Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites.

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.

Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: differential impact of mu and delta receptor activation on proliferation and neurite elongation.

Although opioids are known to affect neurogenesis in vivo, it is uncertain the extent to which opioids directly or indirectly affect the proliferation, differentiation or death of neuronal precursors. To address these questions, the intrinsic role of the opioid system in neurogenesis was systematically explored in cerebellar external granular layer (EGL) neuronal precursors isolated from postnatal mice and maintained in vitro. Isolated neuronal precursors expressed proenkephalin-derived peptides, as well as specific mu and delta, but negligible kappa, opioid receptors. The developmental effects of opioids were highly selective. Morphine-induced mu receptor activation inhibited DNA synthesis, while a preferential delta2-receptor agonist ([D-Ala2]-deltorphin II) or Met-enkephalin, but not the delta1 agonist [D-Pen2, D-Pen5]-enkephalin, inhibited differentiation within the same neuronal population. If similar patterns occur in the developing cerebellum, spatiotemporal differences in endogenous mu and delta opioid ligand-receptor interactions may coordinate distinct aspects of granule neuron maturation. The data additionally suggest that perinatal exposure to opiate drugs of abuse directly interfere with cerebellar maturation by disrupting normal opioid signalling and inhibiting the proliferation of granule neuron precursors.

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 kappa-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 kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-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 kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-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 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). 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 kappa-opioid receptors and suggests 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 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.

Opioid-related changes in nociceptive threshold and in tissue levels of enkephalins after target disruption of the gene for neutral endopeptidase (EC 3.4.24.11) in mice.

Neutral endopeptidase EC 3.4.24.11 (NEP) is localized in peptidergic neurons and various colocalized peptides or other humoral mediators may serve as substrates. Target disruption of the NEP gene was reported to enhance the lethal response to endotoxin shock in mice. We examined thermonociceptive thresholds and enkephalin (ENK) tissue levels in transgenic NEP (-/-) and control wild type NEP (+/+) mice. Hot plate (52 degrees C) latency was 13.1 +/- 1.4 s in NEP (+/+) mice (n = 16) while latency increased significantly (P = 0.031) to 17.7 +/- 1.6 s in NEP (-/-) mice. Naloxone (10 mg/kg) had no effect on hot plate latency in NEP (+/+) mice (12.5 s, n = 8), but significantly decreased the latency in NEP (-/-) mice compared to untreated NEP (-/-) deficient mice (10.5 s, n = 8). Morphine (3 or 10 mg/kg) analgesic response was similar in knockout mice and wild type mice. Methionine-ENK (MET-ENK) and leucine-ENK (LEU-ENK) levels were determined in extracts from cortex, brain stem, hypothalamus, striatum, spinal cord, trigeminal ganglion and heart in treated and untreated mice. ENK-levels varied in a regionally-dependent manner and were significantly decreased in hypothalamus and spinal cord. We conclude that deletion of the NEP gene results in an opioid-related increase in thermonociceptive threshold. Regional differences in opioid metabolism indicate that NEP evokes tissue-specific patterns of ENK-regulation. NEP selectively controls opioid biosynthesis in hypothalamus and spinal cord presumably by feedback regulation.

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

The effects of morphine on the morphogenesis and survival of calbindin-D28k-immunoreactive Purkinje cells were studied in organotypic explant cultures isolated from 1- or 7-day-old mouse cerebella. To reduce experimental variability, bilaterally matched pairs of organotypic cultures were used to compare the effects of opiate drug treatment. One explant within each pair was untreated, while the remaining explant was continuously treated for 7 to 10 days with morphine, morphine plus naloxone, or naloxone alone. In explants derived from 1-day-old mice, morphine treatment significantly reduced Purkinje cell dendritic length compared to symmetrically matched untreated control explants. The concentration of morphine estimated to cause a half-maximal reduction (EC50) in dendritic length was 4.9 x 10(-8) M. At higher concentrations (EC50 = 3.6 x 10(-6) M), morphine also significantly decreased the number of Purkinje cells in explants from 1-day-old mice compared to untreated explants. Electron microscopy identified increased numbers of degenerating Purkinje cells in explants derived from 1-day-old mice. This showed that high concentrations (10(-5) M) of morphine reduced Purkinje cell numbers by decreasing their rate of survival. In explants derived from 7-day-old mice, morphine (10(-5) M) neither affected Purkinje cell dendritic length nor cell numbers compared to symmetrically matched untreated (control) explants. Collectively, these findings suggest that morphine per se, through a direct action on the cerebellum, can affect Purkinje cell differentiation and survival. The results additionally suggest that there is a critical period during development when Purkinje cells are especially vulnerable to the effects of morphine.

An extracellular enzyme from Fusarium solani f. sp. phaseoli which catalyses hydration of the isoflavonoid phytoalexin, phaseollidin.

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) are the prenylated isoflavonoids kievitone and phaseollidin. Two enzyme activities, kievitone hydratase and phaseollidin hydratase, occur in culture filtrates of the bean pathogen, Fusarium solani f. sp. phaseoli, and catalyse similar hydration reactions on the dimethylallyl moieties of the phytoalexins. The enzymes nearly co-purified during hydroxyapatite chromatography followed by preparative native gel electrophoresis. Eluates from successive slices taken from the native gel were assayed for both activities. Although they were not completely separated in the native gel, the activity profiles indicated that the two activities were distinct. The Km of phaseollidin hydratase for phaseollidin was approximately 7 microM.

Induction and purification of kievitone hydratase from Fusarium solani f. sp. phaseoli.

Fusarium solani f. sp. phaseoli is capable of detoxifying the major isoflavonoid phytoalexins produced by its host plant Phaseolus vulgaris. One of the enzymic activities involved is kievitone hydratase (KHase), a secreted glycoprotein which catalyses the conversion of kievitone to the less fungitoxic derivative, kievitone hydrate. Even under conditions of substrate induction, the enzyme is expressed at levels that are too low for satisfactory purification. Therefore, several other isoflavonoids were tested as inducers in culture. Among the phytoalexins produced by the host plant, phaseollinisoflavan was the best inducer, elevating the level of secreted enzyme eight-fold. Treatment with biochanin A, a product of chickpea, resulted in a 16-fold increase of secreted activity. The maximum rate of induction was observed 9-24 hr after addition of biochanin A, during which time several metabolites of the inducer were also present. KHase was purified from filtrates of biochanin A-induced cultures. Denaturing gel electrophoresis indicated that two species of Mr 47,000 and 49,000 copurified with the activity. N-Terminal sequence analysis indicated that the two species possessed related, or identical, polypeptide moieties. Comparison with the size of the non-denatured enzyme, previously determined to be ca 100,000, indicates that its native state is a dimer.