gw-4869 and thermozymocidin

Ceramide, a sphingolipid metabolite, regulates diverse cellular processes including apoptosis, cell senescence, the cell cycle, and cellular differentiation. Exogenously administered ceramide reportedly increased cochlear hair cell death due to gentamicin-induced ototoxicity. Ceramide is mainly generated via a ceramide/sphingomyelin cycle by sphingomyelinase and sphingomyelin synthase or via de novo synthesis by serine palmitoyltransferase and ceramide synthase. This study was designed to investigate the possible involvement of neutral sphingomyelinase, sphingomyelin synthase, or serine palmitoyltransferase in hair cell death due to gentamicin. The basal turns of the organ of Corti of Sprague-Dawley rats were dissected on postnatal days 3-5. Cochlear cultures were exposed to media containing 35 μM gentamicin for 48 h to assess the effects of GW4869 (a neutral sphingomyelinase inhibitor), 2-hydroxyoleic acid (a sphingomyelin synthase activator), and myriocin (a serine palmitoyltransferase inhibitor). Hair cell loss was significantly decreased in the presence of GW4869 or 2-hydroxyoleic acid. Myriocin had no significant effects against gentamicin-induced hair cell loss. In addition, neutral sphingomyelinase was activated by gentamicin exposure. The present findings strongly suggest that the ceramide/sphingomyelin cycle plays an important role in the protection of hair cells against gentamicin-induced ototoxicity.

Topics: Aniline Compounds; Animals; Animals, Newborn; Benzylidene Compounds; Cell Death; Cells, Cultured; Ceramides; Fatty Acids, Monounsaturated; Gentamicins; Hair Cells, Auditory; Oleic Acids; Rats, Sprague-Dawley; Serine C-Palmitoyltransferase; Sphingomyelin Phosphodiesterase; Sphingomyelins; Transferases (Other Substituted Phosphate Groups)

Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

Topics: Aging; Aniline Compounds; Animals; Benzylidene Compounds; Ceramides; Fatty Acids, Monounsaturated; Fumonisins; Glucose; Glycogen; Hepatocytes; Imipramine; Insulin; Insulin Resistance; Male; Oxidoreductases; Rats; Serine C-Palmitoyltransferase; Sphingolipids; Sphingomyelin Phosphodiesterase

Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by approximately 18-23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A.

Topics: Adenylate Kinase; Aniline Compounds; Animals; Benzylidene Compounds; Cell Line; Ceramides; Diet; Ethanol; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression Regulation; Hydrogen Peroxide; Imipramine; Male; Mice; Phosphorylation; Protein Phosphatase 2; Rats

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase-2) is critical for tumor necrosis factor (TNF) alpha-induced trafficking of NMDA receptors and synaptic plasticity. TNFalpha initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFalpha increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory post-synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase-2 prevented TNFalpha-induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA-evoked calcium flux and excitatory post-synaptic currents.

Topics: Aniline Compounds; Animals; Benzylidene Compounds; Calcium; Cells, Cultured; Ceramides; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Fatty Acids, Monounsaturated; Fluorescence Resonance Energy Transfer; Gangliosidosis, GM1; Hippocampus; In Vitro Techniques; Membrane Microdomains; Mutation; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Phosphorylation; Protein Transport; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Serine; Spectrometry, Mass, Electrospray Ionization; Sphingomyelin Phosphodiesterase; Tumor Necrosis Factor-alpha