Athanassia Siafaka-Kapadai

Incorporation of [3H]Valproic Acid into Lipids in GT1–7 Neurons

Valproic acid (2-propylpentanoic acid, valproate, VPA), an 8-carbon, branched chain fatty acid, is effectively used in the treatment of mania and epilepsy. The biochemical mechanisms by which this drug has its therapeutic effects are not yet established. The purpose of this study was to partially characterize the incorporation of [3H]VPA into phospholipids of GT1-7 neurons, an immortalized hypothalamic cell line. GT1-7 neurons were grown to confluence in culture dishes, and then were incubated with various concentrations of [3H]VPA between 10 and 400 microg/mL for various times up to 20 hr. Total lipids were extracted and phospholipids were separated from neutral lipids using TLC. Our results indicate that [3H]VPA (10 microg/mL) was incorporated into phospholipids of GT1-7 neurons in a time-dependent and saturable manner over 300 min. Subsequent separation of the lipid fraction by TLC indicated that 44.4% of the radioactivity taken up by the cells was incorporated into phospholipids and neutral lipids. One of the phospholipids migrated with a slightly lower Rf value than authentic phosphatidylcholine. Our results show that the incorporation of VPA into phospholipids and glycerides was linear with VPA concentrations from 10 to 400 microg/mL. Finally, we synthesized 1-acyl-2-valproyl-sn-glycero-3-phosphocholine and validated its structure with nuclear magnetic resonance and electrospray mass spectrometry to verify the structure of this compound, confirming that this compound is structurally possible. We conclude that VPA is incorporated into lipids in GT1-7 neurons and discuss the possible effects of valproyl phospholipids on neuronal functional properties.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization.

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time‐ and concentration‐dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K m of 3.7 μM and V max 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9–10, was abolished by phenylmethylsulfonyl fluoride and was Ca2+‐ and Mg2+‐independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.

Hepoxilin A3 protects β-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid

Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.

Synthesis of all-trans anandamide: A substrate for fatty acid amide hydrolase with dual effects on rabbit platelet activation

Anandamide (AEA) presents the four double bonds in the cis configuration, deriving from the arachidonic acid moiety. In the context of an antisense strategy based on the double bond configuration, all-trans AEA (t-AEA) was synthesized in high yield starting from all-trans methyl arachidonate and ethanolamine in the presence of KCN. t-AEA was assayed on rabbit platelet aggregation, obtaining effect only at high concentrations (>10(-4) M) after an also concentration-dependent lag phase. At lower concentrations it inhibited PAF-induced rabbit platelet aggregation with an IC(50)=4.6 x 10(-6) M. In contrast to anandamide, the activation of platelets was not due to the conversion of t-AEA to trans arachidonic acid, as ascertained by negative results with FAAH inhibitors. However, t-AEA was found to be a substrate for fatty acid amide hydrolase (FAAH), the enzyme that cleaves anandamide and regulates in vivo the magnitude and duration of the signaling induced by this lipid messenger.

Platelet activating factor and lyso-phosphatidylcholines from strawberry

Lipids from strawberry fruits, leaves, achenes and pollen were separated into classes by TLC, purified by HPLC and tested for biological activity. A lipid fraction from fruits with the same chromatographic behaviour as authentic platelet activating factor (PAF) showed identical biological activity, namely, dose-dependent aggregation of washed rabbit platelets, inhibition of aggregation by CV 3988, platelet desensitization to PAF and vice versa, and loss of activity by alkaline hydrolysis and recovery of activity by reacetylation. The presence of PAF was confirmed by FAB mass spectrometry. Lyso-phosphatidylcholines, including lyso-PAF, were also found in all the plant parts tested.

PAF-acetylhydrolase activity and PAF levels in pancreas and plasma of well-fed, diabetic and fasted rat

PAF-AH activity was determined in pancreas homogenates. The enzyme activity was moderately stable upon storage at -20 degrees C. PAF and lyso-PAF were identified in rat pancreas and their concentrations were determined. PAF levels and PAF-AH activity were compared in the pancreatic tissue and plasma of three different groups of animals: well-fed, STZ-induced diabetic and fasted rats. The concentration of PAF in the pancreas of fasted rats was ten fold lower as compared with that of the well-fed or the diabetic animals. The last two groups had similar pancreatic PAF concentration. PAF levels in the plasma of fasted rats were seven fold lower than those of well-fed or diabetic rats, which were found to be similar. The enzyme PAF-AH had the highest activity in the pancreas of well-fed rats. On the contrary, the enzyme seems to be more active in the plasma of fasted as compared with diabetic and well-fed animals.