Dynorphin A (1-13) neurotoxicity in vitro: Opioid and non-opioid mechanisms in mouse spinal cord neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates k appa-opioid receptors and is antinociceptive at physiological concentration s. Levels of dynorphin A and a major metabolite, dynorphin A (1-13), increa se significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both kap pa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indir ectly) through kappa-opioid and/or NMDA receptor types. Despite these findi ngs, few studies have systematically explored dynorphin toxicity at the cel lular level in defined populations of neurons coexpressing kappa-opioid and NMDA receptors. To address this question, me isolated populations of neuro ns enriched in both kappa-opioid and NMDA receptors from embryonic mouse sp inal cord and examined the effects of dynorphin A (1-13) on intracellular c alcium concentration ([Ca2+](i)) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and duri ng experimental treatments. At micromolar concentrations, dynorphin A (1-13 ) elevated [Ca2+](i) and caused a significant loss of neurons. The excitoto xic effects were prevented by MK-801 (Dizocilpine) (10 mu M), 2-amino-5-pho sphopentanoic acid (100 mu M), or 7-chlorokynurenic acid (100 mu M)-suggest ing that dynorphin A (1-13) was acting (directly or indirectly) through NMD A receptors. In contrast, cotreatment with (-)-naloxone (3 mu M), or the mo re selective kappa-opioid receptor antagonist nor-binaltorphimine (3 mu M), exacerbated dynorphinA(1-13) -induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 mu M). Neuro nal losses were not seen with exposure to the opioid antagonists alone (10 mu M). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided ind irect evidence that dynorphin also stimulates kappa-opioid receptors and su ggests that kappa 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 op ioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, d ynorphinA potentially modulates secondary neurodegeneration in the spinal c ord through complex interactions involving multiple receptors and signaling pathways. (C) 1999 Academic Press.