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Muscarinic cholinergic receptor signal transduction as a potential target for the developmental neurotoxicity of ethanol
No full textCentral nervous system dysfunctions (most notably mental retardation and microcephaly) are among the most significant effects of in utero exposure to ethanol. Ethanol has been shown to cause alterations of both neuronal and glial cells, including cell loss, and changes in their migration and maturation. Here, we propose that one of the potential targets for the developmental neurotoxicity of ethanol may be represented by the signal transduction systems activated by cholinergic muscarinic receptors. Ethanol has been shown to inhibit second messenger systems activated by various G-protein-coupled receptors, including certain subtypes of muscarinic receptors. Although the roles of muscarinic receptors in brain development have not been fully elucidated, two potentially relevant effects have been discovered in the past few years. By activating muscarinic receptors coupled to phospholipid metabolism, acetylcholine can induce proliferation of glial cells, and act as a trophic factor in developing neurons by preventing apoptotic cell death. Ethanol has been shown to inhibit both actions of acetylcholine in vitro. These effects of ethanol may lead to a decreased number of glial cells and to a loss of neurons, which have been observed following in vivo alcohol exposure. In turn, these may be the basis of microencephaly and cognitive disturbances in children diagnosed with Fetal Alcohol Syndrome
Disruption of cholesterol homeostasis in the developing brain as a potential mechanism contributing to the developmental neurotoxicity of ethanol: an hypothesis
No full textWhile excess cholesterol may have deleterious consequences, as in the case of atherosclerosis, too little cholesterol may endanger the development of the brain. Different degrees of mental retardation are often observed in inborn errors of cholesterol synthesis, such as the Smith-Lemti-Opitz syndrome or in maternal phenylketonuria, where the metabolite of accumulating phenylalanine, phenylacetate, is an inhibitor of cholesterol synthesis. Lack of cholesterol during brain development as a consequence of these genetic defects leads to severe brain damage, microencephaly and mental retardation, which are also hallmarks of the fetal alcohol syndrome (FAS). The brain relies on the in situ synthesis of cholesterol, which occurs mostly in astrocytes. Astrocyte-produced cholesterol is utilized for cell proliferation, or is released, via astrocyte-secreted high density lipoprotein-like particles containing apolipoprotein E, outside the cell, where it is taken up and utilized by neurons for dendrite outgrowth and to form synapses. We propose the hypothesis that ethanol may disrupt cholesterol homeostasis during brain development, and that this effect may be responsible, at least in part, for the central nervous system dysfunctions observed in the FAS, which include altered astrocyte proliferation, neuronal death and diminished synaptic contact
Possible role of protein kinase C zeta in muscarinic receptor-induced proliferation of astrocytoma cells
No full textRecent studies have shown that protein kinase C zeta (PKC zeta) is part of a pathway that plays a key role in a wide range of physiological processes including mitogenesis, cell survival, and transcriptional regulation. Most studies on PKC zeta have been done by stimulating cells with tyrosine kinase receptor agonists, or by transfecting the cells with either constitutively active PKC zeta or negative mutants of PKC zeta. Less is known about the ability of endogenous G-protein-coupled receptors to generate a mitogenic signal through activation of endogenous PKC zeta. In the present paper, we showed that in 123-1N1 human astrocytoma cells, which express the G-protein-coupled M2, M3, and M5 muscarinic receptors, PKC zeta is activated by carbachol in a concentration-dependent manner, resulting in the translocation of PKC zeta from the cytoplasm to granules in the perinuclear region. The effect of carbachol was long-lasting (up to 24 hr) and appeared to be mediated by activation of M3 muscarinic receptors. A selective PKC zeta inhibitor peptide (peptide Z) inhibited PKC zeta translocation as well as carbachol-induced DNA synthesis. Inhibition of both phosphatidylinositol 3-kinase and phospholipase D decreased carbachol-induced [(3)H]thymidine incorporation and blocked carbachol-induced PKC zeta translocation, suggesting an involvement of both pathways in these effects
Cholesterol homeostasis in the developing brain: a possible new target for ethanol
No full textCholesterol is an essential component of cell membranes and plays an important role in signal transduction. This brief overview presents evidence from the literature that ethanol may affect cholesterol homeostasis and that, in the developing brain, this may be involved in its developmental neurotoxicity. The effects caused by inborn errors of cholesterol synthesis and by in utero ethanol exposure present several similarities in humans (eg, Smith-Lemli-Opitz syndrome and fetal alcohol syndrome), as well as in animal models. Ethanol has a cholesterol-reducing effect on the cardiovascular system, and a protective effect against Alzheimer's disease, whose pathogenesis has been linked to altered cholesterol homeostasis in the brain. In vitro, ethanol affects several functions that are mediated by cholesterol and important for brain development, such as glial cell proliferation, synaptogenesis, neuronal survival and neurite outgrowth. The brain contains high levels of cholesterol, mostly synthesized in situ. Astrocytes produce large amounts of cholesterol that can be released by these cells and utilized by neurons to form synapses. Ethanol up-regulates the cholesterol transporter ATP binding cassette A1 and cholesterol efflux from primary astrocyte cultures without affecting cholesterol synthesis
Muscarinic receptors, protein kinase C isozymes and proliferation of astroglial cells: effects of ethanol
No full textActivation of cholinergic muscarinic receptors (primarily the M3 subtype) causes proliferation of astroglial cells and this effect is inhibited by low concentrations (10-50 mM) of ethanol. Investigations on the signal transduction pathways activated by muscarinic receptors in a human astrocytoma cell line (1321N1) have focused on protein kinases C (PKC). Among PKC isozymes expressed in this cell line (alpha, epsilon, zeta), the atypical PKCzeta appears to play a primary role in the mitogenic action of muscarinic agonists. We investigated whether activation of these PKC isozymes may be affected by ethanol at concentrations that can inhibit muscarinic receptor-induced proliferation. Carbachol caused an increase in phorbol ester binding and translocation of PKCepsilon, however, these were inhibited only by 100-200 mM ethanol. On the other hand, translocation of the atypical PKCzeta to the perinuclear area by carbachol was inhibited by ethanol in a dose-dependent manner (10-100 mM). These results suggest that activation of PKCzeta may represent a relevant target for the inhibitory effect of ethanol on muscarinic receptor-induced glial cell proliferation
Activation of phosphatidylinositol 3 kinase by muscarinic receptors in astrocytoma cells
No full textStimulation of Gq-coupled acetylcholine muscarinic receptors leads to proliferation of astroglial cells, but the signal transduction pathway(s) that mediate this mitogenic response have not been fully elucidated. In this study, we report on the ability of carbachol to stimulate the phosphorylation of Akt/PKB, an important target of phosphatidylinositol 3 kinase (PI3 kinase) in 1321N1 human astrocytoma cells. Carbachol induced a dose-dependent phosphorylation of Ser473 on Akt, peaking after 15 min. This effect was mediated by activation of the M3 subtype of muscarinic receptors and was inhibited by two PI3 kinase inhibitors. Inhibitors of protein kinase C, mitogen-activated protein kinase and p70S6 kinase, had no effect on carbachol-induced Akt phosphorylation. Carbachol-induced DNA synthesis was strongly inhibited by two PI3 kinase inhibitors, wortmannin and LY294002, suggesting that PI3 kinase activation plays an important role in carbachol-induced proliferation 1321N1 astrocytoma cells
Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol
No full textAcetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3 cholinergic receptors. Ethanol was a potent inhibitor of carbachol-stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50 of 10 mM. On the other hand, basal and serum-stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50 = 200-250 mM). Concentration-response experiments with carbachol, in the presence of 10 mM ethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4-methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5-bromodeoxyuridine-Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 micrograms/ml insulin and 100 ng/ml platelet-derived growth factor BB; on the other hand, the mitogenic effect of 1 mM histamine, 100 U/ml interleukin-1, and 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate were inhibited by approximately 50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of the fetal alcohol syndrome
In vitro neurotoxicology: an introduction
No full textThis introductory Chapter provides a brief overview of the field of neurotoxicology and of the role played by in vitro approaches in investigations on mechanisms of neurotoxicity and of developmental neurotoxicity, and in providing suitable models for neurotoxicity screening
Assessment of cholesterol homeostasis in astrocytes and neurons
No full textCholesterol homeostasis is highly regulated in the nervous system; dysregulation in cholesterol trafficking and content have been involved in the pathogenesis of neurodegenerative diseases (such as Parkinson's and Alzheimer's diseases). Furthermore, low cholesterol levels during brain development are associated with neurodevelopmental deficits and mental retardation. The methods described in this chapter can be used to investigate the effect of neurotoxicants on cholesterol homeostasis. Astrocytes and neurons are two major cell types in the brain in which cholesterol synthesis and efflux are highly regulated to keep a proper cellular cholesterol level. Disruption in cholesterol synthesis and/or cholesterol efflux may result in cholesterol deficiency or accumulation in these cells leading to brain dysfunction
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