Elsevier

Experimental Neurology

Volume 160, Issue 2, December 1999, Pages 361-375
Experimental Neurology

Regular Article
Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

https://doi.org/10.1006/exnr.1999.7235Get rights and content

Abstract

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca2+]i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca2+]i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.

References (96)

  • A. Goldstein

    Binding selectivity profiles for ligands of multiple receptor types: Focus on opioid receptors

    Trends Pharmacol. Sci.

    (1987)
  • G. Grynkiewicz et al.

    A new generation of calcium indicators with greatly improved fluorescence properties

    J. Biol. Chem.

    (1985)
  • K.F. Hauser et al.

    Morphine inhibits Purkinje cell survival and dendritic differentiation in organotypic cultures of the mouse cerebellum

    Exp. Neurol.

    (1994)
  • K.F. Hauser et al.

    Androgen increases the number of cells in fetal mouse spinal cord cultures: Implications for motoneuron survival

    Brain Res.

    (1989)
  • C.J. Hudson et al.

    The kappa opioid-related anticonvulsants U-50488H and U-54494A attenuate N-methyl-d-aspartate induced brain injury in the neonatal rat

    Brain Res.

    (1991)
  • W.D. Hutchison et al.

    Dynorphin A: In vivo release in the spinal cord of the cat

    Brain Res.

    (1990)
  • L. Isaac et al.

    MK-801 blocks dynorphin A (1–13)-induced loss of the tail-flick reflex in the rat

    Brain Res.

    (1990)
  • M. Kolaj et al.

    The opioid peptide dynorphin modulates AMPA and kainate responses in acutely isolated neurons from the dorsal horn

    Brain Res.

    (1995)
  • S.L. Lai et al.

    Dynorphin uses a non-opioid mechanism to potentiate N-methyl-d-aspartate currents in single rat periaqueductal gray neurons

    Neurosci. Lett.

    (1998)
  • T.M. Laughlin et al.

    Spinally administered dynorphin A produces long-lasting allodynia: Involvement of NMDA but not opioid receptors

    Pain

    (1997)
  • J.B. Long et al.

    Intrathecal dynorphin A1–13 and dynorphin A3–13 reduce rat spinal cord blood flow by non-opioid mechanisms

    Brain Res.

    (1987)
  • J.B. Long et al.

    Hindlimb paralytic effects of prodynorphin-derived peptides following spinal subarachnoid injection in rats

    Eur. J. Pharmacol.

    (1988)
  • M.P. Mattson et al.

    Calcium, free radicals, and excitotoxic neuronal death in primary cell culture

    Methods Cell Biol.

    (1995)
  • T.K. McIntosh et al.

    Alterations in regional concentrations of endogenous opioids following traumatic brain injury in the cat

    Brain Res.

    (1987)
  • K.G. Olson et al.

    The effects of dynorphin A (1–13) and U50, 488H on free intracellular calcium in guinea pig cerebellar synaptosomes

    Life Sci.

    (1991)
  • L.A. Opanashuk et al.

    Opposing actions of the EGF family and opioids: Heparin binding-epidermal growth factor (HB-EGF) protects mouse cerebellar neuroblasts against the antiproliferative effect of morphine

    Brain Res.

    (1998)
  • R.W. Oppenheim

    Neurotrophic survival molecules for motoneurons: An embarrassment of riches

    Neuron

    (1996)
  • M. Pohl et al.

    Enkephalinergic and dynorphinergic neurons in the spinal cord and dorsal root ganglia of the polyarthritic rat—In vivo release and cDNA hybridization studies

    Brain Res.

    (1997)
  • R.D. Polakiewicz et al.

    mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control

    J. Biol. Chem.

    (1998)
  • J.D. Porter et al.

    Survival of extraocular muscle in long-term organotypic culture: Differential influence of appropriate and inappropriate motoneurons

    Dev. Biol.

    (1993)
  • B. Przewlocka et al.

    Kappa opioid receptor agonists inhibit the pilocarpine-induced seizures and toxicity in the mouse

    Eur. Neuropsychopharmacol.

    (1994)
  • R.C. Riley et al.

    Spinal release of immunoreactive dynorphin A (1–8) with the development of peripheral inflammation in the rat

    Brain Res.

    (1996)
  • V.K. Shukla et al.

    Non-opioid effects of dynorphins: Possible role of the NMDA receptor

    Trends Pharmacol. Sci.

    (1994)
  • S.R. Skilling et al.

    Selective potentiation of NMDA-induced activity and release of excitatory amino acids by dynorphin: Possible roles in paralysis and neurotoxicity

    Brain Res.

    (1992)
  • J.M. Walker et al.

    Dynorphin (1–17): Lack of analgesia but evidence for non-opiate electrophysiological and motor effects

    Life Sci.

    (1982)
  • J.M. Walker et al.

    Des-tyrosine-dynorphin antagonizes morphine analgesia

    Eur. J. Pharmacol.

    (1982)
  • J.Z. Xiang et al.

    The kappa-opiate agonist U50488H decreases the entry of 45Ca into rat cortical synaptosomes by inhibiting N- but not L-type calcium channels

    Neuropharmacology

    (1990)
  • E.A. Young et al.

    The degradation of dynorphin A in brain tissue in vivo and in vitro

    Peptides

    (1987)
  • U. Arvidsson et al.

    The kappa-opioid receptor is primarily postsynaptic: Combined immunohistochemical localization of the receptor and endogenous opioids

    Proc. Natl. Acad. Sci. USA

    (1995)
  • G. Bakalkin et al.

    Receptor-mediated and nuclear effects of opioids on the cell cycle and apoptosis

    Int. Narcot. Res. Conf. Abstr.

    (1998)
  • R. Bakshi et al.

    Dynorphin A-(1–17) induces alterations in free fatty acids, excitatory amino acids, and motor function through an opiate- receptor-mediated mechanism

    J. Neurosci.

    (1990)
  • G.J. Brewer et al.

    Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination

    J. Neurosci. Res.

    (1993)
  • C. Chavkin et al.

    Dynorphin is a specific endogenous ligand of the kappa opioid receptor

    Science

    (1982)
  • L. Chen et al.

    The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin

    J. Neurosci.

    (1995)
  • L. Chen et al.

    The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat

    J. Physiol. (London)

    (1995)
  • L. Chen et al.

    Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation

    Nature

    (1992)
  • D.W. Choi

    Excitotoxic cell death

    J. Neurobiol.

    (1992)
  • Cited by (0)

    View full text