D-Pro-Endomorphin-1 and D-Pro-Endomorphin-2, Respectively, Attenuate the Antinociception Induced by Endomorphin-1 and Endomorphin-2 Given Intrathecally in the Mouse

First, the antinociception with the tail-flick test of D-Pro-endomorphin-1 and D-Pro-endomorphin-2 given i.t. was compared with that produced by endomorphin-1 and -2 in male CD-1 mice. High doses of D-Pro-endomorphin-1 (0.2–0.4 pmol) and D-Pro-endomorphin-2 (300–800 pmol) given i.t. produced antinociception with low intrinsic activity [about 25% maximum possible effect (MPE)] compared with that of endomorphin-1 (16.4 nmol) and endomorphin-2 (35 nmol) ( 90% MPE). Second, coadministration of a low dose of D-Pro-endomorphin-1 (0.1 pmol), which given alone did not affect the tail-flick latencies, markedly attenuated the antinociception induced by endomorphin-1 (16.4 nmol) but not by endomorphin-2 (35 nmol). Similarly, coadministration of a low dose of D-Pro-endomorphin-2 (200 pmol), which given alone did not affect the tail-flick latencies, significantly attenuated the antinociception induced by endomorphin-2 (35 nmol) and, to a much lesser extent, endomorphin-1 (16.4 nmol). It is concluded that D-Pro-endomorphin-1 and D-Pro-endomorphin-2 at high doses were partial opioid receptor agonists to produce antinociception, and at low doses were opioid receptor antagonists to block selectively the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Furthermore, our results are consistent with the view that the antinociception induced by endomorphin-1 and endomorphin-2 is mediated by the stimulation of different subtypes of -opioid receptors. Endomorphin-1 and endomorphin-2 are two endogenous tetrapeptides isolated from the bovine frontal cortex (Zadina et al., 1997) and human brain (Hackler et al., 1997). These peptides are the first endogenous peptides to be proposed to have high affinity and selectivity for -opioid receptors. Receptor-binding assays and immunocytochemical studies reveal that endomorphin-1 and endomorphin-2 potently compete with 1and 2-receptors and that they are widely located at the sites in the brain and spinal cord abundant in -opioid receptors (Martin-Schild et al., 1997, 1998, 1999; Goldberg et al., 1998; Pierce et al., 1998; Shreff et al., 1998; Wu et al., 1999). Through the stimulation of -opioid receptors, endomorphin-1 and endomorphin-2 inhibit the electrical activity of rostral ventrolateral medulla neurons or spinal substantia gelatinosa neurons (Chu et al., 1999; Wu et al., 1999). The release of endomorphin-2 from the spinal cord can be achieved by electrical stimulation (Williams et al., 1999). The administration of endomorphin-1 and endomorphin-2 given i.c.v. and i.t. produces potent antinociceptive responses that are blocked by -opioid receptors antagonists naloxone, -funaltrexamine, and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-PenThr-NH2 (CTOP) (Zadina et al., 1997; Stone et al., 1997; Narita et al., 1998; Tseng et al., 2000). Neither endomorphin-1 nor endomorphin-2 activates -opioid receptor-coupled G proteins in -opioid receptor knockout mice, and the antinociception induced by endomorphin-1 and endomorphin-2 is attenuated in heterozygous knockout mice and virtually abolished in homozygous knockout mice (Mizoguchi et al., 1999). These findings support the view that antinociception induced by the endomorphin-1 and endomorphin-2 is mediated by the stimulation of -opioid receptors. This work was supported in part by Grant DA 03811 from the National Institute of Health, National Institute on Drug Abuse (to L.F.T.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.038927. ABBREVIATIONS: CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; -FNA, -funaltrexamine; nor-BNI, nor-binaltorphimine; MPE, maximum possible effect; ANOVA, analysis of variance; D-Pro-EM-1, D-Pro-endomorphin-1; D-Pro-EM-2, D-Pro-endomorphin-2. 0022-3565/02/3032-874–879$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 38927/1021569 JPET 303:874–879, 2002 Printed in U.S.A. 874 at A PE T Jornals on A ril 6, 2017 jpet.asjournals.org D ow nladed from However, more recent results illustrate that different subtypes of -opioid receptors may be involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. For example, Sakurada et al. (1999) reported that 1-opioid receptor antagonist naloxonazine was more effective in blocking the antinociceptive effects induced by endomorphin-2 than endomorphin-1 in mice. The antinociception induced by endomorphin-1 is blocked by -opioid receptor antagonists CTOP or -funaltrexamine ( -FNA) but not by -opioid antagonist nor-binaltorphimine (nor-BNI). On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP or -FNA and also by nor-BNI (Tseng et al., 2000; Ohsawa et al., 2001). The findings are taken to indicate that two different subtypes of -opioid receptors are involved in endomorphin-1and endomorphin-2-induced antinociception. D-Pro-Endomorphins, analogs of endomorphins containing D-amino acid isomers, were designed to examine whether these analogs produce antinociception after i.t. administration. Also, the ability of the analogs to modify antinociception induced by i.t. endomorphin-1 and endomorphin-2 was investigated. Materials and Methods Animals. Male CD-1 mice (Charles River Laboratories, Inc., Wilmington, MA), weighing 25 to 30 g were used. Animals were housed five per group in a room maintained at 22 0.5°C with an alternating 12-h light/dark cycle. Food and water were available ad libitum. Drugs. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were purchased from Calbiochem (La Jolla, CA). The peptides were dissolved in sterile saline solution (0.9% NaCl solution) containing 10% hydroxypropyl-cyclodextrin for i.t. injection. D-Pro-Endomorphin-1 (Tyr-D-Pro-Trp-Phe-NH2) and D-Pro-endomorphin-2 (Tyr-D-Pro-Phe-Phe-NH2) were obtained from Dr. T. Sakurada (Department of Biochemistry, Daiichi College of Pharmaceutical Science, Fukuoka, Japan). These D-Pro-endomorphins were first completely dissolved in dimethyl sulfoxide and then 0.9% sodium chloride solution was added to a final concentration of dimethyl sulfoxide at 1%. Assessment of Antinociceptive Response. The antinociceptive response was assessed with the thermal tail-flick test (D’Amour and Smith, 1941). Mice were gently held with the tail positioned in the tail-flick apparatus (model TF6; EMDIE Instrument Co., Maidens, VA) for radiant heat stimulation of the dorsal surface of the tail. The intensity of the heat stimulus was adjusted to cause the animal to flick its tail within 3 to 4 s as the baseline of latency. After measuring the latency, different groups of mice were treated with endomorphin-1, endomorphin-2, or vehicle given i.t., and the tail-flick responses were then measured at different times after injection. The data were expressed as percentage of maximum possible effect (%MPE), which was calculated as [T1 T0)/(T2 T0)] 100. T0 and T1 were predrug and postdrug latency, respectively, and T2 was the cutoff time that was set at 10 s to minimize tissue damage. Drug Administration Protocol. The i.t. injection (5 l) was performed according to the procedure of Hylden and Wilcox (1980) using a 25l Hamilton syringe with a 30-gauge needle. Groups of mice were treated i.t. with various doses of endomorphin-1 (0.82– 16.4 nmol), endomorphin-2 (1.75–35 nmol), D-Pro-endomorphin-1 (0.03–0.4 pmol), D-Pro-endomorphin-2 (50–800 pmol), or vehicle, and the tail-flick tests were performed at 2.5, 5, 7.5, 10, 15, and 20 min thereafter. The effects of D-Pro-endomorphin-1 and D-Proendomorphin-2 on the tail-flick inhibition induced by endomorphin-1 and endomorphin-2 were studied. Groups of mice were coadministered i.t. with various doses of D-Pro-endomorphin-1 (0.03–0.4 pmol) or D-Pro-endomorphin-2 (50–800 pmol) with endomorphin-1 (16.4 nmol) or endomorphin-2 (35 nmol) and the tail-flick response was measured thereafter. In another experiment, groups of mice were coadministered i.t. with D-Pro-endomorphin-1 (0.1 pmol) or D-Pro-endomorphin-2 (200 pmol) with various doses of endomorphin-1 (0.82–16.4 nmol) or endomorphin-2 (1.75–35 nmol), and the tail-flick response was measured thereafter. To establish dose-response curves, at least four doses were used with 8 to 11 mice at each dose. For the calculation of the ED50 values for endomorphin-1and endomorphin-2-induced tail-flick inhibition, the antinociception was assessed using peak effect, which occurred at either 2.5 or 5 min after