Elsevier

Neuroscience

Volume 339, 17 December 2016, Pages 538-547
Neuroscience

Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex

https://doi.org/10.1016/j.neuroscience.2016.10.019Get rights and content

Highlights

  • Methadone causes cellular toxicity in cortical cell cultures.

  • Growth hormone protects cells against methadone-induced toxicity.

  • A protective mechanism of growth hormone is proposed.

Abstract

Human growth hormone (GH) displays promising protective effects in the central nervous system after damage caused by various insults. Current evidence suggests that these effects may involve N-methyl-d-aspartate (NMDA) receptor function, a receptor that also is believed to play a role in opioid-induced neurotoxicity. The aims of the present study were to examine the acute toxic effects of methadone, an opioid receptor agonist and NMDA receptor antagonist, as well as to evaluate the protective properties of recombinant human GH (rhGH) on methadone-induced toxicity. Primary cortical cell cultures from embryonic day 17 rats were grown for 7 days in vitro. Cells were treated with methadone for 24 h and the 50% lethal dose was calculated and later used for protection studies with rhGH. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase release, and caspase activation. Furthermore, the mRNA expression levels of NMDA receptor subunits were investigated following methadone and rhGH treatment using quantitative PCR (qPCR) analysis. A significant protective effect was observed with rhGH treatment on methadone-induced mitochondrial dysfunction and in methadone-induced LDH release. Furthermore, methadone significantly increased caspase-3 and -7 activation but rhGH was unable to inhibit this effect. The mRNA expression of the NMDA receptor subunit GluN1, GluN2a, and GluN2b increased following methadone treatment, as assessed by qPCR, and rhGH treatment effectively normalized this expression to control levels. We have demonstrated that rhGH can rescue cells from methadone-induced toxicity by maintaining mitochondrial function, cellular integrity, and NMDA receptor complex expression.

Introduction

Accumulating evidence indicates that long-term use of opioids such as morphine and methadone can cause cognitive deficits in both animals and humans (Sjogren et al., 2005, Tramullas et al., 2007, Rhodin et al., 2014, Schiltenwolf et al., 2014). These adverse effects are suggested to be associated with neuronal cell death (Mao et al., 2002, Svensson et al., 2008, Perez-Alvarez et al., 2010), reduced neurogenesis (Eisch et al., 2000), and volumetric changes of regions in the brain such as the amygdala, and hypothalamus (Younger et al., 2011). Taken together with the fact that prescription opioid use disorders and frequency of use has increased during 2003–2013 in the US (Han et al., 2015), and that opioid prescription has increased in Scandinavia during 2006–2012 (Mahic et al., 2015), these neurotoxic effects warrant further evaluation.

Recent studies suggest that growth hormone (GH), a somatotrophic hormone released from the anterior pituitary gland, counteracts some of the above-mentioned effects. For instance, GH reduces morphine-induced neurotoxicity in primary hippocampal cell cultures (Svensson et al., 2008), and has been shown to increase overall quality of life and cognitive function in a human patient exposed to chronic methadone treatment (Rhodin et al., 2014). In fact, it is well established that both GH and its mediator insulin-like growth factor-1 (IGF-1) are involved in the protection of the central nervous system (CNS) following injury (see review Nyberg, 2009). The protective effects of GH are demonstrated in various studies, which include GH increasing cell survival after hypoxic-ischemic-induced injury (Scheepens et al., 2001, Alba-Betancourt et al., 2013), preventing apoptosis of lymphocytes (Mitsunaka et al., 2001), and reducing neurological disabilities in mammals (Devesa et al., 2013, Heredia et al., 2013).

In addition to the neuroprotective effects of GH, there is evidence of its role as a cognitive enhancer. First, a relationship between the levels of circulating GH and cognitive function exists, with patients suffering from deficiency of this hormone also having impaired cognitive functions (Falleti et al., 2006). Second, both GH and IGF-1 are strong mediators of both gliogenesis and neurogenesis in the CNS and are thus essential for normal cognitive function (see review Aberg, 2010). Third, GH improves cognitive function in various animal models (Le Greves et al., 2006, Enhamre-Brolin et al., 2013, Gronbladh et al., 2013, Ramis et al., 2013, Studzinski et al., 2015), but also demonstrates promising results in a case study of a human undergoing long-term treatment with methadone (Rhodin et al., 2014).

The relationship between the somatotrophic axis and its beneficial effects in the CNS remains unclear. Nevertheless, evidence indicates that GH increases the transmission of the N-methyl-d-aspartate (NMDA) receptor (see review Nyberg and Hallberg, 2013), a key component for regulating long-term potentiation (LTP) and subsequent facilitation of memory and learning (Tsien et al., 1996). The improved cognitive function following treatment with GH disappears when dizocilpine (MK801), a potent NMDA receptor antagonist, is administered (Ramis et al., 2013). Furthermore, GH increases GluN1, GluN2a, and GluN2b NMDA receptor subunit expression, which has been linked to improved cognitive function (Le Greves et al., 2002, Le Greves et al., 2006, Studzinski et al., 2015). Certain opioids, such as methadone, also have antagonistic affinity to the NMDA receptor (Ebert et al., 1995) marking GH as a promising treatment against methadone-induced injury.

Altogether, evidence suggests that GH is a potent protective agent and acts as a cognitive enhancer in the CNS, but its precise role in these conditions needs to be further evaluated. In this study, we assessed the acute protective effects of recombinant human GH (rhGH) on methadone-induced toxicity in rat primary cortical cell cultures in order to gain further insights into the possible mechanism of rhGH in the brain.

Section snippets

Experimental procedures

All experiments that included animals were approved by the local animal ethics committee at Uppsala University (C14/15) according to the Swedish guidelines regarding animal experiments (Animal Welfare Act SFS1998:56) and the European Communities Council directive (86/609/EEC).

Methadone, MK-801 and morphine, unlike rhGH, induce acute mitochondrial dysfunction

There was an overall effect on mitochondrial function in primary cortical cells for each of the following treatments; methadone, MK801, and morphine (as indicated by ANOVA, p < 0.0001). However, there was no overall effect of rhGH treatment (as indicated by ANOVA, p = 0.1493) shown in Fig. 1A. Post hoc analysis further revealed that methadone significantly reduced the proportion of cells with functional mitochondria at 10–1000 μM in comparison with control (Fig. 1B). The LD50 was calculated to be 60 

Discussion

Our main findings from the present study demonstrate that human GH is protective against acute methadone-induced cellular injury. These findings support previous reports indicating that GH acts as a protective agent in the CNS (Scheepens et al., 2001, Svensson et al., 2008, Alba-Betancourt et al., 2013, Devesa et al., 2013, Heredia et al., 2013, Rhodin et al., 2014).

We have examined the protective effects of rhGH using three assays, each measuring specific toxic events. First, the activity of

Conclusion

Overall, we have demonstrated that rhGH may act as a protective agent against methadone-induced cellular injury. Our results further suggest that rhGH may counteract a caspase-independent-mediated cell death (e.g. necrosis), possibly by normalizing or restoring the NMDA receptor complex. In this study, we have provided evidence for a possible mechanism that may contribute to the protective effects of GH. These data encourage further studies, particularly those associated with glial-neuronal

Acknowledgment

This work was supported by the Kjell and Märta Beijer Foundation; the Swedish Research Council (grant number 9459); and the Swedish Brain Foundation. The authors have no conflict of interest to declare.

References (46)

  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

    J Immunol Methods

    (1983)
  • F. Nyberg

    The role of the somatotrophic axis in neuroprotection and neuroregeneration of the addictive brain

    Int Rev Neurobiol

    (2009)
  • S. Perez-Alvarez et al.

    Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis

    Biochim Biophys Acta

    (2010)
  • S.Y. Proskuryakov et al.

    Necrosis: a specific form of programmed cell death?

    Exp Cell Res

    (2003)
  • M. Ramis et al.

    Cognitive improvement by acute growth hormone is mediated by NMDA and AMPA receptors and MEK pathway

    Prog Neuro-Psychoph

    (2013)
  • A. Scheepens et al.

    Growth hormone as a neuronal rescue factor during recovery from CNS injury

    Neuroscience

    (2001)
  • P. Sjogren et al.

    Neuropsychological assessment of chronic non-malignant pain patients treated in a multidisciplinary pain centre

    Eur J Pain

    (2005)
  • A.L. Studzinski et al.

    Growth hormone (GH) increases cognition and expression of ionotropic glutamate receptors (AMPA and NMDA) in transgenic zebrafish (Danio rerio)

    Behav Brain Res

    (2015)
  • V. Tassain et al.

    Long term effects of oral sustained release morphine on neuropsychological performance in patients with chronic non-cancer pain

    Pain

    (2003)
  • N.A. Thornberry et al.

    A combinatorial approach defines specificities of members of the caspase family and granzyme B – functional, relationships established for key mediators of apoptosis

    J Biol Chem

    (1997)
  • J.Z. Tsien et al.

    The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory

    Cell

    (1996)
  • J.W. Younger et al.

    Prescription opioid analgesics rapidly change the human brain

    Pain

    (2011)
  • D. Aberg

    Role of the growth hormone/insulin-like growth factor 1 axis in neurogenesis

    Endocr Dev

    (2010)
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