Identification of the two new, functional actinoporins, CJTOX I and CJTOX II, from the deep-sea anemone Cribrinopsis japonica.

Actinoporins are pore-forming proteins found in sea anemones. Although we now have a large collection of data on actinoporins, our knowledge is based heavily on those identified in shallow-water anemones. Because the deep sea differs considerably from shallow waters in hydrostatic pressures, temperatures, and the prey composition, the deep-sea actinoporin may have evolved in unique ways. This study, therefore, aimed to obtain new actinoporins in the deep-sea anemone Cribrinopis japonica (Actiniaria, Actiniidae). An actinoporin-like sequence was identified from the previously established C. japonica RNA-Seq database, and the complete length (663 bp) of the deep-sea actinoporin gene, Cjtox I, was obtained. In addition, a similar gene, Cjtox II (666 bp), was also identified from RNA of actinopharynx. CJTOX I and CJTOX II were similar in their primary structures, but CJTOX I lacked one residue in the middle of the protein. There was also a difference in the gene expression in live animals, where only Cjtox I was expressed in tentacles of C. japonica. In the heterologous expression where BL21 (DE3) strain was retransformed with the plasmid containing either Cjtox I or Cjtox II gene, the supernatants of both cell lysates showed hemolytic activity on the equine erythrocytes. Preincubation of the supernatants with sphingomyelin caused reduced activity, implying that the CJTOX I and II would target sphingomyelin as with other actinoporins. Because of the structures similarity to the known actinoporins and the sphingomyelin-inhibitable hemolytic activity, both CJTOX I and II were concluded to be new actinoporins, which were identified for the first time from a deep-sea anemone.

Subcellular localization of sphingomyelin revealed by two toxin-based probes in mammalian cells.

Sphingomyelin (SM) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS-1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin-II (EqtII). Both toxins stained SM in the plasma membrane (PM), and the stains were abolished by sphingomyelin synthase 2 (SMS2) knockdown or sphingomyelinase (SMase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by EqtII, and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes (LEs) among intracellular organelles, whereas EqtII stained recycling endosomes (REs) in addition to LEs. The intracellular SM stains by EqtII were abolished by sphingomyelin synthase 1 (SMS1) knockdown, but not by SMS2 knockdown. These results indicate that lysenin and EqtII label different SM pools and that SMS2 and SMS1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS-1 cells. The use of the two SM-binding probes may provide more insights into various sphingomyelin-mediated processes in different topological domains.

Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme.

We have shown recently that oxidized but not native lipoproteins stimulate the activity of secretory phospholipase A2 group IIA (sPLA(2)(IIA)). Since oxidized lipoproteins potentially contain considerable amounts of oxidized phosphatidylcholine, we examined the effect of oxidized palmitoyl arachidonyl phosphatidylcholine (oxPC) and the competitive effects of oxPC and sphingomyelin (SM) on sPLA(2)(IIA) activity. OxPC either added to the assay medium as separated liposomes or incorporated in varied amounts into LDL progressively enhanced the activity of purified human sPLA(2)(IIA) and abolished the inhibitory effect of LDL-incorporated SM on the enzyme activity. OxPC completely abolished the inhibitory effect of SM at the oxPC/SM concentration ratio 1/2. On the other hand, SM suppressed the activating effect of oxPC in a dose-dependent manner, abolishing it almost completely at a concentration 8 times as high as that of oxPC. Thus, changes in the oxPC/SM concentration ratio in LDL may affect the regulatory mechanisms of sPLA(2)(IIA) activity in human blood, inducing stimulation or inhibition of the enzyme. Influence on regulation of sPLA(2)(IIA) activity can be useful in the development of new therapeutic approaches to the treatment of cardiovascular diseases.

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.

Spinal ceramide modulates the development of morphine antinociceptive tolerance via peroxynitrite-mediated nitroxidative stress and neuroimmune activation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

Equilibrium in lung schistosomula sphingomyelin breakdown and biosynthesis allows very small molecules, but not antibody, to access proteins at the host-parasite interface.

The mechanism by which lung-stage schistosomula expose proteins at the host-parasite interface to nutrient, but not antibody, uptake has been obscure. We have found that Schistosoma mansoni and Schistosoma haematobium larvae emerging from host lung at a pH of around 7.5, and fixed with diluted formaldehyde (HCHO), readily bind specific antibodies in indirect membrane immunofluorescence. Data on inhibitors and activators of parasite tegument-bound, magnesium-dependent, neutral sphingomyelinase (nSMase), and sphingomyelin biosynthesis inhibitors revealed that equilibrium in schistosomular sphingomyelin breakdown and biosynthesis prevents antibody binding, yet permits access of small HO-CH2-OH polymers to interact with and cross-link proteins at the host-parasite interface, allowing for their serological visualization.

Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in ApoE knockout mice.

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death.

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.

Stereoselective synthesis and structure-activity relationship of novel ceramide trafficking inhibitors. (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide and its analogues.

New ceramide trafficking inhibitors, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) and a series of its analogues, were synthesized in diastereomerically and enantiomerically pure forms, and the structure-activity relationship was investigated. These analogues were stereoselectively synthesized via catalytic enantioselective Mannich-type reactions using a Cu(II)-chiral diamine 4 complex. Analysis of HPA-12 analogues having various lengths of the amide side chain showed that the optimal chain length for the inhibition of sphingomyelin biosynthesis is 13 with an IC(50) of approximately 50 nM. Masking of the hydroxy group at the 2'- or 3-position of HPA-12 was carried out by methylation, and it was revealed that these hydroxy groups were essential for the activity. Installation of another hydroxy group onto HPA-12 at the same position as that in the natural ceramide was also conducted, but no enhancement of the activity was observed.

Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyria-independent.

Hexachlorobenzene (HCB) alters phospholipid and heme metabolisms in the liver and Harderian gland. The effects of HCB on phospholipid metabolism, in an organ considered to be non-responsive to its porphyrinogenic effects, remain to be studied. Therefore, as the brain is an organ with this feature, this paper analyzes the effects of HCB on brain phospholipid composition in order to investigate if there is any relationship between HCB-induced porphyrin metabolism disruption and phospholipid alterations. For this purpose, a time-course study of HCB effects on brain phospholipids was performed in two strains of rats differing in their susceptibility to acquire hepatic porphyria: Chbb THOM (low); and Wistar (high). This paper shows for the first time that rat brain phospholipids are affected by HCB exposure. Comparative studies show that HCB-induced disturbances in brain phospholipid patterns are time and strain-dependent. Thus, whereas major phospholipids, phosphatidylcholine and phosphatidylethanolamine were more altered in Wistar rats, minor phospholipids, phosphatidylinositol and phosphatidylserine were more affected in Chbb THOM rats. HCB intoxication led to a sphingomyelin/phosphatidylcholine molar ratio lower than the normal, in both strains. As was expected, brain porphyrin content was not altered by HCB intoxication in either strain. It can be concluded that HCB is able to alter brain phospholipid metabolism in a strain-dependent fashion, and in the absence of alterations in brain heme metabolism. In addition, HCB-induced disturbances in brain phospholipids were not related to the degree of hepatic porphyria achieved by the rats.

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells.

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.

Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine.

The prototype of a new class of antiproliferative phospholipid analogs, hexadecylphosphocholine (HePC), has been shown to inhibit tumor growth and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC, e.g. protein kinase C and CTP:phosphocholine cytidylyltransferase, have been proposed, the mechanisms of HePC-induced anticancer activity are still unclear. Considering that the antiproliferative effect of HePC correlates with inhibition of phosphatidylcholine biosynthesis, which is tightly coupled to sphingomyelin biosynthesis, we tested the hypothesis that treatment of cells with the anticancer drug leads to increased cellular ceramide and subsequently to apoptotic cell death. In the present study, we showed that 25 micromol/liter HePC induced apoptosis. In further experiments, we demonstrated that HePC inhibited the incorporation of radiolabeled choline into phosphatidylcholine and at a later time point into sphingomyelin. This was confirmed by metabolic labeling of the lipid backbone using radiolabeled serine, and it was shown that HePC decreased the incorporation of serine into sphingomyelin by 35% and simultaneously increased the incorporation of serine into ceramide by 70%. Determination of the amount of ceramide revealed an increase of 53% in HePC-treated cells compared with controls. In accordance with the hypothesis that elevated ceramide levels may be the missing link between the metabolic effects of HePC and its proapoptotic properties, HePC-induced apoptosis was blocked by fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, we found that membrane-permeable ceramides additively increased the apoptotic effect of HePC.

Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung.

Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of L-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.

PAF-like activity of O-acetylated sphingomyelin.

O-acetylated sphingomyelin (Ac-SM) was found to cause aggregation of rabbit platelets in vitro. The Ac-SM-induced aggregation was accompanied by subsequent desensitisation of platelets to platelet-activating factor (PAF). The activity of Ac-SM exceeded that of acyl-PAF by about fourfold. BN 52021, a specific PAF-receptor antagonist, was found to inhibit the Ac-SM-induced aggregation. These results, together with earlier reports that sphingomyelin can inhibit the effects of PAF, suggest a new physiological function for sphingomyelin as a regulator of PAF-receptor binding. A search for enzymes to catalyse specifically the acetylation and deacetylation of sphingomyelin is required to confirm the physiological existence of sphingomyelin derivatives.