Enkephalin After Peptidase Inhibition

Great Increase in Antinociceptive Potency of [Leu5]Enkephalin After Peptidase Inhibition

Kazuhito Akahori1, Kenya Kosaka1, Xing Lu Jin1, Yoshiharu Arai1, Masanobu Yoshikawa1, Hiroyuki Kobayashi1, and Tetsuo Oka1,* 1Department of Clinical Pharmacology, School of Medicine, Tokai University, Isehara 259-1143, Japan

Received August 9, 2007; Accepted December 19, 2007

Abstract. Previous in vitro studies have shown that the degradation of [Leu5]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Leu5]enkephalin administered intra-third-ventricularly on the tail-flick response was increased more than 500-fold by the intra-third-ventricular pretreatment with the three peptidase inhibitors. The antinociceptive effect produced by the [Leu5]enkephalin in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with the three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Leu5]enkephalin. The present data, together with those obtained from previous studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of short endogenous opioid peptides, such as penta-, hepta-, and octa-peptides, administered intra-third-ventricularly to rats.

Keywords: [Leu5]enkephalin, opioid peptide, antinociception, peptidase inhibitor

Introduction in vitro isolated preparations, guinea-pig ileum, mouse vas deferens, and rat vas deferens. Results showed that It has been shown that when [Leu5]enkephalin (LE) is AsA played the greatest role in both guinea-pig ileum incubated with an ileal or striatal membrane fraction and rat vas deferens, while it played a similar role to for 60 min at 37°C in the presence of three peptidase either PsE or CsD in mouse vas deferens (2). inhibitors (PIs), amastatin (an aminopeptidase inhibitor), In addition to LE, our previous in vitro experiments captopril (a dipeptidyl carboxypeptidase inhibitor), and also revealed that the mixture of three PIs largely phosphoramidon (an endopeptidase-24.11 inhibitor), prevented the hydrolysis of endogenous opioid peptides approximately 98% of LE remains intact, while in the (EOPs), [Met5]enkephalin (ME), [Met5]enkephalin- absence of the PI, the LE is completely hydrolyzed after Arg6-Phe7 (ME-RF), [Met5]enkephalin-Arg6-Gly7-Leu8 incubation (1). This shows that LE is hydrolyzed, at least (ME-RGL), and dynorphin A (1-8) [dyn (1-8)] by in the cerebral membrane preparation, by only three cerebral membrane preparation (1, 3 – 5). Additionally, types of membrane-bound enzymes: amastatin-sensitive the close proximity of these enzymes to the opioid aminopeptidase(s) (AsA), captopril-sensitive dipeptidyl receptors in isolated preparations such as guinea-pig carboxypeptidase I (angiotensin I-converting enzyme, ileum (6), mouse vas deferens (7), and rat vas deferens kininase II, EC 3.4.15.1) (CsD), and phosphoramidon- (8), suggests that they act to terminate the physiological sensitive endopeptidase-24.11 (“enkephalinase”, EC action of these EOPs. 3.4.24.11) (PsE). The relative importance of the three Because the hydrolysis products of LE by either enzymes in the inactivation of LE was examined in three amastatin-, phosphoramidon-, or captopril-sensitive enzymes such as free Tyr and the [Tyr-Gly-Gly]-, [des- *Corresponding author. [email protected] Tyr]-, and [des-Tyr-Gly-Gly]-fragments are suggested Published online in J-STAGE doi: 10.1254/jphs.FP0071318 to have very low, if any, agonist activity at opioid

295 296 K Akahori et al receptors (9), the potency of the LE should be decreased was calculated for some experiments. by its hydrolysis with these three peptidases. In fact, the in vitro potency of LE in isolated preparations has In vivo apparent pA2 analysis been significantly increased by either amastatin, phos- Tail-flick latency of rats pretreated with the three phoramidon, or captopril (1, 2). PIs was measured before and 15 min after the i.t.v. In the present investigation, effects of the intra-third- administration of LE and converted to %MPE. The ventricular (i.t.v.) injection of three PIs were examined dose-effect curve of an agonist in each rat was on the antinociception induced by the i.t.v. administra- constructed by injecting the rat with two or three tion of LE. doses, such as 0.5, 1 and 2 nmol, with 48-h inter-injection interval. Individual ED50 values were calculated by Materials and Methods least-squares regression of the data in the portion of the dose-effect curve spanning the 50% MPE. The mean Chemicals ED50 value was obtained from individual ED50 values. Captopril and naloxone hydrochloride were kindly Naloxone was given subcutaneously 5 min before the provided by Sankyo (Tokyo). LE, amastatin, and phos- i.t.v. administration of the agonist. Dose-ratio was phoramidon were purchased from Peptide Institute, Inc. calculated by dividing each ED50 value in the presence (Minoh). Chemicals were dissolved in saline. The stock of naloxone by the mean ED50 value in the absence of solution for all peptides used was prepared at concentra- naloxone. The pooled pA2 value was determined by tions of 0.1 – 10 mM in siliconized plastic tubes, entering all the dose-ratio values (12). maintained at −18°C, and then diluted to the desired concentration just before use. In vitro isolated preparations Male ICR-Jcl mice weighing 40 – 50 g and male Microinjection into the third cerebral ventricle Hartley guinea pig weighing 400 – 600 g were used for Male Wistar rats weighing 180 – 220 g were the study. The mouse vas deferens and the myenteric anesthetized with pentobarbital sodium (40 mg/kg, plexus longitudinal muscle strip of guinea-pig ileum intraperitoneally), mounted on a stereotaxic frame, and were set up for electrical stimulation as described implanted with stainless-steel injection cannulae previously (13). The percent inhibition of the stimulated (external diameter of 0.30 mm) 5 – 7 days prior to the muscle twitch produced by an opioid was plotted against day of the experiment. The lower end of the injection the log concentration of the opioid to estimate the IC50 cannula was aimed at the third cerebral ventricle (opioid concentration producing 50% inhibition of the (6.0 mm anterior from lambda and 7.8 mm ventral from twitch). When the effect of peptidase inhibitors on the the surface of the skull) according to the atlas of Paxinos IC50 value of an opioid peptide was studied, these and Watson (10). The injection cannula was attached were given 5 min before the administration of the to a motor-driven 50-µl microsyringe by polyethylene opioid peptide. The ratio of the potency and the tubing. Drugs were injected in volumes of 10 µl for percentage difference, shown in Table 1, were calculated 1 min. The distribution of the drug solution in the from the following formulas: ratio of potency = IC50 cerebroventricular system was verified by infusion of without peptidase inhibitor / IC50 with peptidase methylene blue dissolved in saline after the experiment. inhibitors, and % difference = [(IC50 without peptidase inhibitor − IC50 with peptidase inhibitors) / IC50 without Tail-flick response peptidase inhibitor] × 100. The antinociceptive effect of LE was measured by the tail immersion assay with 55°C as the nociceptive Statistical analyses stimulus (11). The latency to flick the tail from the All values are reported as the mean with S.E.M. of the 55°C water was measured before and 5, 10, 15, 30, data. A statistical analysis was conducted using 45, and 60 min after the opioid administration. The computer software (The SPSS 14.0.1; SPSS, Inc., latency to flick the tail before the administration was Chicago, IL, USA) for comparison across the experi- approximately 1 s (0.5 – 1.7 s). A cut-off time of 5 s was mental conditions. When a significant difference among used to prevent any injury to the tail. The percent of the groups of AUC data was obtained in the one-way maximal possible effect (MPE) for each animal at each analysis of variance (ANOVA), the Bonferroni’s time was calculated using the following formula: multiple comparison test or the Dunnett’s post-hoc test %MPE = [(test latency − baseline latency) / (5 − baseline was applied to define which group contributed to these latency)] × 100. The AUC (area under the curve) value differences. The statistical significance of percent for the antinociceptive action of an opioid on each rat differences between IC50 values of two adjacent groups Analgesia of [Leu5]Enkephalin 297

Table 1. The enhancing effects of the peptidase inhibitors (PIs) on the inhibitory potency of [Leu5]enkephalin in guinea-pig ileum (GPI) and mouse vas deferens (MVD)

Preparations PIs n IC50 (nM) Ratio of potency %Difference

GPI None 5 296 ± 56 1 82.3 ± 1.3** ACP 5 50.5 ± 8.9 5.79 ± 0.50 MVD None 4 10.5 ± 2.5 1 60.0 ± 5.0* ACP 4 3.83 ± 0.51 2.64 ± 0.38

The mixture of three PIs, amastatin (A), captopril (C), and phosphoramidon (P), at the final concentration of 1 µM each was given 5 min before the [Leu5]enkephalin administration. Values are the means ± S.E.M. of n experiments. *P<0.05, **P<0.01. shown in Table 1 was determined by the Wilcoxon smaller than that at the dose of 0.1 nmol in rats treated signed-rank test. The level of statistical significance with three PIs (Fig. 2). Thus, the antinociceptive effect was set at P<0.05. of LE administered i.t.v. on the tail-flick response was increased more than 500-fold by the i.t.v. administration Results of three PIs. However, the possibility that the potency of LE is increased more that 1000-fold by three PIs could Effects of PIs on LE-induced inhibition of tail-flick not be denied, since the potency of LE at the dose more response than 100 nmol in rats not treated with PIs could not be Our previous in vivo study showed that the inhibition estimated because such a high concentration of LE was of the tail-flick response induced by the i.t.v. administra- not available (e.g., 100 nmol in 10 µl) due to the low tion of ME was augmented by increasing the doses of solubility of LE. the three PIs administrated i.t.v., with the maximum inhibition being attained at the dose of 10 nmol each Effects of naloxone on LE-induced inhibition of tail-flick (14). Furthermore, previous in vitro experiments showed response that the concentration of the three PIs required to inhibit The dose–effect curve for LE shifted dose-depen- the hydrolysis of ME was essentially the same as that of dently to the right after s.c. pretreatment with various LE (1, 3). Therefore, each PI at the dose of 10 nmol was doses of naloxone hydrochloride (0.01, 0.02, and employed to inhibit the targeted peptidase in the present 0.05 mg/kg) 5 min before the i.t.v. administration of investigation. LE. Figure 3 shows the Schild plots for naloxone with As reported previously (14), the PIs by themselves values derived from individual dose ratios for each rat. 2 4 did not significantly change the latency of the tail-flick The pA2 value for naloxone against [D-Ala , N-Me-Phe , response. Additionally, 10 nmol of amastatin alone, Gly-ol]enkephalin (DAMGO), a selective µ-agonist, phosphoramidon alone, or captopril alone did not at under the same experimental condition, was reported all enhance the potency of LE (data are not shown). previously (15); and the reported pooled pA2 values and Therefore, the inhibition of the tail-flick response slopes (95% confidence limits shown in parentheses) are induced by the i.t.v. administration of 50 nmol of LE 7.63 (7.49 – 7.79) and −0.99 (1.08 – 0.90), respectively. was examined in rats pretreated i.t.v. with the mixture of The pooled pA2 values and their 95% confidence limits the two or three PIs among amastatin (A), captopril (C), shown in Fig. 3 demonstrate that the effectiveness of and phosphoramidon (P) at the dose of 10 nmol each naloxone to antagonize the antinociceptive effects of LE (Fig. 1). The antinociceptive effects of 50 nmol of LE in is similar to and not significantly different from that of rats pretreated with three PIs (ACP) was significantly DAMGO reported previously. greater than in rats pretreated with any combination of two PIs, AC, AP, CP (Fig. 1). Effects of three PIs on the potency of LE in isolated The effects of the i.t.v. administration of 0.1, 1, 10, guinea-pig ileum and mouse vas deferens and 50 nmol of LE in rats pretreated i.t.v. with three PIs The inhibitory effects of LE on the electrically evoked at the dose of 10 nmol each were investigated (Fig. 2). contractions of both mouse vas deferens and the The potency of LE at the dose of 50, 10, and 1 nmol in myenteric plexus longitudinal muscle preparation of rats pretreated with three PIs was significantly greater guinea-pig ileum were enhanced approximately 2.6- and than LE at the dose of 50 nmol in rats not treated with 5.8-fold, respectively, by pretreatment of the prepara- PIs (Fig. 2). The potency of LE at the dose of 50 nmol in tions with the mixture of three PIs at the concentration rats not treated with PIs was slightly but not significantly of 1 µM each (Table 1). 298 K Akahori et al

Fig. 1. Greater antinociceptive effect of LE in rats pretreated with Fig. 2. Dose-dependent antinociceptive effects of LE in rats the mixture of three PIs (ACP) than in those pretreated with any pretreated with three PIs (ACP) and those in rats not treated with the combination of two PIs (AC, AP, or CP). Panel I: The time course of PI. Panel I: The time course of change in LE-induced antinociceptive change in LE-induced antinociceptive effects in rats pretreated with effects in rats pretreated with the mixture (ACP) of three peptidase the mixture of two or three peptidase inhibitors (PIs), amastatin (A), inhibitors (PIs), amastatin (A), captopril (C), and phosphoramidon (P). The first group of rats received intra-third-ventricularly (i.t.v.) captopril (C), and phosphoramidon (P). The first group of rats = received intra-third-ventricularly (i.t.v.) LE (50 nmol) alone (n = 7). LE (50 nmol) alone (n 7). In the second to fifth group of rats, a In the second to fifth group of rats, the mixture of two or three PIs mixture of three PIs (ACP) at a dose of 10 nmol each was injected = = = = i.t.v. 10 min before the i.t.v. injection of LE (0.1 nmol, n = 4; (AC, n 5; AP, n 6; CP, n 5; ACP, n 9) at the dose of 10 nmol = = = each was given i.t.v. 10 min before the i.t.v. administration of LE 1.0 nmol, n 19; 10 nmol, n 20; 50 nmol, n 9). LE-induced antinociceptive effects in rats were expressed as %MPE. Vertical (50 nmol). Vertical bars represent the S.E.M. Panel II: The AUC0–60 min values for the LE-induced antinociceptive effects, expressed as bars represent the S.E.M. Panel II: The AUC0–60 min values for the %MPE in panel I, in rats pretreated with the mixture of three PIs LE-induced antinociceptive effects, expressed as %MPE in panel I, (ACP) or any combination of two PIs (AC, AP, or CP) at the dose of in rats pretreated with the mixture of three PIs (ACP) at the dose of 10 nmol each. The AUC0–60 min values for the antinociceptive effect of 10 nmol each. The AUC0–60 min values for the antinociceptive effect of LE were calculated for each rat. Vertical bars represent the S.E.M. LE were calculated for each rat. Vertical bars represent the S.E.M. Significantly different from the values of a group that received Significantly different from the values of a group that received 50 nmol LE alone by Dunnett’s post-hoc test following one-way three PIs (ACP) by Bonferroni’s multiple comparison test following < < one-way ANOVA; ***P<0.001. ANOVA; *P 0.05, ***P 0.001.

Discussion antinociceptive effect was increased more than 10,000-, 100-, or 1,000-folds, respectively (15, 16). The present The present study demonstrated that the antinocicep- study, together with previous studies (1, 3 – 5, 14 – 16), tive effect induced by the i.t.v. administration of LE indicated that AsA, PsE, and CsD play an important was increased more than 500-fold by the i.t.v. pretreat- role on the in vivo and in vitro hydrolytic inactivation ment with the three PIs: amastatin, captopril, and of endogenous opioid penta-, hepta-, and octa-peptides phosphoramidon. In our previous experiments with ME, in the brain. dyn (1-8), or ME-RGL under the same condition, the Previous studies indicated that the amastatin-sensitive Analgesia of [Leu5]Enkephalin 299

regulating the concentrations of neuropeptides, includ- ing enkephalins, in cerebrospinal fluid (18). Three reports (1, 17, 18) suggest that the hydrolysis of LE administered i.t.v. is largely prevented in the presence of the three PIs: amastatin, captopril, and phosphoramidon. Therefore, the antinociceptive potency of LE shown in the present study probably largely reflects the real potency of LE itself. The involvement of µ-opioid receptors in the antinociceptive effect of LE in rats pretreated with the three PIs is suggested by the fact that pA2 values and slopes (95% confidence limits shown in parentheses) for naloxone, an opioid antagonist having a preference for µ-opioid receptors (19), against LE are 7.63 (7.49 – 7.79) and −0.99 (1.08 – 0.90) and are not significantly different from those for a selective µ-opioid receptor agonist, DAMGO, that were reported previously (15) − Fig. 3. Schild plots obtained for naloxone in the antinociception and were 7.50 (7.42 – 7.58) and 1.20 (1.40 – 1.00) assay of LE. Abscissae: negative log unit of the molar doses of (15, 20). Although LE is known to act on both µ- and δ- naloxone. Ordinates: log of (dose ratio – 1). Each point was opioid receptors, the antinociceptive effect of δ-opioid converted from individual dose-ratios for each rat. The pooled pA2 receptor agonists is not likely to be measurable under values and slopes are included in each panel. The 95% confidence limits are shown in parentheses. the present experimental conditions since the antinociceptive effect of [D-Pen2,5]enkephalin (DPDPE), a potent selective δ-opioid receptor agonist, at doses up to aminopeptidase plays the most important role among the 10 nmol could not be detected in our preliminary three peptidases in the inactivation of LE in the isolated experiments. These data are in agreement with those guinea-pig ileum preparation (1). However, the present of Heyman et al. (21) showing that the intracerebro- study showed that the antinociceptive potency of the ventricular administration of DPDPE produces anti- i.t.v. administration of 50 nmol LE was not at all nociception in the hot-plate, but not in the tail-flick, augmented by pretreatment of rats with any one PI test with rats. Therefore, only the action of LE at µ- (amastatin, captopril, or phosphoramidon) alone. These opioid receptors could be estimated using the present results suggest that the two non-inhibited peptidases antinociceptive test. among the three peptidases, AsA, PsE, and CsD, can The amount of LE inactivated by the three peptidase rapidly and largely inactivate LE in vivo. Furthermore, during passage of the peptide from the site of administra- the present study showed that the antinociceptive tion to the sites of action (opioid receptors) are likely to potency of the i.t.v. administration of LE in rats pre- be markedly greater in the in vivo experiment than in the treated i.t.v. with the mixture of three PIs were signifi- in vitro experiment, since the antinociceptive potency of cantly greater than that in rats pretreated i.t.v. with any LE administrated i.t.v. was increased more than 500-fold combination of two PIs, AC, AP, or CP. These results by three PIs, while the inhibitory potencies of LE in indicated that any noninhibited peptidase among the isolated mouse vas deferens and guinea-pig ileum were three can inactivate a significant amount of LE. increased approximately 2.6- and 5.8-fold, respectively. Our previous study showed that the hydrolysis of LE This is in good agreement with the facts that the anti- by cerebral membrane preparations is almost completely nociceptive potency induced by the i.t.v. administration prevented by the presence of the three PIs (1). It is not of ME or ME-RGL was increased more than 10,000- or yet known, however, whether or not the hydrolysis of LE 1,000-fold by the three PIs, respectively, whereas the in the cerebrospinal fluid is also completely prevented inhibitory potencies of ME in isolated guinea-pig ileum by the three PIs. Low molecular weight opioid peptides and mouse vas deferens were increased approximately such as ME and dyn (1-7) are shown to be hydrolyzed in 6.8- and 21-fold, respectively, and the potencies of cerebrospinal fluid mainly by aminopeptidase M that is ME-RGL in both preparations were increased approxi- inhibited by amastatin, and the other peptidase activities mately 4.5-fold (16, 22). are suggested to be low in cerebrospinal fluid (17). Since the present and previous studies (14 – 16) Additionally, endopeptidase-24.11, which is inhibited show that the antinociceptive potencies of the low by phosphoramidon, is indicated to play a key role in molecular weight opioid peptides such as ME, dyn (1-8), 300 K Akahori et al

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