Modulation of sensitivity to alcohol by cortical and thalamic brain regions.

The nucleus accumbens core (AcbC) is a key brain region known to regulate the discriminative stimulus/interoceptive effects of alcohol. As such, the goal of the present work was to identify AcbC projection regions that may also modulate sensitivity to alcohol. Accordingly, AcbC afferent projections were identified in behaviorally naïve rats using a retrograde tracer which led to the focus on the medial prefrontal cortex (mPFC), insular cortex (IC) and rhomboid thalamic nucleus (Rh). Next, to examine the possible role of these brain regions in modulating sensitivity to alcohol, neuronal response to alcohol in rats trained to discriminate alcohol (1 g/kg, intragastric [IG]) vs. water was examined using a two-lever drug discrimination task. As such, rats were administered water or alcohol (1 g/kg, IG) and brain tissue was processed for c-Fos immunoreactivity (IR), a marker of neuronal activity. Alcohol decreased c-Fos IR in the mPFC, IC, Rh and AcbC. Lastly, site-specific pharmacological inactivation with muscimol + baclofen (GABAA agonist + GABAB agonist) was used to determine the functional role of the mPFC, IC and Rh in modulating the interoceptive effects of alcohol in rats trained to discriminate alcohol (1 g/kg, IG) vs. water. mPFC inactivation resulted in full substitution for the alcohol training dose, and IC and Rh inactivation produced partial alcohol-like effects, demonstrating the importance of these regions, with known projections to the AcbC, in modulating sensitivity to alcohol. Together, these data demonstrate a site of action of alcohol and the recruitment of cortical/thalamic regions in modulating sensitivity to the interoceptive effects of alcohol.

Metabolism of n -9, n -6 and n -3 fatty acids in hepatoma Morris 7777 cells. Preferential accumulation of linoleic acid in cardiolipin.

The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids. The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.

Role of oxidative damage and IL-1 beta-converting enzyme-like proteases in Fas-based cytotoxicity exerted by effector T cells.

The implication of oxidative damage and/or intact mitochondrial function in physiological Fas-based cytotoxicity has been tested using the cytolytic hybridoma d11S and the CD8(+) CTL clone KB5.C20, previously stimulated to express Fas ligand (FasL) on their surface, as effectors and U937 or U937-rho0 cells (depleted of mitochondrial DNA) as targets. Immobilized anti-Fas mAb, which induced death of U937 cells, inhibited the growth of U937-rho0 cells but without inducing cell death. By contrast, FasL-expressing effectors readily killed both targets, with induction of DNA fragmentation, in 20 h assays. These results demonstrate the lack of involvement of mitochondrial-derived free radicals and/or intact mitochondrial function in physiological Fas-based cytotoxicity. Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas) with a polyunsaturated fatty acid, which induces cell death through the generation of lipid free radicals, resulted in the potentiation of Fas-based cytotoxicity. This potentiating effect, but not Fas-based cytotoxicity itself, was eliminated by the physiological antioxidant vitamin E. On the other hand, the IL-1beta-converting enzyme (ICE)-like protease tetrapeptide inhibitor Ac-YVAD-cmk partially inhibited Fas-based cytotoxicity, while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was a much more effective inhibitor of Fas-induced apoptosis. It was concluded that Fas-induced cytotoxicity was clearly dependent on ICE-like protease activation, and especially on that of CPP32 in Fas-sensitive cells, including mitochondrial DNA-depleted ones.

Biosynthesis of unsaturated fatty acids in the main cell lineages of human leukemia and lymphoma.

Unsaturated fatty acids are essential for the proliferation of many haematopoietic cells, but little is known about their biosynthetic pathways in these cells. We have studied the activity of the main desaturation-elongation enzymes in human B-(Reh-6, Raji, Ramos) and T-(CEM, Jurkat) lymphocytic, promonocytic (U937), promyelocytic (HL-60) and pluripotent myeloid (K562) cell lineages, as well as the changes induced by cell differentiation. Cells were incubated with 14C-labelled 18:0, 18:2(n - 6) and 18:3(n - 3) or supplemented with the corresponding unlabelled fatty acid and synthesis of polyunsaturated fatty acids (PUFA) was evaluated by argentation-TLC and GLC. The main activity present in most cells was delta 9-desaturase (range between 200-1000 pmol/24 h per 10(6) cells) that was regulated by the type of free fatty acids in culture media. A great variability in the activities of delta 6- and delta 5-desaturase was observed. They were virtually absent in B-cells and only one (Jurkat) T-cell line synthesized significant amounts of (n - 6) and (n - 3) PUFA. The main PUFA formed by Jurkat cells were 20:3 and 20:4(n - 6) (30 and 40%, respectively, of cell lipid radioactivity) and 20:5, 22:5 and 22:6(n - 3) (60, 20 and 10%, respectively, of cell radioactivity). Cell differentiation caused complex changes in desaturase activities. The activity of delta 9-desaturase increased with the degree of differentiation of B-cells. Differentiation of U937 cells to macrophages with PMA caused a 2-3-fold increase in the activity of (delta 6 + delta 5)- and delta 9-desaturases and no changes and a 2-fold decrease, respectively, if the inducer was DMSO. Differentiation of HL-60 cells to granulocytes with DMSO virtually abolished delta 9-desaturase activity and greatly reduced that of delta 6- and delta 5-desaturases. delta 9-Desaturase activity increased (2.5-fold) in myeloid K562 cells differentiated to erythroblasts with hemin. No induction of delta 6-desaturase, absent in K562 cells, occurred after differentiation to erythroblasts or megakaryoblasts and they synthesized alternative PUFA through sequential elongation and delta 5-desaturation of 18:2(n - 6) and 18:3(n - 3). The activities of delta 6- and delta 5-desaturase in HL-60 and U937 cells increased when differentiation also stimulated the synthesis of eicosanoids and extracellular release of PUFA.

Increased cytotoxicity of polyunsaturated fatty acids on human tumoral B and T-cell lines compared with normal lymphocytes.

Epidemiological and experimental data suggest that fatty acids may modulate the growth of tumor cells. We have analyzed the effect of different types of fatty acids, bound to serum proteins in physiological conditions, on the lipid composition and growth of human neoplastic B and T-cell lines and compared their effect on normal lymphocyte proliferation. Fatty acids with 0 to 2 unsaturations (stearic, oleic, and linoleic), at concentrations up to 50 or 100 microM did not significantly affect the proliferation of leukemic cells. However, long-chain polyunsaturated fatty acids (PUFA), and mainly docosahexaenoic (22:6, n-3), were cytotoxic at concentrations greater than or equal to 20 microM after 48-72 h in culture. Simultaneous supplementation with vitamin E restored normal cell growth. The amount of end-products of lipid peroxidation in cells correlated with the observed toxicity but the amount of superoxides did not. Fatty acid supplementations increased cell triacylglycerol content but did not affect the degree of unsaturation of phospholipids, cholesterol/phospholipids molar ratio, or membrane fluidity. Glutathione-S-transferase activity was low in Raji and CEM cells, moderate in lymphocytes and high in Ramos cells and did not increase with supplementations. The proliferation of normal lymphocytes, which produced lower amounts of end-products of lipid perodixation, was not inhibited, but in some cases stimulated, by PUFA (with the exception of 30 microM 22:6). The extension of these results to situations in vivo could lead to use of PUFA for delaying leukemia progression or in adjuvant chemotherapy.

Fatty acid metabolism in human lymphocytes. II. Activation of fatty acid desaturase-elongase systems during blastic transformation.

The fatty acid desaturation-elongation ability of human T-lymphocytes during blastic transformation was determined both by gas-liquid chromatography and incubation with radiolabeled precursors. Human peripheral blood mononuclear cells (PBMC) were activated with phytohemagglutinin (PHA) and cultured in media supplemented with different fatty acids (18:0, 18:1(n - 9), 18:2(n - 6), 18:3(n - 3) and 20:4(n - 6)) at a final concentration of 30 microM. All the fatty acids added were elongated by activated PBMC and the maximal activity was observed on 20:4(n - 6) (a 25% of conversion to 22:4(n - 6)). Supplementation with stearic acid increased the proportion of oleic (from 21.4% to 23.7%) and eicosaenoic (from 3.1% to 5.7%) acids in cellular lipids, indicating the existence of a delta 9-desaturase activity. Supplementation with linoleic and linoleic acids increased slightly the cell content in their more unsaturated derivatives. Direct measurement of desaturase activities was performed by incubating quiescent and activated PBMC with [1-14C]stearic, [1-14C]linoleic and [1-14C]linolenic acids. Quiescent cells exhibited a very low delta 9-desaturase and no sign of delta 6-desaturase activity. A moderate and progressive activation of delta 9-, delta 6- and delta 5-desaturases was observed during blastic transformation of human PBMC. Up to 8% of 18:0 was converted to monoenes, 4% and 1.5% of 18:2(n - 6) was converted to trienes and tetraenes, respectively, and 14.5% of 18:3(n - 3) was converted to pentaenes. The maximal relative activities were found after 48 h of PHA-stimulation for delta 9-desaturase (around 90 pmol of 18:0 converted per 10(6) cells in the last 24 h) and at 72 h for delta 6- and delta 5-desaturases (around 75 and 140 pmol of 18:2 and 18:3, respectively, converted per 10(7) cells in the last 24 h). Although these activities are not enough to explain all the changes in fatty acid composition of human PBMC during blastic transformation, they may contribute to a more controlled cell phospholipid composition.