Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.     

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.     

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

The effect of myo-inositol on ethanol-induced metabolic changes and insulin concentration in the rat

Myo-inositol was found to possess several beneficial effects on the organism. The effect of myo-inositol on ethanol-induced metabolic changes and insulin concentration was investigated in growing rats. The increase in liver triglycerides induced by ethanol drinking (10% ethanol solution as the only drinking fluid for 10 days) was completely abolished by simultaneous treatment with myo-inositol (0. 1 g/100 g b.w., every day given intragastrically). The ethanol-evoked decrease in blood insulin and the increase in liver glycogen were also partially prevented by myo-inositol. Myo-inositol did not cause any undesirable metabolic changes in the rats. The results indicate that myo-inositol may be useful in the treatment of some metabolic consequences of alcohol drinking.     

Prostaglandin E2 protects gastric mucosal cells from apoptosis via EP2 and EP4 receptor activation

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.     

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.     

Abnormal myo-inositol and phospholipid metabolism in cultured fibroblasts from patients with ataxia telangiectasia

Ataxia telangiectasia (AT) is a complex autosomal recessive disorder that has been associated with a wide range of physiological defects including an increased sensitivity to ionizing radiation and abnormal checkpoints in the cell cycle. The mutated gene product, ATM, has a domain possessing homology to phosphatidylinositol-3-kinase and has been shown to possess protein kinase activity. In this study, we have investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. In six fibroblast lines from patients with AT, myo-inositol accumulation over a 3-h period was decreased compared to normal fibroblasts. The uptake and incorporation of myo-inositol into phosphoinositides over a 24-h period, as well as the free myo-inositol content was also lower in some but not all of the AT fibroblast lines. A consistent finding was that the proportion of 32P in total labeled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Turnover studies revealed that AT cells exhibit a less active phospholipid metabolism as compared to normal cells. In summary, these studies demonstrate that two manifestations of the AT defect are alterations in myo-inositol metabolism and phospholipid synthesis. These abnormalities could have an effect on cellular signaling pathways and membrane production, as well as on the sensitivity of the cells to ionizing radiation and proliferative responses.     

Modulation of ethanol effect on hepatocyte proliferation by polyamines

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.     

Ethanol modulates the growth of human breast cancer cells in vitro

The role of ethanol or its metabolites on breast neoplasm has not been characterized. We hypothesized that ethanol may alter the growth rate of human breast tumor epithelial cells by modulating putative growth-promoting signaling pathways such as p44/42 mitogen-activated protein kinases (MAPKs). The MCF-7 cell line, considered a suitable model, was used in these studies to investigate the effects of ethanol on [(3)H]thymidine incorporation, cell number, and p44/42 MAPK activities in the presence or absence of a MAPK or extracellular signal-regulated kinase ERK-1, and (MEK1) inhibitor (PD098059). Treatment of MCF-7 cells with a physiologically relevant concentration of ethanol (0.3% or 65 mM) increased p44/42 activities by an average of 400% (P < 0.02), and subsequent cell growth by 200% (P < 0.05) in a MEK1 inhibitor (PD098059)-sensitive fashion, thus suggesting that the Ras/MEK/MAPK signaling pathways are crucial for ethanol-induced MCF-7 cell growth.     

Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.     

Ethanol induces cell cycle arrest and triggers apoptosis via Sp1-dependent p75NTR expression in human neuroblastoma cells

Ethanol exposure has deleterious effects on the central nervous system. Although several mechanisms for ethanol-induced damage have been suggested, the precise mechanism underlying ethanol-induced neuronal cell death remains unclear. Recent studies indicate that the p75 neurotrophin receptor (p75NTR) has a critical role in the regulation of neuronal survival. This study was designed to examine the role of p75NTR in ethanol-induced apoptotic signaling in neuroblastoma cells. Ethanol caused highly increased level of p75NTR expression. The use of small interfering RNA to inhibit p75NTR expression markedly attenuated ethanol-induced cell cycle arrest and apoptosis. DNA binding activity of Sp1 was increased by ethanol, whereas inhibition of Sp1 activity by mithramycin, a Sp1 inhibitor, or short hairpin RNA suppressed ethanol-induced p75NTR expression. In addition, inhibitors of casein kinase 2 (CK2) and extracellular signal-regulated kinase (ERK) augmented ethanol-induced p75NTR expression. Our results also demonstrate that inhibition of ERK and CK2 caused a further increase in the activation of the p75NTR proximal promoter induced by ethanol. This increased activation was partially suppressed by the deletion of the Sp1 binding sites. These results suggest that Sp1-mediated p75NTR expression is regulated at least in part by ERK and CK2 pathways. The present study also showed that treatment with ethanol resulted in significant increases in the expression of p21, but not the levels of p53 and p53 target genes such as Bax, Puma, and Bcl-2. Furthermore, the inhibition of p75NTR expression or Sp1 activity suppressed ethanol-induced p21 expression, cell cycle arrest, and apoptosis. These data suggest that ethanol increases p75NTR expression, and CK2 and ERK signaling inversely regulate Sp1-mediated p75NTR expression in ethanol-treated neuroblastoma cells. Thus, our study provides more insight into the mechanisms underlying ethanol actions.     

Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter

Chronic exposure to ethanol results in heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory guanine nucleotide regulatory protein. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization (Nagy, L. E., Diamond, I., Collier, K., Lopez, L., Ullman, B., and Gordon, A. S., Mol. Pharmacol., in press). To understand the mechanism underlying ethanol-induced increases in extracellular adenosine, we examined the interaction of ethanol with the adenosine transport system in S49 lymphoma cells. We found that ethanol inhibited nucleoside uptake without affecting deoxyglucose or isoleucine transport. Inhibition of adenosine uptake was due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake. Consequently, ethanol no longer increased extracellular adenosine concentrations. Taken together with our previous studies, these results suggest that ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which causes an initial increase in intracellular cAMP levels and subsequent development of heterologous desensitization of cAMP signal transduction.     

Lipid-induced filamentous growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis is obligately dependent on infection of maize to complete the sexual phase of its life cycle. Mating interactions between haploid, budding cells establish an infectious filamentous cell type that invades the host, induces large tumours and eventually forms large masses of black spores. The ability to switch from budding to filamentous growth is therefore critical for infection and completion of the life cycle, although the signals that influence the transition have not been identified from the host or the environment. We have found that growth in the presence of lipids promotes a filamentous phenotype that resembles the infectious cell type found in planta. In addition, the ability of the fungus to respond to lipids is dependent on both the cAMP signalling pathway and a Ras/MAPK pathway; these pathways are known to regulate mating, filamentous growth and pathogenesis in U. maydis. Overall, these results lead us to hypothesize that lipids may represent one of the signals that promote and maintain the filamentous growth of the fungus in the host environment.     

cAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism

The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.