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M Nikolova-Karakashian

Publications and source records attributed to M Nikolova-Karakashian.

15 recordsLinked to original sources
PubMed2002

Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor P75.

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within 10 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-phosphate (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) 1/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of sphingosine-kinase blocked the neuroprotective effect of NGF, suggesting that sphingosine-1-phosphate was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.

Animals↗
PubMed2001

Ceramide modulates nicotinic receptor-dependent Ca(2+) signaling in rat chromaffin cells.

Ceramide, which is an integral component of the sphingomyelin signaling pathway, can attenuate voltage-gated Ca(2+) channel (VGCC) activity in a number of cell types. The aim of the present study was to determine whether ceramide can also modulate VGCC activity, and as a consequence nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion, in rat adrenal chromaffin cells. Short-term C(6)-ceramide (CER) treatment dose-dependently inhibited nicotine (NIC)-induced peak intracellular Ca(2+) transients. Sphingomyelinase elicited similar responses, whereas the inactive ceramide analog C(2)-dihydroceramide had no effect on NIC-induced Ca(2+) transients. CER suppressed KCl- and NIC-induced Ca(2+) transients to a similar extent, suggesting that the voltage-gated Ca(2+) channel was a primary site of inhibition. In direct support of this concept, whole-cell patch-clamp analysis demonstrated that CER and sphingomyelinase significantly reduced peak Ca(2+) currents. Pretreatment with staurosporine significantly attenuated CER-dependent inhibition of both NIC-induced Ca(2+) transients and peak Ca(2+) current, suggesting that the effects of CER are mediated at least in part by protein kinase C. Consistent with suppressed Ca(2+) signaling, CER also significantly inhibited NIC-induced catecholamine secretion measured at the single-cell level by carbon fiber amperometry. This effect of CER was also significantly attenuated by pretreatment with staurosporine These data demonstrate that the sphingomyelin signaling pathway can modulate nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion in rat chromaffin cells.

Adrenal Medulla↗
PubMed2000

Ceramidases.

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4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Pivotal role for acidic sphingomyelinase in cerebral ischemia-induced ceramide and cytokine production, and neuronal apoptosis.

Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. The signaling cascades activated by cerebral ischemia that may either promote or protect against neuronal death are not well understood. One injury-responsive signaling pathway that has recently been characterized in studies of non-neural cells involves cleavage of membrane sphingomyelin by acidic and/or neutral sphingomyelinase (ASMase) resulting in generation of the second messenger ceramide. We now report that transient focal cerebral ischemia induces large increases in ASMase activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase. Neurons lacking ASMase exhibit decreased vulnerability to excitotoxicity and hypoxia, which is associated with decreased levels of intracellular calcium and oxyradicals. Treatment of mice with a drug that inhibits ASMase activity and ceramide production reduces ischemic neuronal injury and improves behavioral outcome, suggesting that drugs that inhibit this signaling pathway may prove beneficial in stroke patients.

Animals↗
PubMed2000

Apoptosis and dysregulated ceramide metabolism in a murine model of alcohol-enhanced lipopolysaccharide hepatotoxicity.

BACKGROUND: The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity. METHODS: Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified. RESULTS: Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-alpha levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function. CONCLUSIONS: These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.

Alanine Transaminase↗
PubMed1999

Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.

Amidohydrolases↗
PubMed1999

Regulation of cytochrome P450 expression by sphingolipids.

Sphingolipids modulate many aspects of cell function, including the expression of cytochrome P450, a superfamily of heme proteins that participate in the oxidation of a wide range of compounds of both endogenous (steroid hormones and other lipids) and exogenous (e.g. alcohol, drugs and environmental pollutants) origin. Cytochrome P450-2C11 (CYP 2C11) is down-regulated in response to interleukin-1beta (IL-1beta), and this response involves the hydrolysis of sphingomyelin to ceramide as well as ceramide to sphingosine, and phosphorylation of sphingosine to sphingosine 1-phosphate. Activation of ceramidase(s) are a key determinant of which bioactive sphingolipid metabolites are formed in response to IL-1beta. Ceramidase activation also appears to account for the loss of expression of CYP 2C11 when hepatocytes are placed in cell culture, and the restoration of expression when they are plated on Matrigel; hence, this pathway is influenced by, and may mediate, interactions between hepatocytes and the extracellular matrix. Recent studies using inhibitors of sphingolipid metabolism have discovered that sphingolipids are also required for the induction of CYP1A1 by 3-methylcholanthrene, however, in this case, the requirement is for de novo sphingolipid biosynthesis rather than the turnover of complex sphingolipids. These findings illustrate how changes in sphingolipid metabolism can influence the regulation of at least several isoforms of cytochrome P450.

Animals↗
PubMed1997

Dihydroceramide biology. Structure-specific metabolism and intracellular localization.

This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-DH-Cer (C6-NBD-DH-Cer) was incubated with HT29, NRK, BHK, or HL-60 cells, it was efficiently converted to dihydrosphingomyelin and dihydroglucosylceramide, and a number of other sphingolipids, with the nature of the products depending on the cell line. In addition, complex sphingolipids were formed that contained a desaturated (sphingosine) backbone, indicating that DH-Cer (and/or its metabolites) were substrates for the desaturase(s) that introduce the 4,5-trans double bond. Based on the kinetics and inhibitor studies, double bond addition did not appear to occur with the complex sphingolipids directly, but rather, during turnover and resynthesis. The conversion of C6-NBD-DH-Cer to more complex sphingolipids was highly stereoselective for the natural D,erythro isomer of C6-NBD-DH-Cer. Interestingly, the stereochemistry of the sphingoid base backbone also affected the localization of fluorescent sphingolipids: the D,erythro species appeared in the Golgi apparatus, whereas other stereo-isomers accumulated in the endoplasmic reticulum. In addition to C6-NBD-Cer and C6-NBD-DH-Cer, C6-NBD-4-D-hydroxy-DH-Cer gave rise to formation of complex sphingolipids and localized at the Golgi apparatus. These studies indicate that dihydroceramide is used as the initial backbone of complex (glyco)sphingolipids, perhaps to avoid build up of ceramide as an intermediate since this is such a potent bioactive compound. The stereoselectivity in transport and metabolism suggests that trafficking of ceramide is protein-directed rather than simply a consequence of vesicular membrane flow.

Animals↗
PubMed1997

Bimodal regulation of ceramidase by interleukin-1beta. Implications for the regulation of cytochrome p450 2C11.

Interleukin 1beta (IL-1beta) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Cer has been proposed to play a role in the down-regulation of cytochrome P450 2C11 (CYP2C11) and induction of alpha1-acid glycoprotein (AGP) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (1995) J. Biol. Chem. 270, 25233-25238). Nonetheless, some of the features of the down-regulation of CYP2C11 do not fit a simple model of Cer as a second messenger as follows: N-acetylsphinganine (C2-DHCer) is as potent as N-acetylsphingosine (C2-Cer) in suppression of CYP2C11; the IL-1beta concentration dependence for SM turnover is different from that for the increase in Cer; and the increase in Cer mass is not equivalent to the amount of SM hydrolyzed nor the time course of SM hydrolysis. In this article, we report that these discrepancies are due to activation of ceramidase by the low concentrations of IL-1beta ( approximately 2.5 ng/ml) that maximally down-regulate CYP2C11 expression, whereas higher IL-1beta concentrations (that induce AGP) do not activate ceramidase and allow Cer accumulation. This bimodal concentration dependence is demonstrated both by in vitro ceramidase assays and in intact hepatocytes using a fluorescence Cer analog, 6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-Cer (NBD-Cer), and following release of the NBD-fatty acid. IL-1beta increases both acid and neutral ceramidase activities, which appear to be regulated by tyrosine phosphorylation because pretreatment of hepatocytes with sodium vanadate increases (and 25 microM genistein reduces) the basal and IL-1beta-stimulated ceramidase activities. Since these findings suggested that sphingosine (and, possibly, subsequent metabolites) is the primary mediator of the down-regulation of CYP2C11 by IL-1beta, the effects of exogenous sphingosine and C2-Cer on expression of this gene were compared. Sphingosine was more potent than C2-Cer in down-regulation of CYP2C11 when added alone or with fumonisin B1 to block acylation of the exogenous sphingosine. Furthermore, the suppression of CYP2C11 by C2-Cer (and C2-DHCer) is probably mediated by free sphingoid bases, rather than the short chain Cer directly, because both are hydrolyzed by hepatocytes and increase cellular levels of sphingosine and sphinganine. From these observations we conclude that sphingosine, possibly via sphingosine 1-phosphate, is a mediator of the regulation of CYP2C11 by IL-1beta in rat hepatocytes and that ceramidase activation provides a "switch" that determines which sphingolipids are elevated by this cytokine to produce multiple intracellular responses.

Amidohydrolases↗
PubMed1995

Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes.

Interleukin-1 triggers the down-regulation of several hepatic cytochrome P450 gene products, but the cellular signaling pathways involved are not known. We have examined the role of sphingomyelin hydrolysis to ceramide in the suppression of CYP2C11, a major constitutive form of cytochrome P450, by interleukin-1. Treatment of rat hepatocytes cultured on matrigel with interleukin-1 beta caused a rapid turnover of sphingomyelin and an increase in cellular ceramide, with no change in cellular phosphatidylcholine. The ceramide was composed mainly of a D-erythro-sphingosine backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of CYP2C11 and induced the expression of the interleukin-1-responsive alpha 1-acid glycoprotein mRNA. In contrast, the acute-phase gene beta-fibrinogen, which is induced by interleukin-6 but not by interleukin-1, did not respond to C2-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the effect of C2-ceramide on CYP2C11, but not on alpha 1-acid glycoprotein expression. These results are consistent with a role for ceramide or a related sphingolipid in mediating the down-regulation of CYP2C11, the induction of alpha 1-acid glycoprotein, and perhaps other cellular effects of interleukin-1 in hepatocytes.

Acute-Phase Proteins↗
PubMed1995

Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very low density lipoprotein secretion.

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.

Animals↗
PubMed1992

Effect of a sunflower oil-supplemented diet on protein kinase activities of rat liver plasma membranes.

1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter (rs) of the fluorescence probe DPH and SDPH decreased strongly in the experimental group. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.

Animals↗