Elsevier

Brain Research

Volume 881, Issue 1, 20 October 2000, Pages 1-8
Brain Research

Research report
Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone

https://doi.org/10.1016/S0006-8993(00)02770-0Get rights and content

Abstract

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of β-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu1-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [d-Ala2, MePhe4, Gly(ol)5]enkephalin (DAMGO) was completely reversed by pretreatment with β-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID50 values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6β-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose–response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6β-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.

Introduction

Recently two novel peptides have been isolated from bovine brain [41] that have high affinity and selectivity for the mu-opioid receptor, and have been termed endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2). Furthermore, both peptides have been isolated from human brain [6] and immunoreactivity for endomorphin-1 and endomorphin-2 were widely and densely distributed throughout human and rat brain whereas endomorphin-2-immunoreactivity is present in the superficial laminae of the spinal cord dorsal horn where primary afferent nociceptors terminate. These findings indicate that endomorphin-2 plays a physiological role in regulating nociceptive information in the spinal cord [16], [17], [18]. Both endomorphin-1 and endomorphin-2 significantly increase nociceptive thresholds after both spinal and supraspinal administration [4], [31], [35], [41] which are blocked by mu-receptor-specific antagonists, naloxone and β-funaltrexamine [41]. Neither supraspinal endomorphin-1 or -2 elicited antinociception in CXBK mice which are also insensitive to morphine [4].

There is biochemical and pharmacological evidence supporting the existence of mu-opioid receptor subtypes [5], [20], [40] which are localized in spinal and supraspinal structures involved in the modulation of nociception [19]. At least two mu-opioid receptor subtypes have been proposed: mu1 and mu2 opioid receptor subtypes [23], [24]. β-Funaltrexamine irreversibly antagonizes both mu1 and mu2 opioid receptors and inhibits both supraspinal and spinal antinociception, whereas naloxonazine selectively antagonizes the mu1-opioid receptor. It has been suggested that these receptor subtypes have different physiological roles, with mu1-opioid receptors mediating supraspinal antinociception, whereas mu2-opioid receptors mediate spinal antinociception measured by the tail-flick test.

Recent report suggests that heroin and morphine-6β-glucuronide in the family of mu opioids both act through a novel third mu-opioid receptor subtype, distinct from those mediating morphine's actions. 3-methoxynaltrexone selectively competes for [3H]morphine-6β-glucuronide binding antagonizes the antinociceptive actions of heroin and morphine-6β-glucuronide without interfering with mu (morphine and DAMGO), delta- or kappa-opioid receptor agonist-induced antinociception [2].

The objective of the present study was to determine whether antinociceptive activities of i.t. administered endomorphin-1 and endomorphin-2 are mediated through mu-opioid receptor subtypes, mu1, mu2, or a novel mu-opioid receptor in the mouse paw-withdrawal test.

Section snippets

Animals

Adult male ddY mice weighing 22–25 g were housed in a light- and temperature-controlled room (lights on 09:00–21:00 h; 24°C) and had free access to food and water. The experiments were performed with the approval of the Committee of Animal Experiments in Tohoku Pharmaceutical University.

Injection procedure

The procedure for intrathecal (i.t.) injections was adapted from the method of Hylden and Wilcox [11] with a constant injection volume of 2 μl/mouse. For i.t. administration, a 29-gauge needle connected to a

Potency and time course of i.t. injection of endomorphin-1 and -2

The time course of antinociceptive activity for i.t. endomorphin-1, -2 and DAMGO is shown in Fig. 1. Groups of 10 mice were tested for antinociception at 1, 5, 10, 15, 20, 30 and 45 min. Endomorphin-1 and -2 at 1 min post-injection produced dose-dependent antinociception with ED50 values of 0.14 (0.06–0.32) and 0.24 (0.12–0.49) nmol, respectively (figure not shown).

Endomorphin-1 and -2 after 5 min of i.t. injection induced antinociception with ED50 values of 1.9 (0.59–6.11) and 2.6 (1.51–4.47)

Discussion

Both endomorphin-1 and -2 induced dose-dependent antinociception after spinal administration in the paw-withdrawal test. The peak effects of endomorphins-induced antinociception occurred rapidly, within 1 min of the i.t. injection, and disappeared at 10–15 min after injection. The present results of i.t. injected endomorphins are in agreement with those of Stone et al. [36] and Sakurada et al. [31] who reported that the antinociceptive effect of the endomorphins is short-lasting and is absent

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