Potentiation of okadaic acid-induced ceramide elevation but not apoptosis by inhibition of glucosylceramide synthase in human neuroepithelioma cells

Caspase-dependent apoptosis induced by okadaic acid (OA) in CHP-100 neuroepithelioma cells has previously been shown to associate with a rapid and sustained elevation in intracellular ceramide concentration. We now report that treatment of CHP-100 cells with OA also evoked a rapid elevation in glucosylceramide levels that was maintained at steady state as cells underwent apoptosis; moreover, as observed for ceramide, OA-induced glucosylceramide accumulation was not blocked by fumonisin B-1. Remarkably, when cell death was prevented by caspase inhibition, glucosylceramide accumulation was potentiated and ceramide elevation reduced, thus suggesting that, during apoptosis completion, accumulation of ceramide was partly driven by impairment of its glucosylation through a caspase-dependent mechanism. We studied whether ceramide glucosylation provided a mechanism for negative modulation of OA-induced apoptosis. We observed that the blocking of glucosylceramide synthesis markedly potentiated OA-induced ceramide elevation, but neither accelerated apoptosis onset nor potentiated the apoptotic response. These results indicate that modulation of ceramide glucosylation does not affect the apoptotic response to okadaic acid and suggest that caution must be exercised concerning the possibility that ceramide plays a key role in apoptosis induction. (C) 2001 Elsevier Science Inc. All rights reserved

Age-dependent changes of rat liver plasma membrane composition

The chemical composition of liver plasma membrane was studied in Wistar rats aged between 3 and 24 months. Results obtained indicate a significant age-dependent positive correlation of both the protein:phospholipid and cholesterol:phospholipid ratios, whereas the protein:cholesterol ratio seems to remain unaffected. Phospholipid analysis of liver plasma membrane reveals that only the phosphatidylcholine content has a significant negative correlation with age; all other phospholipid species remain basically unchanged

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase

Phosphoinositide-derived diacylglycerol conversion to phosphatidic acid is a receptor-dependent and compartmentalized phenomenon in human neuroblastoma

We report that upon muscarinic stimulation of SI(-N-BE(2) human neuroblastoma cells, the extent of phosphoinositide-derived diacylglycerol (DG) conversion to phosphatidic acid (PA), operated by a DG kinase, is dependent on the potency of receptor stimulation and correlates with the reduction of phosphatidylinositol 4,5-bisphosphate mass. Evidence is provided that agonist-evoked Ca2+ mobilisation or protein kinase activation are not key events in triggering receptor-generated DG conversion to PA; furthermore, the phenomenon is compartmentalized, namely it occurs within a topologically restricted area that is poorly accessible to DG artificially generated by cell treatment with bacterial phosphatidylinositol-specific phospholipase C. Possible mechanisms driving regulation of the DG kinase operating in the transduction system investigated are discussed

Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis

We report that apoptosis induced by N-hexanoylsphingosine (C-6-Cer)in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on berate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C-6-Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis

Phenothiazines inhibit acetylcholinesterase by concentration-dependent-type kinetics. A study with trifluoperazine and perphenazine

The properties of perphenazine (PPZ) and trifluoperazine (TFP) as fluorescent dyes were exploited to calculate their critical micellar concentrations. The relative fluorescence quantum yield of the two amphiphiles was dependent on their concentration, abruptly decreasing above 30-40 muM PPZ and 20-30 muM TFP. Evidence is presented that this phenomenon is driven by the formation of nonfluorescent drug aggregates. The type of inhibition kinetics displayed by PPZ and TFP on human erythrocyte acetylcholinesterase (AChE) was also dependent on drug concentration, turning from noncompetitive to a "mixed" inhibition type at concentrations at which PPZ and TFP were demonstrated to undergo micelle formation. Results support the notion that phenothiazines may interact with AChE both as monomers and micellar aggregates, producing different inhibitory effects