Ethanol-induced translocation of protein kinase A occurs in two phases: control by different molecular mechanisms

BACKGROUND: Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) regulates cellular functions. The specificity of PKA-mediated phosphorylation is determined primarily by PKA localization to sub-cellular sites. Chronic exposure to ethanol causes sustained translocation of the PKA catalytic subunit (C) from the Golgi to the nucleus in NG108-15 cells. Here we find that this is preceded by a transient short-term ethanol-induced translocation of PKA C. Different molecular mechanisms appear to underlie early and late phases of ethanol-induced translocation of PKA subunits. METHODS: The time course and localization of PKA C and regulatory (RII) subunits was assessed by immunocytochemistry in NG108-15 cells in the presence of ethanol, adenosine receptor (A2) blockade, and inhibitors of PKA activity and RNA and protein synthesis. RESULTS: Ethanol induces an early phase (<30 min) of C translocation to the cytoplasm and nucleus. This requires cAMP via adenosine A2 receptor activation. C then returns to the Golgi area after 60 min. A second phase of C translocation occurs during continuing exposure to ethanol (>12 hr). Re-accumulation of nuclear C no longer requires A2 or cAMP. RII also translocates to the nucleus during chronic treatment with ethanol. Both C and RII remain in the nucleus as long as ethanol is present. Unlike the early phase of ethanol induced translocation, the second phase of PKA subunit translocation requires protein and RNA synthesis. CONCLUSIONS: We identify two distinct phases of ethanol-induced PKA translocation which appear to be regulated by different molecular mechanisms. The first requires A2 signaling and cAMP; the later phase requires RNA and protein synthesis. The two phases of ethanol-induced PKA translocation observed in cell lines may contribute to changes in PKA signaling, cAMP-dependent gene expression, and the initiation and maintenance of sustained drinking behavior in experimental animals.     

Alcohol reduces the number of pheochromocytoma (PC12) cells in culture.

Pheochromocytoma (PC12) cells were used as an in vitro neuronal cell model to examine detrimental effects of alcohol on cell numbers. Alcohol exposure (100, 200, 400, and 800 mg/dl) reduced PC12 cell numbers in a dose-dependent manner. Cells that were treated with nerve growth factor (NGF) incurred less severe reductions in numbers compared with cells that were never treated with NGF. Because NGF stops proliferation of many of the PC12 cells and differentiates them into neuronal-like cells, these data suggest that differentiated, nonproliferating cells are less vulnerable to alcohol-induced reductions in cell numbers. In a subsequent experiment using only undifferentiated PC12 cells, alcohol reduced cell number of both proliferating and nonproliferating cultures; however, the reductions in proliferating cultures were more severe than in nonproliferating cultures. Two mechanisms may account for alcohol-induced reductions of PC12 cell numbers--inhibition of proliferation and killing of cells. PC12 cell cultures are a useful model system to examine mechanism(s) underlying alcohol's depletion of neuronal-like cells.     

NMDA Prevents Alcohol-Induced Neuronal Cell Death of Cerebellar Granule Cells in Culture

Neuronal cell loss is one of the most debilitating effects of alcohol exposure during development of the nervous system. In this study, primary cultures of neuronal cells (cerebellar granule cells) were wed to examine mechanisms of alcohol-induced neuronal cell death. Previously, we established that (Pantazis et al., Alcohol Clin Exp Re8 17:1014–1021,1993): (1) alcohol exposure caused neuronal cell death in cultures of cerebellar granule cells and this cell loss was both time-dependent and dose-dependent; and (2) the vulnerability of cerebellar granule cells to alcohol-induced loss changed with the length of time the cells were in culture before Initiating alcohol exposure—that is, younger cultures (1 day in vitro) were much more susceptible to alcohol-induced neuronal cell death than older cultures (4 or 7 days in vitro). The primary goal of the present study was to examine the potential role of the NMDA receptor in alcohol-induced death of cerebellar granule cells in culture. Experiments were performed to test the hypothesis that the alcohol-induced death of cerebellar granule cells can be prevented or reduced by NMDA treatment Our results Indicate that stimulation of the NMDA receptor has a neuroprotective effect and can significantly reduce the alcohol-induced neuronal cell death of newly established cerebellar granule cell cultures. This neuroprotective effect of NMDA is blocked by 2-amlno-5-phosphonovalerate, a come inhibitor of the NMDA receptor, Confirming that this neuroprotective effect is mediated via the NMDA receptor. This is the first report that alcohol's neurotoxic effect can be ameliorated by activation of the NMDA receptor.     

Melatonin inhibition of nicotine-stimulated dopamine release in PC12 cells

Melatonin, a pineal hormone, modifies numerous physiologic processes including circadian rhythms and sleep. In specific tissues, melatonin appears to have an inverse relationship with dopamine. To examine this relationship, a pheochromocytoma cell line (PC12) was used to determine the extent of melatonin's ability to inhibit nicotine-stimulated dopamine release. Multiple experiments were conducted that examined: (1). the dose response of acute melatonin (5 min); (2). the effects of chronic melatonin (16 h pre-exposure); (3). the effects of prior nicotine or melatonin exposure (5 min) on melatonin's ability to alter dopamine release from a second 5-min nicotine exposure; and (4). the role of melatonin receptors (by pertussis toxin inhibition) on nicotine-stimulated dopamine release. In the dose response studies, melatonin inhibited nicotine-stimulated dopamine release with an ED50 of 8.6 microM. Chronic exposure to melatonin had no effect on melatonin's acute inhibition of nicotine-stimulated dopamine release. Prior nicotine or melatonin exposure had little effect on subsequent melatonin or nicotine exposure, except that the cells exposed to nicotine were not responsive to a second exposure to nicotine. Blockade of melatonin receptor function by pre-exposure to pertussis toxin (16 h) did not prevent melatonin's inhibition of nicotine-stimulated dopamine release. However, the toxin-treated cells were less inhibited by melatonin when compared to control cells suggesting a partial role for melatonin receptors. These results indicate that melatonin can acutely inhibit nicotine-stimulated dopamine release in PC12 cells. This model system allows detailed examination of melatonin's cellular actions as well as supporting a role for melatonin on neuronal dopamine release.     

Vulnerability of Cerebellar Granule Cells to Alcohol-Induced Cell Death Diminishes with Time in Culture

This study examined the effects of alcohol exposure on the viability of cerebellar granule cells in culture. Continuous alcohol exposure, starting 1 day after the cultures were established, significantly reduced granule cell numbers, even with a single day of exposure to an alcohol concentration as low as 100 mg/dl. The depletion of cerebellar granule cells by alcohol was concentration-dependent (greater loss of cells at higher alcohol concentrations) and duration-dependent (greater loss of cells at longer exposure durations). The loss of granule cells also depended on the number of days the granule cells were in culture before alcohol exposure. Alcohol was significantly more effective in reducing the cell numbers of newly established granule cell cultures (1 day in vitro) compared with older cultures (4 or 7 days in vitro). Cell cycle analysis established that the cerebellar granule cells did not proliferate in culture, indicating that alcohol exposure did not reduce cell numbers by interfering with cell proliferation in this system. Instead, alcohol-induced killing of the granule cells was the most likely mechanism to account for the depletion of granule cells in vitro. Granule cell cultures are a useful in vitro model system to study the cellular and molecular aspects of neuronal cell depletion associated with fetal alcohol exposure. The potential role of the JV-methyl-D-aspartate receptor in this alcohol-induced neuronal cell death is discussed.