The Enhancing Effects of Amastatin, Phosphoramidon and Captopril on the Potency of [Met5]-Enkephalin in Rat Vas Deferens Suying CUI", Midori KAJIWARA,Kaori ISHII, KazukoAOKI, Junshi SAKAMOTO,Teruhiko MATSUMIYAand Tetsuo OKA* Departmentof Pharmacology,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 aminopeptidase, endopeptidase-24.11 and peptidyl dipeptidase 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-phenylalanine-sensitive carboxypeptidase 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 thiorphan and phosphoramidon-sensitive ileal or striatal membrane fractions of guinea endopeptidase-24.11 ("enkephalinase", 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 opioid receptors (1, or that in the brain (5). Additionally, the lung 2). Moreover, enkephalin has been shown to and brain peptidyl dipeptidases 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 Peptide 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