Tolerance to morphine at the μ-opioid receptor differentially induced by cAMP-dependent protein kinase activation and morphine

https://doi.org/10.1016/S0014-2999(99)00881-XGet rights and content

Abstract

Human neuroblastoma SH-SY5Y cells express endogenous μ-opioid receptor and develop cellular tolerance to morphine after prolonged (≥4 h) treatment with morphine. Treatment with forskolin (25 μM, 12 h), an adenylyl cyclase activator, also desensitized μ-opioid receptor response to morphine (10 μM) by 38% (P<0.001), which was reversed by the cyclic AMP (cAMP) dependent kinase inhibitor N-(2-aminoethyl)-5-isoquinolinesulfonamide (H8) (100 μM). Treatment with both morphine and forskolin appeared to cause an additive effect in desensitizing μ-opioid receptor. In μ-opioid receptor stably transfected human embryonic kidney 293 (HEK-μ) cells, morphine treatment produced cAMP upregulation, yet failed to induce μ-opioid receptor tolerance. However, treatment with forskolin (25 μM) or 8-bromo-cAMP (1mM) led to profound μ-opioid receptor tolerance, which was reversed by H8. These results demonstrate that cAMP-dependent kinase activation causes μ-opioid receptor tolerance. However, morphine-induced μ-opioid receptor tolerance in SH-SY5Y cells is not mediated by cAMP-dependent kinase activation. In addition, our results indicate that cAMP-upregulation does not necessarily lead to μ-opioid receptor tolerance.

Introduction

Agonist-induced receptor desensitization is a common phenomenon associated with activation of many neurotransmitter receptors, leading to reduced biological responses during continuous or repeated agonist exposure. Among G-protein coupled receptors, mechanisms of desensitization have been extensively characterized for the β2-adrenergic receptor. Phosphorylation of β2-adrenoceptor by the cyclic AMP (cAMP) dependent protein kinase and the G-protein coupled receptor kinase 2 are, among other mechanisms, involved in rapid desensitization of β2-adrenergic receptor (Freedman and Lefkowitz, 1996).

Opioid receptors belong to the family of G-protein coupled receptors. Activation of all three major (μ, δ, κ) opioid receptor types lead to inhibition of adenylyl cyclase, activation of K+ channels, as well as inhibition of Ca2+ conductance Loh et al., 1988, Mestek et al., 1995, Koob and Nestler, 1997. Morphine and other clinically used opioids act primarily on the μ-opioid receptor to produce their analgesic and rewarding effects. Tolerance (including attenuated analgesic effect) after repeated administration of opioids is well documented in clinical practice; however, the mechanisms remain to be elucidated. Receptor downregulation, internalization, and uncoupling from the second messenger system of μ-opioid receptor have all been speculated to contribute to opioid tolerance, yet numerous studies from different laboratories have so far failed to identify consistent changes of receptor numbers or affinity, or G-protein levels after prolonged treatment with morphine Loh et al., 1988, De Vries et al., 1991, Koob and Nestler, 1997. In addition, unlike the peptide agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), morphine does not internalize μ-opioid receptor Arden et al., 1995, Keith et al., 1998.

Using a reconstituted in vitro system, activation of cAMP-dependent kinase was shown to disrupt coupling of the μ-opioid receptor to Gi Harada et al., 1989, Harada et al., 1990. Although morphine acutely inhibits adenylyl cyclase, chronic exposure to morphine results in a compensatory upregulation of adenylyl cyclase activity and cAMP production Sharma et al., 1975, Yu and Sadée, 1988, which can lead to the activation of cAMP-dependent kinase. Therefore, it is possible that enhanced cAMP-dependent kinase activity, after prolonged morphine treatment, can contribute to μ-opioid receptor tolerance. Phosphorylation of the μ-opioid receptor is enhanced during chronic morphine exposure (Wang et al., 1996). Moreover, activation of cAMP-dependent kinase can phosphorylate and activate cAMP response element binding protein (CREB), which regulates the expression of numerous genes (Koob and Nestler, 1997). Contradicting these results, activation of cAMP-dependent kinase was found to prevent DAMGO-induced μ-opioid receptor desensitization in Xenopus oocytes expressing the μ-opioid receptor and a G-protein-activated K+ channel (Chen and Yu, 1994).

To study cAMP/cAMP-dependent kinase pathway mediated μ-opioid receptor desensitization, we examined μ-opioid receptor tolerance in human neuroblastoma SH-SY5Y cells, and human embryonic kidney (HEK) 293 cells stably transfected with a cloned μ-opioid receptor (HEK-μ) (Arden et al., 1995). HEK-μ cells express only the cloned μ-opioid receptor. SH-SY5Y cells natively express both μ and δ opioid receptors (ratio∼4.5:1). In both cell lines, μ-opioid receptor tolerance can be assessed by the decreased effect of morphine in inhibiting cAMP accumulation. The cAMP-dependent kinase can be activated by treating cells with forskolin, an adenylyl cyclase activator that increases cAMP levels and cAMP-dependent kinase activity (Sibley et al., 1998). More directly, cell permeable cAMP analogs such as 8-bromo-cAMP can activate the cAMP-dependent kinase Itano et al., 1996, Sibley et al., 1998.

In this study, we report that tolerance to morphine developed in SH-SY5Y cells either by treatment with morphine or by activation of cAMP-dependent kinase, but through different mechanisms. In HEK-μ cells, tolerance occurred after treatment with forskolin or 8-bromo-cAMP, but not with morphine.

Section snippets

Materials

Human neuroblastoma SH-SY5Y cells were provided by Dr. June L. Biedler of the Sloan–Kettering Institute for Cancer Research (Rye, NY). N-(2-aminoethyl)-5-isoquinolinesulfonamide (H8) was from Seikagaku America (Rockville, MD). Morphine sulfate was provided by the National Institute on Drug Abuse (Bethesda, MD). All other chemicals were purchased from Sigma (St. Louis, MO). The rat μ-opioid receptor cDNA in vector pRC/CMV was a generous gift from Dr. Lei Yu, and stably transfected into HEK 293

Tolerance to morphine after treatment with morphine in SH-SY5Y cells

Upon prolonged exposure to morphine, SH-SY5Y cells develop a moderate degree tolerance to morphine while the cAMP second messenger system is upregulated Sharma et al., 1975, Yu et al., 1990, Wang et al., 1994. To determine the time course of the development of tolerance and cAMP upregulation, SH-SY5Y cells were pretreated with 1 μM morphine for 0–24 h. At the end of incubation, cells were thoroughly washed to remove morphine. At 1 μM, morphine was able to induce full cAMP upregulation and

Discussion

Exposure of SH-SY5Y cells to μ-opioid receptor agonist morphine led to reduced ability of subsequent morphine to inhibit adenylyl cyclase activity and cAMP accumulation. Unlike the rapid desensitization seen with other G-protein coupled receptors including the δ-opioid receptor (Freedman and Lefkowitz, 1996), μ-opioid receptor tolerance to morphine developed over 4 h in SH-SY5Y cells. The delayed on-set of tolerance paralleled to the development of tolerance in vivo (Grumbach et al., 1974).

Acknowledgements

This work was supported by The National Institute on Drug Abuse grant DA04166. Z.W. was supported in part by The National Institute of Health grant GM07175.

References (34)

  • B.J Van Vliet et al.

    mu-Opioid receptor-regulated adenylate cyclase activity in primary cultures of rat striatal neurons upon chronic morphine exposure

    Eur. J. Pharmacol.

    (1991)
  • Z Wang et al.

    Basal phosphorylation of mu-opioid receptor is agonist modulated and Ca2+-dependent

    FEBS Lett.

    (1996)
  • Z Wang et al.

    3-Isobutyl-1-methylxanthine inhibits basal mu-opioid receptor phosphorylation and reverses acute morphine tolerance and dependence in mice

    Eur. J. Pharmacol.

    (1999)
  • K.O Aley et al.

    Multiple receptors involved in peripheral alpha 2, mu, and A1 antinociception, tolerance, and withdrawal

    J. Neurosci.

    (1997)
  • H Ammer et al.

    Chronic morphine treatment increases stimulatory beta-2 adrenoceptor signaling in A431 cells stably expressing the mu-opioid receptor

    J. Pharmacol. Exp. Ther.

    (1997)
  • J.R Arden et al.

    Phosphorylation and agonist-specific intracellular trafficking of an epitope-tagged mu-opioid receptor expressed in HEK 293 cells

    J. Neurochem.

    (1995)
  • E.J Bilsky et al.

    Effects of naloxone and d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance in mice

    J. Pharmacol. Exp. Ther.

    (1996)
  • Cited by (34)

    • Mesenchymal stem cells from bone marrow attenuated the chronic morphine-induced cAMP accumulation in vitro

      2019, Neuroscience Letters
      Citation Excerpt :

      The cAMP upregulation induced by chronic morphine was regarded as the marker of its tolerance and dependence [17]. Consistent with previous research [17], acute morphine treatment significantly inhibited cAMP accumulation of RA-differentiated SH-SY5Y cells while chronic morphine treatment significantly increased the cAMP accumulation of RA-differentiated SH-SY5Y cell (Fig. 2). Thus, chronic morphine treatment successfully and effectively led to μ receptor tolerance to morphine in vitro.

    • Effects of perinatal exposure to lead (Pb) on purine receptor expression in the brain and gliosis in rats tolerant to morphine analgesia

      2016, Toxicology
      Citation Excerpt :

      This phenomenon is defined as a progressive need to increase doses of the drug to obtain the same pharmacological effect, and is induced by adaptive changes within the mesolimbic system (Koob et al., 2004; Nestler, 2004; Robinson and Kolb, 2004), which may directly lead to a state of dependence. As literature data have shown, morphine tolerance has been repeatedly described (Crain and Shen, 2004; Gunduz et al., 2010; Heinzen and Pollack, 2004; Itoh et al., 2000; Liu and Anand, 2001; Raghavendra and Kulkarni, 2000; Wang and Sadée, 2000) and various relationships with neurotransmitters and their receptors have already been demonstrated (Allen and Dykstra, 2000; Cook et al., 2000; Li et al., 2012; Ozdemir et al., 2012; Ozdoğan et al., 2003; Ranjbar-Slamloo et al., 2012; Smith et al., 2004; Stoller et al., 2007). On the other hand, despite common knowledge on the neurotoxic activity of various environmental toxins, including lead (Pb), the involvement of such environmental factors in the addictions to various drugs is poorly described.

    • A new method to effectively and rapidly generate neurons from SH-SY5Y cells

      2016, Neuroscience Letters
      Citation Excerpt :

      For example, RA-treated SH-SY5Y cells can express adult splicing forms of tau with neuronal localization as well as tyrosine hydroxylase (TH) [1,10], making these cells suitable for in vitro cellular models of Alzheimer’s disease (AD) and Parkinson’s disease (PD) research. In addition, they can also serve as a cell model for research in morphine tolerance, since the differentiated SH-SY5Y cells are able to express the opioid receptor μ [19]. It would be interesting to investigate the expression of these specific markers in neurons differentiated from SH-SY5Y cells under these culture conditions with a combination of RA and CM-hNSC applications.

    • Role of neuroinflammation in morphine tolerance: Effect of tumor necrosis factor-α

      2012, Acta Anaesthesiologica Taiwanica
      Citation Excerpt :

      The within-system tolerance occurs by eliciting an opposite action within the same system; the between-systems tolerance is characterized by alterations in the primary drug-sensitive system adaptation not directly involved in the drug's primary action system. Investigations of the mechanisms of within-system included modulation of intracellular adenylyl cyclase (AC) and cAMP-dependent protein kinase A (PKA),2,3 uncoupling of G-protein signaling,4,5 increased binding of β-arrestin to opioid receptors,6,7 and μ-opioid receptor oligomerization.8,9 As to the mechanisms of the between-systems, N-methyl-d-aspartate (NMDA) receptors,10,11 glutamate transporters (GTs),12 and glial activation with the release of proinflammatory cytokines stand out.13,14

    • Cytokine biomarkers and chronic pain: Association of genes, transcription, and circulating proteins with temporomandibular disorders and widespread palpation tenderness

      2011, Pain
      Citation Excerpt :

      These cells express a wide variety of growth factor/cytokine receptors including GM-CSF, IL-1, IL-4, VEGF, TNFα, and TGFβ [6,9,15,32,64]. Undifferentiated human neuroblastoma cells (SH-SY5Y cells) were selected as they express pain-relevant receptors and molecules (eg, opioids and their receptors) [95] and possess native neuronal machinery and response characteristics [72,92]. Human hepatoma cells (HepG2 cells) and human glioma cells (U87MG cells) were selected because they provide model systems within which to study cytokine-relevant inflammatory pathways [12,48,50].

    View all citing articles on Scopus
    View full text