Original article
Naloxone-precipitated morphine withdrawal evokes phosphorylation of heat shock protein 27 in rat heart through extracellular signal-regulated kinase

https://doi.org/10.1016/j.yjmcc.2011.04.002Get rights and content

Abstract

Heat shock protein 27 (Hsp27) is a well-known stress response protein that becomes phosphorylated through extracellular signal-regulated kinase (ERK). Different drugs of abuse, such as morphine and/or its withdrawal, induce severe stress situations. In this study, we investigated Hsp27 and phospho-Hsp27 expression during morphine dependence and withdrawal and evaluated the involvement of ERK in the phosphorylation of Hsp27 in the rat right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg/kg, s.c.). ERK1/2, Hsp27 and phospho-Hsp27 at Ser15 were determined by quantitative blot immunolabeling using specific antibodies. Hsp27 expression was increased 30, 60, 90 and 120 min (144.5 ± 14.2%, P < 0.0001; 128.9 ± 4.6%, P = 0.04; 177.4 ± 12.7, P < 0.0001; and 136.2 ± 11.0%, P = 0.042, respectively) after saline injection to rats dependent on morphine. Naloxone-precipitated morphine withdrawal also increased the phosphorylation of Hsp27 at Ser15 at those time points (146.8 ± 19.8%, P = 0.034; 143.9 ± 17.9%, P = 0.032; 161.2 ± 33.3%, P = 0.029; and 152.2 ± 25.5%, P = 0.008, respectively). However, there were no changes in Hsp27 phosphorylation in the morphine dependent group injected with saline. In addition, there was an increase in the phosphorylation of ERK 60 min after naloxone injection in morphine dependent rats (pERK1: 116.3 ± 4.2%, P = 0.015 and pERK2: 117.2 ± 1.5%, P = 0.05). Pretreatment with SL327, an inhibitor of ERK phosphorylation, decreased activation (phosphorylation) of both ERK and Hsp27 (pERK1: 4.5 ± 3.6%, P < 0.0001; pERK2: 42.3 ± 3.3%, P < 0.0001; and pHsp27: 97.6 ± 1.5%, P = 0.008), suggesting that ERK activation triggers Hsp27 phosphorylation. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of Hsp27 in the heart and suggest that phosphorylation of Hsp27 is closely linked to and also dependent on the ERK pathway.

Research highlights

► Opioid addiction. ► Adaptive changes in the heart after morphine withdrawal. ► Role of CRF receptors in the negative state of opioid withdrawal. ► Chaperones and morphine dependence and withdrawal.

Introduction

Repeated morphine administration appears to activate the hypothalamo–pituitary–adrenocortical (HPA) axis and thus is able to induce considerable stress response in both experimental and in clinical situations [1], [2]. Activation of HPA axis in stressful situations is quite comparable to that seen in human using drugs of abuse and probably involves recruitment of brain stress neurocircuitry (e.g. induction of ACTH, glucocorticoids and corticotrophin-releasing factor (CRF) activity) [3], [4]. Furthermore, the profound cellular stress induced by chronic morphine treatment and withdrawal is also evidenced by the overexpression of heat shock proteins (Hsps) as well as massive alterations in the neuronal and glial cell function [5].

Hsps were identified primarily on the basis of their fast and typically protective response to cellular stressors. Hsps are divided into multiple subfamilies based on molecular weight, domain conservation and function. Hsps are rapidly induced at the transcriptional level following stress, but also undergo several post-translational modifications that alter their functional roles for being used as immediate response elements [6]. Hsp27, a member of the small-Hsp family, acts as an endogenous cytoprotective stress response protein, eliciting cardioprotection to ischemic injury, via its role as a molecular chaperone and in phosphorylation-dependent stabilization of actin [7]. Hsp27 can be reversibly phosphorylated on three serine residues by the mitogen-activated protein kinases 2 and 3 (MK2/3), which are themselves activated by phosphorylation through either the p38 or the extracellular signal-regulated protein kinase (ERK) signaling pathway [8], [9], [10], [11], [12]. ERK transduces a broad range of extracellular stimuli to yield diverse intracellular responses. The ERK signaling pathway could be important as a regulator of cardiac function [13]. Recently, several studies have shown that this pathway contributes to naloxone-precipitated withdrawal in morphine dependent rats [14], [15], [16], [17]. Moreover, ERK has been proposed to be involved in a cardioprotective program through its interaction with reperfusion injury salvage kinase (RISK) [18]. However, many differences in signal transduction by RISK have been revealed between species, thus it has been shown that RISK is not important for cardioprotection in large mammals such as pigs [19].

Because the mechanism implicated in the adaptive changes that occur during morphine dependence and withdrawal remains unclear, the aim of the present study was to examine Hsp27 and phospho-Hsp27 expression in order to determine the magnitude and severity of cellular stress during chronic morphine treatment and withdrawal and to evaluate the interaction between Hsp27 and ERK, investigating the effects of SL327, a selective inhibitor of ERK activation [5], on the levels of Hsp27 phosphorylated at Ser15. Since the adaptive changes in opioid receptors and associated signaling systems after morphine dependence can occur in specific brain regions and in other tissues and cells expressing opioid receptors, such as in the heart [20], this study was performed in the right ventricle.

Section snippets

Animals and treatments

Male Sprague–Dawley rats (220–240 g at the beginning of the experiments) were housed four-to-five per cage under a 12-h light/dark cycle (light: 8:00–20:00 h) in a room with controlled temperature (22 ± 2 °C), humidity (50 ± 10%), food and water available ad libitum and prehandled for several days preceding the experiment to minimize stress, as previously described [21]. All surgical and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals published

Results

Before performing the immunodetection assays, we assessed the efficacy of chronic treatment with morphine by pellet implantation. For this purpose, the weight of the animals was recorded on the days of pellet implantation and on the day of killing (day 8), before receiving any injections. Rats treated with morphine showed a significantly lower (P < 0.01; t-test) body weight gain (17.90 ± 2.00 g, n = 40) than animals receiving placebo pellets (43.50 ± 3.30 g, n = 40). Administration of naloxone to control

Discussion

Although it is known that opioid receptor activation attenuates both contractile dysfunction and development of myocardial infarction [29], [30], there are only a few data about the potential cardiac effects of chronic opioid receptor stimulation. Chronic opiate exposure induces cardioprotective effects [31] and numerous neurochemical adaptations in the noradrenergic system at heart levels [1], [2], [12].

Previous studies have demonstrated that chronic μ-opioid receptor stimulation decreases

Conflict of interest

The authors state no conflict of interest.

Acknowledgment

This work was supported by Ministerio de Educación y Ciencia Spain, (grants SAF/FEDER 2007-62758; 2009-07178; 2010-17907).

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