Forskolin Delays the Ethanol-Induced Desensitization of Hypothalamic β-Endorphin Neurons in Primary Cultures

Ethanol and its metabolite acetaldehyde have been shown to stimulate immunoreactive β-endorphin (IR-β-EP) secretion from hypothalamic neurons in primary cultures. Also, chronic ethanol and acetaldehyde have been shown to cause the development of tolerance and desensitization of these neurons. In this study, we determined some of the cellular events leading to desensitization of the function of β-endorphin (p-EP) Secretory neurons. The fetal hypothalamic cells were treated with various doses of ethanol (W and 50 mM) or acetaldehyde (6.25,12.5, and 25 mM) for 6 hr or treated with these drugs at 12 hr intervals for 72 hr. Determination of IR-β-EP concentrations in the media revealed that ethanol increased IR-β-EP secretion from these cultures for 12 hr; after this period, the cultured cells did not respond to ethanol. Acetaldehyde stimulated IR-β-EP secretion from this culture for a period of 48 hr, but the IR-β-EP secretory response to acetaldehyde reduced gradually with time during the first 48-hr period and reached the basal level at 72 hr. The desensitization of β-EP neurons 12 hr after treatment with alcohol did not seem to be related to the loss of viable cells, because chronic ethanol exposures did not produce any effect on cell viability. However, reduced IR- β-EP secretory response to acetaldehyde with time was associated with the time-dependent increase in cell death. Pretreatment of cultures with a cAMP analog, forskolin, increased the activity of functional β-EP neurons and delayed the ethanol desensitization effects on these neurons. Pretreatment of forskolin did not delay the acetaldehyde desensitization of β-EP neurons, but protected these cells from acetaldehyde toxicity. These results suggest that (i) chronic treatment with ethanol desensitizes β-EP-secreting neurons due to reduced cellular functions and (ii) chronic acetaldehyde reduces β-EP neurotransmission due to cell death. Furthermore, data suggest for the first time that cAMP pretreatments delay the ethanolinduced desensitization of opioid neurons and partly protect against the neurotoxic action of acetaldehyde on opioid neurons.     

Effect of Ethanol, Propanol, Butanol, and Catalase Enzyme Blockers on β-Endorphin Secretion from Primary Cultures of Hypothalamic Neurons: Evidence for a Mediatory Role of Acetaldehyde in Ethanol Stimulation of β-Endorphin Release

Previously, we have shown that low closes of ethanol (12.5–100 mm ) and acetaldehyde (12.5–50 μm ), but not salsolinol, enhanced immunoreactive β-endorphin (IR-β-EP) secretion from fetal hypothalamic neurons in primary culture. In this study, the effects of ethanol, propanol, and butanol, as well as the effect of catalase inhibitors on IR-β-EP secretion were studied in vitro to determine the role of membrane fluidization and ethanol metabolism on ethanol-induced IR-β-EP secretion. The primary cultures of fetal hypothalamic neurons were maintained for 8–9 days in chemically defined medium and treated for 5 hr with ethanol (50 mm ), propanol (25 and 50 mm ), and butanol (25 and 50 mm ). Determination of hourly secretion of IR-β-EP from the cultures revealed that only 50 mm ethanol caused stimulation of IR-β-EP secretion, whereas propanol and butanol did not alter IR-β-EP response at any given concentration. Pretreatment of these cultures with the catalase inhibitors, 3-amino-1,2,4 -triazole (3-AT; 1, 5, and 10 mm ), caused a dose-dependent inhibition of ethanol-stim-ulated IR-β-EP secretion, but did not inhibit dibutyryl cAMP (dcAMP)-stimulated IR-β-EP secretion. Another catalase inhibitor, sodium azide (5 mm ), also inhibited ethanol-stimulated IR-β-EP secretion. Measurement of acetaldehyde production in cultured cells and media after ethanol or dcAMP treatments revealed that cultured cells produce acetaldehyde only after ethanol treatment and at levels of acetaldehyde (8–24 μm ) that are known to evoke IR-β-EP release. The catalase inhibitor 3-AT (10 mm ) treatment reduced ethanol-evoked acetaldehyde production. These results suggest for the first time that the catalase-H₂O₂ system is functional in the cultured hypothalamic neurons and is involved in mediation of ethanol's effects on IR-β-EP secretion via conversion of alcohol to acetaldehyde.     

Effect of Alcohol, Acetaldehyde, and Salsolinol on β-Endorphin Secretion from the Hypothalamic Neurons in Primary Cultures

The effect of ethanol, acetaldehyde, and salsolinol on hypothalamic β -endorphin secreting neurons is studied by using rat fetal hypothalamic neurons in primary culture. Exposure of these neuronal cells to different concentrations of ethanol (12.5–50 mM) and acetaldehyde (12.5-50 μm) caused a concentration-dependent increase in the secretion of β -endorphin. Salsolinol (12.5–50 μm) did not cause any significant change in the secretion of β -endorphin. Ethanol's effect was short-lasting (2 hr). Acetaldehyde's effect on β -endorphin secretion was greater and longer lasting, as compared with ethanol. Ethanol and salsolinol do not have any effect on cell viability, whereas higher concentrations of acetaldehyde appear to reduce the number of viable cells after 6 hr of treatment. None of the above treatments has any effect on cellular DNA content. These results suggest that ethanol is a potent stimulator of hypothalamic β -endorphin. These results also show for the first time that ethanol's metabolite acetaldehyde is more potent in stimulating β -endorphin secretion and may be significant in the ethanol regulated β -endorphin secretion.     

Characterization of the Production of Acetaldehyde by Astrocytes in Culture after Ethanol Exposure

The nervous system is one of the main targets of ethanol toxicity. Astrocytes might play an important role in ethanol-induced brain toxicity, because their integrity is essential for the normal growth and functioning of neurons. On the other hand, acetaldehyde has been implicated as a mediator in some of the biochemical, pharmacological, and behavioral effects of ethanol. The present study aimed at demonstrating the ability of astrocytes in culture to produce acetaldehyde from ethanol. Significant metabolization of ethanol with production of acetaldehyde was demonstrated in the primary culture of astrocytes. This production was quite low, compared with that usually observed in hepatocytes, but was in the same range as that measured in whole brain homogenates and corresponded to biologically active levels. Such a demonstration could bring new elements for understanding of ethanol neurotoxicity.     

srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1

Objective:To explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons.Methods:In this study,expression of Slit1 was observed predominantly in the glia.while expression of Robo2 and srCAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion(DRG).Furthermore,upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting.Results:It was observed that inhibition of nenrite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively(1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody.It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-CTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3.These data demonstrated a role for srGAP3 in nenrite outgrowth ol DRG sensory neurons.Conclusions:Our observations suggest that srGAP3 promotes neurile outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.     

Acetaldehyde Exposure Causes Growth Inhibition in a Chinese Hamster Ovary Cell Line That Expresses Alcohol Dehydrogenase

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.     

Acetalde hyde-Induced Stimulation of Collagen Synthesis and Gene Expression Is Dependent on Conditions of Cell Culture: Studies with Rat Lipocytes and Fibroblasts

Acetaldehyde has been proposed as a mediator of fibrogenesis in alcoholic liver disease, based in part on its ability to stimulate collagen synthesis by hepatic lipocytes in late primary or passaged culture. In this study, we examined the effect of acetaldehyde on rat lipocytes and fibroblasts at various stages of culture, in an effort to determine whether culture-related events influence responsiveness to this compound. Lipocytes from normal rat liver were studied in primary culture at 3 and 7 days after plating; fibroblasts were studied in subculture, at subconfluent and confluent densities. Both cell types were incubated with 100 μM acetaldehyde for 24 hr followed by measurement of collagen synthesis and type I collagen gene expression. Acetaldehyde had no effect on lipocytes at either 3 or 7 days in primary culture. The inability of acetaldehyde to stimulate collagen synthesis in primary culture was not attributable to toxicity, because cell morphology and total protein synthesis were identical in both treated and untreated cultures. Fibroblasts exhibited a variable response to acetaldehyde that was dependent on cell density: subcon-fluent cells contained similar amounts of type I collagen mRNA in both the presence and absence of acetaldehyde, whereas confluent cells exhibited a 2- to 3-fold increase in collagen mRNA levels upon acetaldehyde exposure. To determine whether quiescent lipocytes would respond to acetaldehyde in a culture system that mimics the hepatic environment in vivo, lipocytes were plated in coculture with hepatocytes on a basement membrane gel and incubated with 20 mM ethanol for 72 hr. Direct communication between these two cell types did not provoke lipocyte activation, even in the setting of ethanol oxidation. We conclude from these experiments that acetaldehyde is not a primary stimulus to lipocyte activation in vivo. Acetaldehyde may enhance collagen synthesis by lipocytes, but its activity appears restricted to cells that have undergone a prior priming event in culture or in vivo. Of the many phenotypic changes that occur in lipocytes during the first week of primary culture, none sensitizes them to the fibrogenic effects of acetaldehyde.     

Neurotrophin 3 supports the survival of developing muscle sensory neurons in culture.

Target-dependent cell death of different sub-populations of sensory neurons may be regulated by different trophic factors. To investigate this possibility, we have taken advantage of the fact that the fractions of muscle sensory and cutaneous sensory neurons in chicken dorsal root ganglia (DRG) are probably different at different segmental levels, and we have compared the responses of chicken DRG from levels that do and do not innervate limb tissue to various growth factors in vitro. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) both supported neurite outgrowth from DRG explanted from all segmental levels. In contrast, neurotrophin 3 (NT-3) supported robust neurite growth only from DRG explanted from the cervical or lumbar levels, which innervate limb muscles. Similarly, NGF and BDNF both promoted survival of dissociated neurons from limb and nonlimb segmental levels, whereas NT-3 promoted survival of more neurons from limb compared to nonlimb levels. This suggests that muscle sensory neurons, which are probably more prevalent at the cervical and lumbar levels, may be specifically affected by NT-3. To evaluate this possibility directly, we compared the survival of retrogradely labeled muscle and cutaneous neurons in NGF, BDNF, and NT-3. Identified muscle sensory neurons survived best in vitro in the presence of NT-3, while the survival of identified cutaneous sensory neurons was greatest in NGF. This work provides direct evidence for a potential role of NT-3 versus NGF in the survival of a specific subpopulation of DRG neurons.     

Acute and chronic exposure to ethanol and the electrophysiology of the brush border membrane of rat small intestine.

In this study we have investigated the effects of (a) chronic ethanol intake on glucose and galactose absorption across the rat jejunum in vivo and on the potential difference across the isolated brush border membrane (Vm) and (b) acute exposure to ethanol (4% or 8%) and acetaldehyde (0.25%) on changes in Vm associated with Na(+)-dependent galactose absorption across the jejunum and ileum. Chronic ethanol intake was associated with hyperpolarization of Vm and an enhanced galactose but not glucose transport. Acute ethanol and acetaldehyde were without effect on Vm whether or not galactose was present. We conclude that while a greater electrochemical gradient across the brush border membrane is a likely explanation for the stimulation of galactose absorption induced by ethanol feeding, factors other than changes in Vm are responsible for the inhibitory effects of acute ethanol.     

Inhibition of Testosterone Production by Rat Leydig Cells with Ethanol and Acetaldehyde: Prevention of Ethanol Toxicity with 4-Methylpyrazole

The toxic effects of ethanol and acetaldehyde on testosterone biosynthesis were examined in vitro using isolated Leydig cells prepared from adult rat testes. The ability of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, to prevent the toxic effects of ethanol on testosterone production was investigated. Ethanol was found to inhibit gonadotropin-stimulated testosterone production in a dose dependent fashion. Concentrations of ethanol (25 mg/100 reduce testosterone levels by 44% as compared to the controls. Acetaldehyde at micromolar concentrations also inhibited testosterone biosynthesis. The addition of 4-methylpyrazole to the culture medium prevented the toxic effects of ethanol as determined by testosterone production. These studies suggest that ethanol per se may not directly inhibit testosterone biosynthesis. Rather, it would appear that acetaldehyde, the first product of ethanol metabolism, may be responsible for the toxic effects of ethanol upon Leydig cells at least in vitro.     

Cultured adult animal neurons and schwann cells give us new insights into diabetic neuropathy

Since we introduced cultured dorsal root ganglia (DRG) neurons from streptozotocin (STZ)-induced diabetic mice as "an in vitro model to study diabetic neuropathy" (Sotelo et al., 1991), more than 30 papers have been devoted to the study of diabetic neuropathy with culture systems of neurons and Schwann cells derived from adult animals. So far, methods for dissociated cell culture of peripheral neurons (mainly DRG neurons) and Schwann cells, and for explant culture of peripheral ganglia and retinas have been applied to diabetic animals or patients. In addition to these diabetic cells, adult animal neurons and Schwann cells cultured under high glucose conditions and adult animal neurons exposed to diabetic serum have been utilized. The findings from these culture models clearly show that the exposure of mature neurons and Schwann cells to hyperglycemic conditions in vivo or in vitro can alter their biophysical and biochemical properties (e.g., cell viability, neurite outgrowth activity, polyol metabolism and electrophysiological features). Therefore, the cultured neurons and Schwann cells can be useful tools for investigating the precise mechanisms leading to diabetic neuropathy and the efficacy of therapeutic agents for the prevention and treatment of that condition.     

Acetaldehyde Causes a Prolongation of the Doubling Time and an Increase in the Modal Volume of Cells in Culture

The effects of culturing four human cell lines--Raji, MOLT-4, WI-L2, and K562--in the presence of 10-360 microM acetaldehyde for 3-18 days have been investigated. Concentrations of 45-360 microM caused a prolongation of the cell doubling time, and those of 90-360 microM caused an increase in the modal cell volume and in the protein content per cell. The results indicate that relatively low concentrations of acetaldehyde cause an impairment of cell proliferation and an abnormality of cell growth in vitro and support the possibility that ethanol-derived acetaldehyde may be responsible for some aspects of tissue damage in chronic alcoholics, including the increase in the mean cell volume of erythrocytes. Three of the four cell lines studied showed a reduction and the fourth showed no change in modal cell volume after culture with 100 mM ethanol, suggesting that the macrocytosis of red cells induced by chronic alcoholism is not caused via some direct effect of ethanol on the erythron.     

Ethanol withdrawal influences survival and morphology of developing rat hippocampal neurons in vitro

BACKGROUND: Previous studies in this laboratory have shown that, like their counterparts in vivo, fetal rat hippocampal pyramidal neurons in culture develop abnormally small dendritic arbors when exposed to ethanol. This study asked whether ethanol's inhibitory effects on dendritic development differ when the duration of ethanol exposure and timing of withdrawal are varied to correspond with early versus later stages of development and whether ethanol withdrawal influences survival of these neurons. METHODS: We compared neurons exposed continuously for 6 or 14 days to ethanol (70 mM) with neurons transferred from ethanol-containing medium to control medium either 1 day after adding ethanol (before dendrites elongated) or 6 days after adding ethanol (after dendrites began elongating). We then performed morphometric and cell density analyses at 6 and 14 days using digital images of neurons immunostained with microtubule-associated protein 2 (MAP2) to visualize dendrites. RESULTS: Continuous exposure to ethanol decreased the length and number of dendrites formed but had no effect on neuron survival compared with controls without ethanol. Dendritic length was less inhibited when ethanol was withdrawn after 1 day, but the number of dendrites per cell was unchanged compared with neurons continuously exposed to ethanol. Withdrawal from ethanol at 1 day slightly enhanced the survival of neurons assessed at 14 days compared with neurons in control medium and with neurons exposed continuously to ethanol. In contrast, withdrawal from ethanol at 6 days severely decreased the number of neurons at 14 days. CONCLUSIONS: These results suggest that dendrites can achieve normal length when ethanol exposure is limited to only 1 day and withdrawal occurs before dendrites begin elongating. However, a persistent reduction in dendrite number results in smaller overall dendritic arbor size. Although continuous exposure to ethanol has little effect on neuron survival in these cultures, and exposure limited to 1 day followed by withdrawal can be neuroprotective against cell death associated with increased time in culture, longer exposure before withdrawal can trigger cell death.     

Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion

Estrogen is known to influence different functions in brain tissue ranging from neuronal development to plasticity and survival, but the mechanisms involved have not been defined clearly. Previous studies have shown the presence of the two estrogen receptors (ERs), ERalpha and ERbeta, in several brain areas, but less is known about the role of estrogen in the peripheral nervous system. Here we demonstrate that dorsal root ganglion (DRG) neurons express ERalpha and ERbeta during early postnatal development and in culture, and that the ERs localize mainly to neuronal cell nuclei. Studying the role of estrogen in DRG, we observed that low concentrations of 17beta-estradiol increased survival of cultured DRG neurons deprived of nerve growth factor. 17beta-Estradiol up-regulated the expression of the antiapoptotic molecule Bcl-x without affecting that of Bax, suggesting a mechanism by which the hormone counteracted neuronal death. Antiestrogens abolished the action of 17beta-estradiol in the DRG neurons, which demonstrates an involvement of ERs. The results show that estrogen and ERs play an important role in the development and survival of DRG neurons.     

Ethanol Inhibits Muscarinic Receptor-Induced Axonal Growth in Rat Hippocampal Neurons

Background: In utero alcohol exposure can lead to fetal alcohol spectrum (FAS) disorders characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. One mechanism through which ethanol has been shown to exert its effects is the perturbation of activated signaling cascades. The cholinergic agonist carbachol has been shown to induce axonal outgrowth through intracellular calcium mobilization, protein kinase C (PKC) activation, and ERK1/2 phosphorylation. This study investigated the effect of ethanol on the differentiation of rat hippocampal pyramidal neurons induced by carbachol as a possible mechanism involved in the developmental neurotoxicity of ethanol. Methods: Prenatal rat hippocampal pyramidal neurons were treated with ethanol (50 to 75 mM) in the presence or absence of carbachol for 24 hours. Neurite outgrowth was assessed spectrophotometrically; axonal length was measured in neurons fixed and immunolabeled with the neuron‐specific βIII tubulin antibody; cytotoxicity was analyzed using the thiazolyl blue tetrazolium bromide assay. The effect of ethanol on carbachol‐stimulated intracellular calcium mobilization was assessed utilizing the fluorescent calcium probe, Fluo‐3AM. The PepTag® assay for nonradioactive detection of PKC from Promega was used to measure PKC activity, and ERK1/2 activation was determined by densitometric analysis of Western blots probed for phospo‐ERK1/2. Results: Ethanol treatment (50 to 75 mM) caused an inhibition of carbachol‐induced axonal growth, without affecting neuronal viability. Neuron treatment for 15 minutes with ethanol did not inhibit the carbachol‐stimulated rise in intracellular calcium, while inhibiting PKC activity at the highest tested concentration and ERK1/2 phosphorylation at both the concentrations used in this study. On the other hand, neuron treatment for 24 hours with ethanol significantly inhibited carbachol‐induced increase in intracellular calcium. Conclusions: Ethanol inhibited carbachol‐induced neurite outgrowth by inhibiting PKC and ERK1/2 activation. These effects may be, in part, responsible for some of the cognitive deficits associated with in utero alcohol exposure.     

An effect of nerve growth factor on parasympathetic neurite outgrowth.

Addition of nerve growth factor (NGF) to dissociated parasympathetic ciliary ganglion neurons resulted, within 60 min of its addition, in a 2-fold increase in average neurite length and an accompanying enlargement and spreading of neuronal growth cones. These effects occurred over a concentration range of NGF of 0.1-10 ng/ml and were blocked by affinity-purified antibody to NGF. Epidermal growth factor, fibroblast growth factor, and angiotensin did not have these effects, although insulin at high concentrations was able to induce a response similar to that of NGF. Dissociated sympathetic chain neurons also responded to NGF with increased neurite lengths, and, in addition, NGF considerably extended the survival time of these neurons in culture. However, the effect of NGF on ciliary ganglion neurons was limited to neurite outgrowth, and NGF did not promote the survival of these parasympathetic neurons.     

Role of the GTP-Binding Protein Go in the Suppressant Effect of Ethanol on Voltage-Activated Calcium Channels of Murine Sensory Neurons

Whole-cell and single-channel recording techniques were used to investigate the acute, in vitro effects of ethanol on the function of voltage-activated Ca²⁺ channels in cultured neurons derived from dorsal root ganglia (DRG) of embryonic mice. Although 5.4 MM ethanol produced a sustained increase of the amplitude of the whole-cell Ca²⁺ current (I_Ca), 43.2 mM ethanol had a time-dependent biphasic effect. That is, within 0.5 min of exposure to 43.2 MM ethanol, the maximal amplitude of I_Ca initially increased before declining to a new steady-state value. As anticipated, the facilitatory and inhibitory effects of ethanol on I_Ca were associated with an increase and decrease, respectively, in the probability of single-channel open events. Pretreatment of DRG with 200 ng/ml of pertussis toxin abolished the inhibitory, but not the facilitatory, effect of 43.2 mM ethanol on I_Ca Pretreatment with pertussis toxin also prevented the reduction of the probability of single-channel opening caused by 43.2 mM ethanol. Similarly, dialysis of neurons with polyclonal antibodies against the a-subunit of Go but not G., abolished the inhibitory effect of 43.2 mM ethanol on I_Ca These data demonstrate concentration- and time-dependent biphasic effects of ethanol on the activity of Ca²⁺ channels. The inhibitory effect of ethanol requires activation of the a-subunit of Go, which then decreases the probability of Ca²⁺ channel opening.     

Early Events in the Development of Neuronal Polarity In Vitro Are Altered by Ethanol

Among the neuropathological effects of prenatal exposure to ethanol is the disruption of neuromorphogenesis. The effects of ethanol on early events in the development of axons and dendrites were studied using cultured embryonic rat hippocampal neurons, which develop in vitro in a stereotypical sequence of events that mimics their development in vivo. During the first 24 hr in culture, hippocampal neurons attach to the substrate and develop into one of three stages identified by phase-contrast microscopy: (i) neurons having lamellipodia and no processes (stage 1); (ii) neurons developing minor processes (<40 μm) that subsequently become the cell's axon or dendrites (stage 2); or (iii) polarized neurons with at least one axon (process with length ≥40 μm) (stage 3). Exposure to ethanol (300 mg/dl or 800 mg/dl) in the culture medium resulted in an increase in both the number of minor processes per neuron and the number of stage 3 neurons having more than the typical single axon. In addition, ethanol exposure significantly altered the proportion of neurons in the three early stages of development at 18 to 24 hr in vitro, without affecting overall neuron survival. With ethanol, there was a smaller proportion of neurons in the first stage of development, and a greater proportion of polarized stage 3 neurons. These findings suggest that ethanol alters the normal establishment of neuronal polarity, disrupting mechanisms that ensure the formation of the appropriate number of processes and that regulate the timing of process outgrowth.     

Estrogen elicits dorsal root ganglion axon sprouting via a renin-angiotensin system

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-alpha agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-beta agonist diarylpropionitrile was ineffective. 17beta-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.     

Evaluation of the Role of Acetaldehyde in the Actions of Ethanol on Gluconeogenesis by Comparison with the Effects of Crotonol and Crotonaldehyde

Liver cells from fasted rats oxidized ethanol and crotonol at identical rates. Ethanol and crotonol increased the cytosolic lactate/pyruvate ratio to the same extent, however only ethanol increased the mitochondrial B-hydroxybutyrate/acetoacetate ratio. The rate of oxidation of crotonaldehyde by liver cells was 30% to 50% of the rate of oxidation of acetaldehyde. Cyanam-ide, which is especially inhibitory towards the low Km mitochondrial aldehyde dehydrogenase, inhibited the oxidation of acetaldehyde to a greater extent than it inhibited the oxidation of crotonaldehyde. Acetaldehyde, but not crotonaldehyde, increased the B-hydrox-ybutyrate/acetoacetate ratio; both aldehydes produced some increase in the lactate/pyruvate ratio. Consequently, differences between the effects of ethanol and crotonol on the mitochondrial redox state may relate to differences between the metabolism of their respective aldehydes. Intact mitochondria oxidized crotonaldehyde at about 10% the rate found with acetaldehyde. In deox-ychoiate-disrupted mitochondria, in the presence of external NAD+, crotonaldehyde was oxidized at rates less than one-fourth the rate found with acetaldehyde. Cyanamide inhibited the oxidation of acetaldehyde to a greater extent than the oxidation of crotonaldehyde. These results suggest that crotonaldehyde is a poor substrate for the low Km mitochondrial aldehyde dehydrogenase. In isolated hepatocytes, acetaldehyde stimulated glucose production from pyruvate, but inhibited gluconeogenesis from glycerol, xylitol, and sorbitol. Crotonaldehyde had no effect on glucose production from these substrates, indicating that the effects of acetaldehyde were due to the metabolism of acetaldehyde in the mitochondria. Ethanol stimulated glucose production from pyruvate, whereas crotonol was without effect. The stimulation by ethanol, and the lack of effect by crotonol, appears to be due to changes in the mitochondrial redox state produced as a consequence of the further oxidation of acetaldehyde, but not crotonaldehyde. Crotonol and ethanol inhibited glucose production from glycerol, xylitol, and sorbitol, suggesting that changes in the cytosolic redox state play the major role in the effect of the alcohols on glucose production from these substrates. However, the inhibition by ethanol was consistently 10% to 15% greater than the inhibition by crotonol, suggesting that acetaldehyde contributes to the effects of ethanol. These differences between ethanol and crotonol are consistent with a role for acetaldehyde metabolism in the actions of ethanol. Crotonol may be a useful aid in determining the mechanism whereby ethanol alters henatic functions.