Elsevier

Brain Research

Volume 859, Issue 2, 24 March 2000, Pages 217-223
Brain Research

Research report
Chronic fentanyl treatments induce the up-regulation of μ opioid receptor mRNA in rat pheochromocytoma cells

https://doi.org/10.1016/S0006-8993(00)01961-2Get rights and content

Abstract

Chronic activation of adenylate cyclase-cAMP-cAMP-dependent protein kinase (PKA) systems by administration of opioid receptor agonists has been considered as one of the mechanisms of opioid tolerance and dependence. Although analysis of the μ opioid receptor (MOR) gene suggests that cAMP-related signal transduction systems regulate the expression of this gene, which transcription factors affect the MOR gene expression in brain and neural cells has not been clarified. This study deals with the effects of fentanyl on MOR mRNA levels in the rat pheochromocytoma cell line (PC12 cells). PC12 cells were cultured in medium with clinically relevant concentrations of fentanyl. The quantitative reverse transcription and polymerase chain reaction (RT–PCR) method was used for determination of MOR mRNA. Treatment of PC12 cells with fentanyl induced the MOR mRNA up-regulation in a concentration- and time-dependent manner. A cAMP analogue also up-regulated MOR mRNA. The intracellular cAMP level increased after fentanyl treatment. A PKA inhibitor blocked the MOR mRNA up-regulation by fentanyl and the cAMP analogue. Expression of a dominant inhibitory Ras also inhibited the MOR mRNA up-regulation. Fentanyl-induced up-regulation of MOR mRNA via activation of cAMP signaling may be important in compensating for the MOR reduction during long-term treatment of PC12 cells with fentanyl. The present study could be relevant to understanding the molecular mechanisms of opioids in a state of drug tolerance or dependence, and in patients under anesthesia or being treated for pain.

Introduction

Opioid receptors belong to the superfamily of G protein-coupled receptors. Acute treatment of opioids leads to an inhibition of adenylate cyclase (AC) activity through the coupling of inhibitory Gi/Go classes of G protein and AC, and subsequently to a reduction of cAMP formation. The reduction of cAMP level affects cAMP-mediated second messenger systems mostly via a decrease in cAMP-dependent protein kinase (PKA) activity 8, 17, 23. In contrast, long-term administration of agonists causes an uncoupling between opioid receptors and AC systems, and consequently, a reduction in the inhibition of cAMP formation, which is termed desensitization 8, 17, 23. Furthermore, prolonged opioid exposure increases levels of cAMP and PKA 8, 14, 17, 23. Consistent with these previous findings, a recent study has shown that long-term μ opioid receptor (MOR) agonist administration induces AC superactivation in African green monkey kidney COS-7 cells co-transfected with AC and MOR [3]. Thus, chronic activation of AC systems with opioids has been considered as one of the mechanisms underlying opioid tolerance, dependence and withdrawal 3, 8, 14, 17, 23. Despite extensive investigation, the regulation of opioid receptor gene expression by opioids themselves is poorly understood. The analysis of promoter regions of the MOR gene 19, 20, 22suggests that cAMP and cAMP-related intracellular signal transduction systems regulate the expression of the receptor by activating several gene transcription factors including cAMP response element-binding protein (CREB) and AP-1. However, whether these transcription factors affect the MOR gene expression in neural tissue is not known.

Mitogen-activated protein kinase (MAPK) plays an important role in intracellular signaling pathways that lead to the division or differentiation of many cell types [4]. Stimulation of G protein-coupled receptors such as those for thrombin, acetylcholine and α-adrenergic agonists, can activate the MAPK cascade via small G protein Ras 6, 19. Opioid treatment has been shown to activate MAPK 12, 13, and that the MAPK cascade is connected with the cAMP related-pathway 9, 10, 33. In cultured cortical neurons, activation of MAPK results in an activation CREB [10]. An increase in the intracellular cAMP formation leads to an activation of MAPK. Moreover, PKA activates the transport of activated MAPK from the cytosol to the nucleus, and then MAPK activates a number of transcription factors [16]. These findings provide new insights concerning the regulation of opioid receptor and AC systems via the Ras-MAPK cascade.

Fentanyl is the most popular opioid anesthetic, and is used in many situations including in cardiovascular surgery. Although a vast amount of pharmacological knowledge of fentanyl has been accumulated, the effects of fentanyl on the gene expression of opioid receptors have not been established. In this study, we have determined MOR mRNA levels by the quantitative reverse transcription and polymerase chain reaction (RT–PCR) method, and precisely investigated the effects of fentanyl on levels of MOR mRNA in the rat pheochromocytoma cell line (PC12 cells). We have found that fentanyl up-regulates MOR mRNA, and cAMP- and Ras-related intracellular signal transduction systems mediate the up-regulation of MOR mRNA in PC12 cells.

Section snippets

Reagents

Culture medium was purchased from Nikken Biomedical Laboratory (Kyoto, Japan). Calf serum was obtained from Mitsubishi Kasei (Tokyo, Japan), and horse serum, from Gibco-BRL (Grand Island, NY). Fentanyl, morphine and naloxone were from Sankyo (Tokyo, Japan), and [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) and chlorophenylthio-cAMP (CPTcAMP), from Sigma (St. Louis, MO). H-89 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA), and β-funaltrexamine (βFNA) was purchased from

Quantitative RT–PCR analysis for mRNA in PC12 cells

To determine the amount of MOR mRNA in PC12 cells, we studied the linear relationship between the number of PCR cycles and the amounts of reaction products (Fig. 1). β-actin mRNA was used as an internal control. The amplification curve was linear between 13 and 22 cycles (β-actin) or between 31 and 40 cycles (MOR). Over 25 cycles for β-actin or 43 cycles for MOR, the curve reached a plateau. Consequently, the PCR run consisted of 34 cycles for MOR, and of 16 cycles for β-actin. Using this assay

Discussion

Opioid receptors are pharmacologically classified into at least three subtypes (δ, μ and κ). It is considered that μ and δ opioid receptors are associated with drug tolerance and dependence [26]. Using PC12 cells as a model of neuronal cells, we tried to determine PCR products from the three opioid receptor subtypes. The RT–PCR technique permits quantifying small amounts of the MOR mRNA under clinically relevant conditions. Using subtype-selective primers, we failed to detect significant

Acknowledgements

This work was in part supported by a grant from the Smoking Research Foundation, a Nakajima Memorial Research Grant and grants-in-aid from the Ministry of Education, Science, and Culture of Japan.

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