. 1. Naloxone (Nal) -reversible inhibitory action results obtained with guinea-pig ileum (1 ), of amastatin (Ama) on electrically-evoked con mouse vas deferens (2), pig caudate synaptic tractions of rat vas deferens in the presence of membranes (12) and purified endopeptidase [Met5]-enkephalin (ME). The mixture of captopril and phosphoramidon at the final concentration of 24.11 from pig kidney (13). 1 ,uM each was given 10 min before the enkephalin Thirdly, the effect of captopril, an inhibitor administration. Compounds were added to the bath of peptidyl dipeptidase A, on the inhibitory at the dot. Note that amastatin at the final concen potency of [Met5]-enkephalin in the rat vas tration of 10-' M enhances, and that of 10-6 M deferens pretreated with two peptidase further enhances, but that of 10-5 M no more inhibitors, amastatin, and phosphoramidon , augments the inhibition of contractions . at the final concentration of 1 ;€M each was

Table 3. Enhancing effect of phosphoramidon on the inhibitory potency of [Met'] -enkephalin in rat vas deferens pretreated with both amastatin and captopril

The mixture of two peptidase inhibitors, amastatin and captopril , was added 10 min before the enkephalin administration at the final concentration of 1 pM each . Phosphoramidon was given immediately after the mixture administration. Each value represents the mean±S .E. of 4 experiments. ***P<0.01. Table 4. Enhancing effect of captopril on the inhibitory potency of [Met5]-enkephalin in the rat vas deferens pretreated with both amastatin and phosphoramidon

The mixture of two peptidase inhibitors, amastatin and phosphoramidon, was added 10 min before the enkephalin administration at the final concentration of 1 uM each. Captopril was given immediately after the mixture administration. Each value represents the mean±S.E. of 4 experiments. ***P<0.01.

Table 5. Negative enhancing effect of L-tyrosyl-L-tyrosine (Tyr-Tyr) or D-phenylalanine (D-Phe) on the inhibitory potency of enkephalin in the rat vas deferens pretreated with three peptidase inhibitors, amastatin (Ama). ohosohoramidon (Pho) and caotooril (Cao)

The final concentration of Ama, Pho or Cap was 1 uM while that of Tyr-Tyr or D-Phe was 0.1 mM. The peptidase inhibitor was given 10 min before the [Met5]-enkephalin ad:Anistration. aNumber of experiments. examined. The IC50 value of enkephalin in tyrosyl-L-tyrosine or D-phenylalanine at the the presence of 1 ,uM of captopril was not final concentration of 0.1 mM each (Table 5). significantly different from that of 2 -tM of captopril, but significantly lower than that of Discussion 0.1 ,uM of captopril (Table 4). This suggested Three enzymes, amastatin-sensitive amino that the enkephalin-hydrolyzing peptidyl peptidase, phosphoramidon-sensitive endo dipeptidase A in rat vas deferens was inhibited peptidase-24.11 and captopri l-sensitive maximally with captopril at the final concen peptidyl dipeptidase A, are indicated to be tration of 1 tiM, being consistent with the involved in the inactivation of [Met5] previous results obtained with guinea-pig enkephalin in rat vas deferens in the present ileum (1 ), mouse vas deferens (2) and a investigation, since any of three peptidase membrane preparation from the electric inhibitors, amastatin, phosphoramidon and organ of Torpedo marmorata (14). captopril, is shown to enhance significantly Finally, the effect of either L-tyrosyl-L the inhibitory potency of [Met5]-enkephalin tyrosine, an inhibitor of an aminoterminal in the rat vas deferens pretreated with the directed dipeptidylpeptidase (15), or D residual two peptidase inhibitors. Additionally, phenylalanine, a carboxypeptidase inhibitor the enkephalin-hydrolyzing aminopeptidase, (16), on the inhibitory potency of [Met5] endopeptidase-24.11 and peptidyl dipep enkephalin in the rat vas deferens pretreated tidase A in rat vas deferens are suggested to with three peptidase inhibitors, amastatin, be inhibited maximally with 1 tiM of amas phosphoramidon and captopril, at the final tatin, 1 ,iM of phosphoramidon and 1 uuM concentration of 1 ,uM each was estimated. of captopril, respectively, since the enhancing The IC50 value of enkephalin was not sig effect of each peptidase inhibitor on the nificantly altered by the addition of either L enkephalin-induced inhibition is shown to reach maximum at the dose of 1 uM. In magnitude of the enhancement of the contrast to these three peptidases, both inhibitory potency of [Met5]-enkephalin by dipeptidyl aminopeptidase and carboxy the mixture of three peptidase inhibitors, peptidase seem not to be involved sig amastatin, phosphoramidon and captopril, at nificantly in the inactivation of exogenously the final concentration of 1 uM each, in added enkephalin in rat vas deferens since the mouse vas deferens has been shown to be IC50 values of [Met5]-enkephalin in the approximately three-fold higher than that in preparations pretreated with three peptidase guinea-pig ileum, but two to three-fold inhibitors, amastatin, phosphoramidon and lower than that in rat vas deferens (7). captopril, are not significantly different from Additionally, the mixture of these three those pretreated with four peptidase in peptidase inhibitors has been shown not to hibitors, amastatin, phosphoramidon, cap enhance significantly the inhibitory potency topril and either L-tyrosyl-L-tyrosine, which of 9-endorphin in both guinea-pig ileum and has been demonstrated to be an inhibitor of rat vas deferens but augment significantly the an amino-terminal-directed dipeptidylpep potency of 3-endorphin in mouse vas tidase (15), or D-phenylalanine, which has deferens (7). been shown to be a carboxypeptidase In conclusion, the rat vas deferens, like the inhibitor (16). other three preparations, guinea-pig ileum The results obtained with rat vas deferens (1), mouse vas deferens (2) and striatal mem in the present investigation coupled with branes of guinea-pig brain (3), is shown to those obtained with both guinea-pig ileum contain three distinct enkephalin-inactivating (1) and mouse vas deferens (2) in the enzymes. Additionally, the susceptibility of previous studies show that three distinct the enkephalin-hydrolyzing enzymes to in enzymes, amastatin-sensitive aminopeptidase, hibitors in rat vas deferens is found to be phosphoramidon-sensitive endopeptidase similar to that in both guinea-pig ileum (1) 24.11 and captopril-sensitive peptidyl dipep and mouse vas deferens (2), although the tidase A, play critical roles in the inactivation peptidases in rat vas deferens have been of enkephalin in three isolated preparations, shown to be different from those in mouse vas guinea-pig ileum, mouse vas deferens and deferens in terms of the ability of the l9 rat vas deferens. Additionally, the data endorphin hydrolysis (7). obtained with these three isolated pre parations indicate that both L-tyrosyl-L References tyrosine-sensitive dipeptidyl aminopeptidase 1 Aoki, K., Kajiwara, M. and Oka, T.: The role of and D-phenylalanine-sensitive carboxypep bestatin-sensitive aminopeptidase, angiotensin tidase are not involved significantly in the converting enzyme and thiorphan-sensitive inactivation of enkephalin in three isolated ""enkephalinase" in the potency of preparations, guinea-pig ileum (1), mouse in the guinea-pig ileum. Japan. J. Pharmacol. 36, vas deferens (2) and rat vas deferens. 59-65 (1984) Moreover, the finding that [Met5]-enkephalin 2 Aoki, K., Kajiwara, M. and Oka, T.: The remains intact almost wholly after it is inactivation of [Met5]-enkephalin by bestatin incubated with either ileal or striatal sensitive aminopeptidase, captopril-sensitive membrane fractions of guinea-pig in the peptidyl dipeptidase A and thiorphan-sensitive presence of three peptidase inhibitors, endopeptidase-24.11 in mouse vas deferens. amastatin, thiorphan and captopril, at the Japan. J. Pharmacol. 40. 297-302 (1986) final concentration of 1 PM each, for 60 min 3 Hiranuma, T. and Oka, T.: Effects of peptidase at 37°C (3), strongly indicates that inhibitors on the [Met']-enkephalin hydrolysis enkephalin is exclusively inactivated by three in ileal and striatal preparations of guinea-pig: Almost complete protection of degradation by enzymes. the combination of amastatin, captopril and However, the total activity of three enke thiorphan. Japan. J. Pharmacol. 41, 437-446 phalin-inactivating enzymes in one isolated (1986) preparation is likely to be significantly 4 Fulcher, I.S., Chaplin, M.F. and Kenny, A.J.: different from that in the other, since the Endopeptidase-24.11 purified from pig intestine is differently glycosylated from that in kidney. 11 McKnight, A.T., Corbett, A.D. and Kosterlitz, Biochem. J. 215, 317-323 (1983) H.W.: Increase in potencies of opioid 5 Relton, J.M., Gee, N.S., Matsas, R., Turner, A.J. after peptidase inhibition. Eur. J. 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Res. 13 Matsas, R., Kenny, A.J. and Turner, A.J.: The Commun. 128, 317-324 (1985) metabolism of neuropeptides: the hydrolysis of 7 Kuno, Y., Aoki, K., Kajiwara, M., Ishii, K. and peptides, including enkephalins, tachykinins and Oka, T.: The relative potency of enkephalins and their analogues, by endopeptidase-24.11. f3-endorphin in guinea-pig ileum, mouse vas Biochem. J. 223, 433-440 (1984) deferens and rat vas deferens after the adminis 14 Turner, A.J. and Dowdall, M.J.: The metabolism tration of peptidase inhibitors. Japan. J. Phar of neuropeptides: both phosphoramidon macol. 41, 273-281 (1986) sensitive and captopril-sensitive metallopep 8 Oka, T., Negishi, K., Suda, M., Sawa, A., Fujino, tidases are present in the electric organ of M. and Wakimasu, M.: Evidence that Torpedo marmorata. Biochem. J. 222, 255-259 (1-13) acts as an on opioid K-receptors. (1984) Eur. J. Pharmacol. 77, 137-141 (1982) 15 Lee, C.-M. and Snyder, S.H.: Dipeptidyl 9 Oka, T., Negishi, K., Suda, M., Matsumiya, T., aminopeptidase III of rat brain: Selective Inazu, T. and Ueki, M.: Rabbit vas deferens: A affinity for enkephalin and angiotensin. J. Biol. specific bioassay for opioid K-receptor . Chem. 257, 12043-12050 (1982) Eur. J. Pharmacol. 73, 235-236 (1981) 16 Hartsuck, J.A. and Lipscomb, W.N.: Carboxy 10 Corbett, A.D., Paterson, S.J., McKnight, A.T., peptidase A. In The Enzymes, Edited by Boyer, Magnan, J. and Kosterlitz, H.W.: Dynorphin1-8 P.D., Vol. 3, p. 1-56, Academic Press, New York and dynorphin1-9 are ligands for the KK-subtype (1971) of receptor. Nature 199, 79-81 (1982)