J Pharmacol Sci 106, 295 – 300 (2008)2 Journal of Pharmacological Sciences ©2008 The Japanese Pharmacological Society Full Paper 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-injec- tion 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.