Albena Momchilova

Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity

Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis.

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C(12)-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L(o) domains but induces the formation of a gel-like phase. The activation of phospholipase A(2) by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

Three‐dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion‐mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three‐dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady‐state levels of ERK1/2 activity in primary human fibroblasts, induced by cell‐derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5‐fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo‐like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.

Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes

The influence of the phospholipid composition and the physico-chemical properties of rat liver plasma membranes on the activity of membrane-bound phospholipase A2 has been investigated. The plasma membrane composition was modified by the aid of exogenous phospholipases A2, C and D, and by butanol treatment. The partially delipidated membranes thus obtained were enriched with different phospholipids. The steady-state fluorescent anisotropy of 1,6-diphenyl-1,3,5-hexatriene and the lipid order parameter-SDPH in the modified membranes were calculated. It was established that the activity of the membrane-bound phospholipase A2 was higher in rigid membranes and was decreased when the membrane lipid bilayer was fluidized.

Ha‐ ras‐TRANSFORMATION ALTERS THE METABOLISM OF PHOSPHATIDYLETHANOLAMINE AND PHOSPHATIDYLCHOLINE IN NIH 3T3 FIBROBLASTS

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha‐ ras transformation. All phospholipid fractions were reduced in ras ‐transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras ‐cells. Acyl‐CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine‐cytidylyltransferase and CTP:phosphoethanolamine‐chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline‐ and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP‐choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP‐ethanolamine pathway.

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.

Age-related changes in rat liver plasma membrane phospholipase A2 activity

The alterations of the lipid composition and fluidity of liver plasma membranes as well as the activity of membrane-bound phospholipase A2 (EC 3.1.1.4) in male and female rats of different ages are investigated. An age-induced increase of the protein/phospholipid and cholesterol/phospholipid ratios is observed, as well as a decrease in the unsaturation index of plasma membrane phosphatidylcholine. These changes induce an augmentation of the steady-state fluorescent anisotropy-rs and structural order parameter for 1,6-diphenyl-1,3,5-hexatriene-SDPH in plasma membranes. The membrane-bound phospholipase A2 activity is enhanced with aging, correlating well with the reduced membrane fluidity. The sex-determined alterations in all these parameters is also investigated.

Effect of sphingosine on domain morphology in giant vesicles

Sphingosine is a bioactive molecule which is known to participate in the regulation of a number of cellular processes such as apoptosis, cell differentiation, growth, etc. Sphingosine was observed to exhibit different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Our current results showed that in a glycerophospholipid matrix sphingosine segregated in gel leaf-like domains whereas cholesterol presence increased its miscibility by melting gel domains in a concentration-dependent manner. Sphingosine and cholesterol did not form merging liquid domains on the micron scale as observed for sphingomyelin and cholesterol. However, we were able to visualize that sphingosine appears as a stabilizer and amplifier of domains in liquid-ordered phase by increasing the temperature of their formation and fraction. These results imply that sphingosine acts as a modulator of the lipid domain formation and thus it could exert its biological role, not only through direct binding to proteins, but also indirectly by influencing their sorting in membranes and modulating the processes of signal transduction.

Phospholipase A2 activity in ram spermatozoa plasma membranes.

A highly active phospholipase A2 in ram spermatozoa plasma membranes has been established. Phospholipase A2 was optimally active at pH 7.4 and was Ca2+ dependent. Low concentrations of K+ (25 mM) were found to inhibit phospholipase A2 activity, whereas higher concentrations induced enzyme reactivation. An inhibitory effect of Zn2+ has also been observed.

Rat liver microsomal phospholipase A2 and membrane fluidity

The influence of the physical state and the lipid composition on microsomal membrane-bound phospholipase A2 activity has been investigated. It was established that the decrease of membrane fluidity expressed by the alterations of the steady-state fluorescent anisotropy (rs) and the structural order parameter for DPH (SDPH) leads to augmentation of phospholipase A2 specific activity. Phosphatidylinositol is the only phospholipid having a specific activating effect on microsomal membrane-bound phospholipase A2.