Elsevier

Neuropeptides

Volume 42, Issue 3, June 2008, Pages 319-330
Neuropeptides

The differential effect of morphine and β-endorphin administered intracerebroventricularly on pERK and pCaMK-II expression induced by various nociceptive stimuli in mice brains

https://doi.org/10.1016/j.npep.2008.01.003Get rights and content

Abstract

The present study was performed to characterize the differential molecular mechanisms of morphine and β-endorphin which are injected intracerebroventiricularly in mice. In the immunoblot assay, the increases of phosphorylated extracellular signal-regulated protein kinase (pERK) as well as phosphorylated calcium/calmodulin-dependent protein kinase IIα (pCaMK-IIα) expression induced by noxious stimuli were attenuated by intracerebroventricular (i.c.v.) β-endorphin pretreatment in the hypothalamus, but not by i.c.v. morphine pretreatment. In addition to these immunoblot results, immunohistochemical study also showed that the attenuation of pERK or pCaMK-IIα immunoreactivity elicited by i.c.v. pretreatment of β-endorphin mainly occurred in the paraventricular nucleus of the hypothalamus (PVN). We also investigated the effect of morphine and β-endorphin on pERK and pCaMK-IIα expression in the locus coeruleus (LC). I.c.v. injection of morphine significantly increased pERK as well as pCaMK-IIα expression in the locus coeruleus, while β-endorphin increased only pCaMK-IIα in the LC. In addition, β-endorphin significantly attenuated pERK expression induced by SP i.t. injection. These results suggest that the antinociceptive effects of supraspinally administered morphine and β-endorphin are involved with differentially intracellular signal transduction molecules-pERK, pCaMK-IIα in the PVN and the LC.

Introduction

β-Endorphin is an endogenous opioid peptide synthesized by the cells in the arcuate nucleus of the hypothalamus and the nucleus tractus solitarius in the brain stem (Bloom et al., 1978, Bronstein et al., 1992) and which contain neurons projecting widely throughout the brain. Although β-endorphin was known to have moderate affinity for endogenous μ/δ-opioid receptors and about 10-fold lower affinity for κ-opioid receptors (Raynor et al., 1994), it was suggested that the analgesic effect of β-endorphin administered i.c.v. may be mediated by the activation of μ- or pupative ε-opioid receptor (Sun et al., 2003); for review see reference (Tseng, 2001).

Several lines of evidence have supported the antinociceptive effect of ε-receptor activation. Suh and Tseng (1990b) have reported the absence of cross-tolerance between supraspinally injected morphine and β-endorphin in the tail-flick test in mice. In addition, it was also demonstrated that β-endorphin and morphine activate different sites of the pain modulatory system in the supraspinal regions (Tseng and Wang, 1992), which are also involved with different neurotransmitters to modulate nociceptive processing in the spinal cord (Suh and Tseng, 1990a, Tseng et al., 1985, Tseng et al., 1986, Tseng and Suh, 1989). The existence of epsilon opioid receptor, however, still remains controversial and the differential effect of morphine and β-endorphin was also not clearly demonstrated yet. Because, the putative epsilon opioid receptor has not yet been cloned and selective agonists and antagonists for epsilon opioid receptor are not available.

Inflammation or injuries to peripheral tissues cause long-lasting increases in the responses of central nociceptive neurons to innocuous and noxious stimuli (central sensitization) (Ji and Woolf, 2001, Ma and Woolf, 1996, Willis, 2001), by which the excitability of spinothalamic tract cells depends on the activation of ERK1/2 (Dai et al., 2002, Ji et al., 2002) or CaMK-II located at the spinal dorsal horn (Willis, 2001). Extracellular signal-regulated protein kinase (ERK1 and ERK2) is one of the mitogen-activated protein kinase families. It has been reported that the phosphorylated ERK was increased in the spinal cord induced by noxious, but not innocuous, stimuli (Dai et al., 2002, Ji et al., 2002), which involved the activation of NMDA or metabotropic glutamate receptors followed by transcriptional activation such as NK-1 or prodynorphin, leading to central or peripheral sensitization (Galan et al., 2002, Ji et al., 1999, Ji et al., 2002, Karim et al., 2001). It is also well known that Ca2+/calmodulin dependent protein kinase II (CaMK-II) plays an important role in the regulation of calcium signaling in synaptic transmission by phosphorylating various proteins, including neuronal membrane receptors and intracellular transcription factors. Previously, we reported that phosphorylation of ERK1/2 or CaMK-IIα protein in various supraspinal regions may be implicated in the nociceptive processing. Especially, in the hypothalamic paraventricular nucleus, which was projected from the spinal cord directly or indirectly involved in autonomic, neuroendocrine, and emotional responses to somatosensory stimulation, the increase of pERK1/2 and pCaMK-IIα play a critical role in the nociceptive processing induced by formalin s.c. or substance P i.t. injection (Choi et al., 2005a, Choi et al., 2005b, Choi et al., 2006). However, it was little known whether morphine and β-endorphin could modurate the pERK or pCaMK-IIα expression induced by nociceptive stimuli or not.

Pharmacological and behavioral efforts to reveal the existence of the ε-opioid receptor were widely investigated. However, the molecular mechanisms of the differential effects of morphine and β-endorphin are not yet fully understood. Particularly, pERK and pCaMK-IIα previously known to be involved with supraspinal nociceptive processing were not yet characterized. In the present study, we suppose that the effects of morphine and β-endorphin, which has been known to exert its action by μ- and ε-opioid receptors, respectively, affect differently on pERK or pCaMK-IIα expression induced by various nociceptive stimulus in the hypothalamic paraventricular nucleus. We therefore examined the effect of morphine and β-endorphin on the expression of pERK and pCaMK-IIα elicited by various nociceptive stimuli.

Section snippets

Materials and methods

These experiments were approved by the University of Hallym Animal Care and Use Committee. All procedures were conducted in accordance with the ‘Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.

The effects of intracerebroventricular injected morphine and β-endorphin on nociceptive behaviors induced by s.c. formalin or intrathecal substance P injection

To determine the ideal doses of morphine and β-endorphin for the molecular study, we investigated the effects of morphine and β-endorphin on the nociceptive behaviors induced by s.c. formalin or i.t. SP at the several dose (0.1–2.0 μg/5 μl). The s.c. injection of 5% formalin into the left hind paw caused an acute, immediate nociceptive response, i.e., licking, shaking and biting the injected paw, which lasted for 5 min (1st phase). The 2nd phase formalin response began about 20 min after formalin

Discussion

Several lines of evidence of pharmacological studies have suggested that the putative episilon opioid receptors activate the descending pain inhibitory system, which is distinctive μ-opioid receptor mediated anitinociception (Mizoguchi et al., 2000, Suh and Tseng, 1990b, Tseng and Wang, 1992). We demonstrated the molecular clue of the differential antinociceptive effect of μ- and ε-opioid receptor mediated antinociception by comparing the effects of morphine and β-endorphin on the pERK and

Acknowledgements

This research was supported by research grants from the Korean Ministry of Science and Technology under Brain Fronteir (M103KV010014-06K2201-01410) and Medical Research Center program of MOST/KOSEF (R13-2005-022-01001-0).

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