Cyclic AMP protects against staurosporine and wortmannin-induced apoptosis and opioid-enhanced apoptosis in both embryonic and immortalized (F-11κ7) neurons

Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 05/1998; 70(4):1376-82.
Source: PubMed


The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11kappa7 cell line (an immortalized mouse neuroblastoma x dorsal root ganglion hybrid stably transfected to overexpress kappa-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the mu-specific opioid agonist morphiceptin (E7CH) or the kappa-specific opioid agonist U69,593 (F-11kappa7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with non-pretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16-24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp-adenosine 3',5'-cyclic monophosphothioate and increased killing with the antagonist Rp-adenosine 3',5'-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.

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    • "A large number of in vitro studies with neuronal and glial primary cultures as well as with different cell lines have shown that morphine and other related opioids can inhibit cell proliferation and induce cell death, either by apoptosis or necrosis (Dawson et al., 1997; Goswami et al., 1998; He et al., 2011; Hu et al., 2002; Kugawa et al., 1998; Lazarczyk et al., 2010; Oliveira et al., 2002, 2003; Shoae-Hassani et al., 2011; Stiene-Martin and Hauser, 1993; Stiene-Martin et al., 1991; Yin et al., 1999; Zheng et al., 2010). In addition, there is evidence showing opioid-induced autophagy and cell death mediated by beclin-1 and ATG5 (Zhao et al., 2010). "
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    ABSTRACT: Short-term incubation with pharmacologically relevant concentrations of morphine has been shown to transiently affect the metabolism and redox status of NG108-15 cells through δ-opioid receptor stimulation, but apparently did not provoke cell death. The present work tries to determine if incubation with morphine at longer time intervals (24 h) provokes apoptosis and/or necrosis, as it has been described in other cell lines. We have also checked the potential modulatory role of yohimbine on these effects, on the basis of the previously described interactions between this drug and opioid receptor ligands. Incubation with morphine 0.1 and 10 μM provoked the appearance of images compatible with apoptosis (bebbling, pyknotic cells with cytoplasmic and nuclear condensation) and necrosis (cells swollen with vacuolated cytoplasm lacking cell processes) that could be observed directly and/or after staining with methylene blue, crystal violet and propidium iodide/4',6-diamidino-2-phenylindole (IP/DAPI). Quantification of apoptosis by activation of caspases 3 and 7 and DNA fragmentation with the Tunel assay revealed a modest but significant increase after incubation with the two concentrations of morphine used. Co-incubation with 10 μM yohimbine prevented all these effects of the opioid. The results extend previous findings of a yohimbine-sensitive, neurotoxic effect of morphine on NG108-15 cells. Copyright © 2012 John Wiley & Sons, Ltd.
    Journal of Applied Toxicology 03/2014; 34(1). DOI:10.1002/jat.2817 · 2.98 Impact Factor
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    • "Apoptosis, a genetically determined mechanism of programmed cell death triggered by a variety of internal and external stimuli, is also a pathologic feature in certain inflammatory diseases of the brain and CNS infections, such as HAD [58]. Opiates have been shown to induce apoptosis of all types of cells including lymphocytes [6], [7], [8], [59], [60], [61], [62] through the activation of caspase-3. These findings are important, considering that inhibitors of caspase-3 activity can rescue neurons from the apoptotic cycle. "
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    ABSTRACT: Parenteral use of drugs; such as opiates exert immunomodulatory effects and serve as a cofactor in the progression of HIV-1 infection, thereby potentiating HIV related neurotoxicity ultimately leading to progression of NeuroAIDS. Morphine exposure is known to induce apoptosis, down regulate cAMP response element-binding (CREB) expression and decrease in dendritic branching and spine density in cultured cells. Use of neuroprotective agent; brain derived neurotropic factor (BDNF), which protects neurons against these effects, could be of therapeutic benefit in the treatment of opiate addiction. Previous studies have shown that BDNF was not transported through the blood brain barrier (BBB) in-vivo.; and hence it is not effective in-vivo. Therefore development of a drug delivery system that can cross BBB may have significant therapeutic advantage. In the present study, we hypothesized that magnetically guided nanocarrier may provide a viable approach for targeting BDNF across the BBB. We developed a magnetic nanoparticle (MNP) based carrier bound to BDNF and evaluated its efficacy and ability to transmigrate across the BBB using an in-vitro BBB model. The end point determinations of BDNF that crossed BBB were apoptosis, CREB expression and dendritic spine density measurement. We found that transmigrated BDNF was effective in suppressing the morphine induced apoptosis, inducing CREB expression and restoring the spine density. Our results suggest that the developed nanocarrier will provide a potential therapeutic approach to treat opiate addiction, protect neurotoxicity and synaptic density degeneration.
    PLoS ONE 04/2013; 8(4). DOI:10.1371/journal.pone.0062241 · 3.23 Impact Factor
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    • "Disruption of physiological apoptotic cell death during development leads to brain malformations and premature death in rodent models (Kuida et al., 1996). Morphine has been shown to induce apoptotic cell death in vitro studies (Goswami et al., 1998; Singhal et al., 1998, 1999, 2000, 2002; Tegeder et al., 2003; Yin et al., 2000). In contrast to the abundance of in vitro studies , only a small number of studies reported potential neurotoxic effect of opioids in vivo (Emeterio et al., 2006; Mao et al., 2002). "
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    ABSTRACT: Prolonged neonatal opioid exposure has been associated with: antinociceptive tolerance, long-term neurodevelopmental delay, cognitive, and motor impairment. Morphine has also been shown to induce apoptotic cell death in vitro studies, but its in vivo effect in developing rat brain is unknown. Thus, we hypothesized that prolongued morphine administration in neonatal rats in a model of antinociceptive tolerance and dependence is associated with increased neuroapoptosis. We analyzed neonatal rats from the following groups (1) naïve group (n=6); (2) control group (normal saline (NS), n=5), and (3) morphine group (n=8). Morphine sulfate or equal volume of NS was injected subcutaneously twice daily for 6.5 days starting on postnatal day (PD) 1. Development of antinociceptive tolerance was confirmed by Hot Plate test on the 7th day. Evidence of neuronal and glial apoptosis was determined by cleaved caspase-3 immunofluorescence combined with specific markers. At PD7, morphine administration after 6½ days significantly increased the density of apoptotic cells in the cortex and amygdala, but not in the hippocampus, hypothalamus, or periaqueductal gray. Apoptotic cells exhibited morphology analogous to neurons. Irrespective of the treatment, only a very few individual microglia but not astrocytes were caspase-3 positive. In summary, repeated morphine administration in neonatal rats (PD1-7) is associated with increased supraspinal apoptosis in distinct anatomical regions known to be important for sensory (cortex) and emotional memory processing (amygdala). Brain regions important for learning (hippocampus), and autonomic and nociceptive processing (hypothalamus and periaqueductal gray) were not affected. Lack of widespread glial apoptosis or robust glial activation following repeated morphine administration suggests that glia might not be affected by chronic morphine at this early age. Future studies should investigate long-term behavioral sequelae of demonstrated enhanced apoptosis associated with prolonged morphine administration in a neonatal rat model.
    International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 03/2013; 31(4). DOI:10.1016/j.ijdevneu.2013.02.009 · 2.58 Impact Factor
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