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Antioxidant-rich diets exert a protective effect in diseases involving oxidative damage. Among dietary components, green tea is an excellent source of antioxidants. In this study, cultured neonatal rat cardiomyocytes were used to clarify the protective effect of a green tea extract on cell damage and lipid peroxidation induced by different periods of hypoxia followed by reoxigenation. Cultures of neonatal rat cardiomyocytes were exposed to 2--8 hr hypoxia, eventually followed by reoxygenation, in the absence or presence of alpha-tocopherol or green tea. LDH release and the production of conjugated diene lipids were measured, and appeared linearly related to the duration of hypoxia. During hypoxia, both LDH release and conjugated diene production were reduced by alpha-tocopherol and, in a dose dependent manner, by green tea, the 50mgr;g/ml being the most effective dose. Reoxygenation caused no further increase in LDH leakage, while it caused a significant increase in conjugate dienes, which absolute value was lower in antioxidant supplemented cells. Anyway, the ratio between conjugated diene production after hypoxia and after reoxygenation was similar in all groups, indicating that the severity of free radical-induced reoxygenation injury is proportional to the severity of previous hypoxic injury. Since hypoxic damage is reduced by alpha-tocopherol and green tea, our data suggest that any nutritional intervention to attenuate reoxygenation injury must be directed toward the attenuation of the hypoxic injury. Therefore, recommendations about a high dietary intake of antioxidants may be useful not only in the prevention, but also in the reduction of cardiac injury following ischemia.

The antioxidant activities of three different green tea extracts were investigated and compared by two different methods. By the first method, which evaluated the direct protective effect of the green tea extracts on lipid peroxidation, the extracts were added, at different concentrations, to a lipid model system, made by refined peanut oil, freshly submitted to a further bleaching and subjected to forced oxidation at 98 °C, by an oxidative stability instrument. By the second method, the effectiveness of the same extracts was checked in cultures of neonatal rat cardiomyocytes exposed to a free radical-generating system by evaluating conjugated diene production and lactate dehydrogenase release. All of the extracts revealed a strong antioxidant activity by both the methods, and a particular effectiveness was demonstrated by the extracts having higher amounts of (−)-epigallocathechin-3-gallate and (−)-epigallocathechin, as analyzed by reverse-phase HPLC analysis. Keywords: Green tea extracts; catechins; oxi...

The clinical use of the antitumoral doxorubicin (DOX) is limited by its cardiotoxicity, which is mediated through different mechanisms. The membrane lipid peroxidation induced by DOX may cause disruption of the unsaturated fatty acyl chains; in the endoplasmic reticulum, containing the system catalyzing the desaturation/elongation of fatty acids, DOX could interfere with the metabolism of linoleic and alpha-linolenic acids. Using primary cultures of neonatal rat cardiomyocytes we demonstrated that the exposure to different concentrations of DOX (10(-5) and 10(-7) M) for 24 h caused an increase in the production of conjugated dienes, an impairment in the desaturation/elongation of essential fatty acids, and a reduction in the cellular content of highly unsaturated fatty acids. Conversely, 1 h exposure to 10(-5) M DOX was sufficient to induce alterations in the desaturation/elongation of linoleic and alpha-linolenic acids, but did not cause either formation of conjugated dienes or modification of the fatty acyl pattern. Therefore, DOX has a dual negative effect, depending on its concentration and on the time of exposure, one directed against the membrane highly unsaturated fatty acids, the other against the system which is required for the synthesis of these fatty acids themselves. These two effects synergically act in causing heart cell damage.

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M  2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4.

Delta-6-desaturase (D6D) activity is influenced by many nutritional and non-nutritional factors, among which one of the most important is aging. D6D activity could be susceptible to the dual influence of aging itself and of nutritional deficiencies, due to the reduced intake and/or absorption of essential nutrients. Particularly, vitamin B6 deficiency might be a crucial factor for D6D activity in aged people. Using 20 month old Sprague-Dawley rats fed a diet with a subnormal level of vitamin B6, we evaluated D6D activity for linoleic acid (LA) and alpha-linolenic acid (ALA) in liver microsomes, and the fatty acid composition of microsomal total lipids. We observed a diminished D6D activity for LA and also for ALA in vitamin B6-deficient animals, being approximately 63% and 81% respectively of the corresponding activity in control rats. As a consequence, significant modifications in the relative molar content of microsomal fatty acids were observed. The content of arachidonic and docosahexaenoic acid, the main products of the conversion of LA and ALA respectively, decreased, LA content increased and a decrease in the unsaturation index was observed in liver microsomes of B6-deficient rats. The foregoing results suggest that the impairment of D6D activity by vitamin B6 deficiency might be an important factor in decreasing the synthesis of n-6 and n-3 PUFAs. This may be particularly important in aging, where D6D activity is already impaired.

We previously demonstrated that cultured neonatal rat myocytes have the capacity to desaturate/elongate essential fatty acids, α-linolenic acid conversion being higher than linoleic acid conversion. The whole process of highly unsaturated fatty acid formation from linoleic and α-linolenic acids slows with aging. In this study we grew heart myocytes in culture for different periods of time, and we observed a decrease in the desaturating/elongating activities for both substrates as the cells aged in culture. Alpha-linolenic acid conversion into highly unsaturated fatty acids was less impaired by aging than linoleic acid conversion. These modifications are correlated to the age-dependent alterations observed in the total lipid fatty acid composition, which caused a decrease in the unsaturation index. Changes in the lipid composition that occur in aging cultures parallel those reported for several tissues upon aging in the whole animal. The data herein reported may suggest the possibility of counteracting the effects of aging on lipid metabolism by supplementing cultures with appropriate amounts of highly unsaturated fatty acids.

We have evaluated the effects of three different 18:3n-6, 20:5n-3 and 22:6n-3 fatty acid combinations on essential fatty acid (EFA) metabolism in rat cultured cardiomyocytes. The desaturating/elongating activities for linoleic (LA) and alpha-linolenic acid (ALA) were evaluated by radiolabeling the cells with 1-[14C]LA or 1-[14C]ALA and the fatty acid pattern of cardiomyocytes was assessed by gas chromatography. LA and ALA conversion to more unsaturated metabolites was reduced by increasing respectively n-3 and n-6 fatty acid concentration in the media. The all three combinations used reduced the saturated and increased the polyunsaturated fatty acid content of cardiomyocytes. The n-6/n-3 fatty acid ratio did not change compared to control cells in cardiomyocytes receiving the highest amount of 18:3n-6 and the lowest amounts of n-3 fatty acids. This combination may be suitable for modifying EFA desaturating/elongating activities without altering the physicochemical parameters which are related to the correct balance between n-6 and n-3 fatty acid content.

In human airway epithelial cell lines 9HTEo- and CFNPE9o, histamine causes a transient elevation of intracellular free calcium concentration ([Ca2+]i) detected by fura 2 fluorescence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca(2+)-ATPase inhibitor thapsigargin. Histamine also stimulates inositol phosphate accumulation. Changes in [Ca2+]i and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10(-4) M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca2+]i are abolished by a short-term preincubation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regulation by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phospholipase C through a pertussis toxin-insensitive G protein.

Intracellular free calcium concentration, phosphoinositide turnover, and inositol phosphate production were analyzed in peripheral blood lymphocytes from seven well-nourished alcoholic patients without severe acute or chronic liver disease, before and after stimulation with anti-CD3 antibody. Seven comparable nondrinkers were studied as controls. A lower increase in intracellular free calcium concentration was detected in alcoholics, after anti-CD3 stimulation of lymphocytes, than in control subjects. Lymphocyte activation generated inositol phosphates in both controls and alcoholics, but inositol phosphate production was significantly lower in alcoholics. The agreement between these findings indicates that the reduction in inositol phosphates is one of the most important events in the early phases of lymphocyte activation in alcoholics.

In this study we demonstrate that cultured rat cardiomyocytes possess the capacity to desaturate/elongate essential fatty acids (EFAs). Alpha-linolenic acid (ALA) conversion to higher metabolites was greater than linoleic acid (LA) conversion, according to the higher affinity of the delta-6-desaturase enzyme for the n-3 than for the n-6 EFAs. Gamma-linolenic acid (GLA) supplementation to the culture medium had no influence on LA conversion; but the addition of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids significantly decreased the formation of interconversion products from LA. The conversion of ALA to higher metabolites was greatly affected by GLA; EPA had no effect on ALA conversion, while DHA significantly inhibited it. Both GLA (converted mostly to dihomo-gamma-linolenic acid) and EPA can be removed from phospholipids and addressed to prostanoid biosynthesis, so avoiding their potential accumulation and the inhibition of their own production. Our data clearly indicate that supplementation of the culture medium with either n-6 or n-3 fatty acids can cause reduced levels of the other series of fatty acids. This effect may be undesirable, since both n-6 and n-3 fatty acids are important in the prevention of coronary diseases.

In many cell types, agonists can stimulate both phosphoinositide (PtdIns) and phosphatidylcholine (PC) hydrolysis by activating specific phospholipases. Using cultures of neonatal rat cardiomyocytes we have verified the existence of an α 1 -adrenoceptor mediated hydrolysis of PtdIns and PC. PtdIns breakdown, evaluated as inositol phosphate production, occurred in the early phase of cell stimulation, while PC hydrolysis, evaluated as choline metabolite production, was evidenced at longer stimulation times. The appearance of a delayed peak of choline phosphate and the invariance of free choline in the intracellular water phase strongly suggest the involvement of a specific PC-phospholipase C, generating choline phosphate and diacylglycerol, the activator of protein kinase C. Since it is plausible that various metabolites of signal-induced degradation of membrane phospholipids may take part in long term physiological responses, PC breakdown could be involved in cellular mechanisms that require prolonged protein kinase C activation.

Polyenylphosphatidylcholine is a choline-glycerophospholipid containing up to 80% of total fatty acids as linoleic acid and may be an important factor in ensuring normal functioning of cell membranes. We tested the effect of a polyenylphosphatidylcholine-supplemented diet and compared it with both a trilinolein-supplemented and a laboratory chow diet on the fatty acid composition, microviscosity, and delta-6-desaturase activity of liver microsomal membranes of 12-month-old rats, in the absence or presence of oxidative stress induced by adriamycin. Polyenylphosphatidylcholine- and trilinolein-supplemented diets showed a similar increase in linoleic acid content and delta-6-desaturase activity in liver microsomes, indicating that low amounts of linoleic acid are able to partially restore the enzyme activity in old rats, independent of the source of linoleic acid. After adriamycin treatment, delta-6-desaturase activity increased in polyenylphosphatidylcholine and trilinolein groups, indicating a protective mechanism against the damage induced by polyunsaturated fatty acid peroxidation. The measurement of malondialdehyde production showed a protective effect on adriamycin-induced lipid peroxidation by polyenylphosphatidylcholine supplementation only. Microsomal membrane microviscosity did not change independent of diet and adriamycin treatment, suggesting that the response of microsomes to lipid peroxidation might be the maintenance of a given membrane order. Administration of polyenylphosphatidylcholine can prevent or minimize the liver damage induced by adriamycin treatment.

Fatty acid composition of phosphatidylinositols was analyzed in peripheral blood lymphocytes from nine alcoholic patients who were well nourished and without severe acute and chronic liver disease, before and after stimulation with anti-CDs antibody. Six comparable nondrinkers were studied as controls. A reduction in unsaturated fatty acid (mainly arachidonic) and an increase in palmitic and stearic acid molar content were observed in phosphatidylinositol (PI), phos-phatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bis-phosphate (PIP2) in unstimulated samples from alcoholic patients in comparison with normal subjects, leading to a significant decrease in the saturated/unsaturated ratio. In controls, anti-CD3 stimulation caused a marked decrease in arachidonic acid relative molar content counterbalanced by an increase in other polyunsaturated fatty acid relative molar content in PI, PIP, and PIP2 fractions. Interestingly, after anti-CD3 stimulation, alcoholic patients show the same trend of modification in the fatty acid composition resulting in a sharp reduction of arachidonic acid relative molar content. These results support the hypothesis of an alteration in nutrients being responsible for immune derangement in alcoholics.

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after alpha 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the "in vivo" ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.

Previous studies showed no differences in the phospholipid content of human meningiomas compared to normal leptomeninges, but only a higher unsaturation degree in the individual phospholipid fractions of tumors. Inasmuch as phosphoinositides play a role in the membrane responsiveness to numerous effectors, we studied the fatty acid pattern of the different phosphoinositide fractions of 14 human meningiomas of different histological origin. The fatty acid analysis revealed remarkable differences among the histological types, and, above all, among the different phosphoinositide fractions of a single tumor class. The phosphoinositides derived from transitional meningiomas appeared to be the most saturated ones, because of their low arachidonic acid content. Furthermore, in all the meningiomas, long chain polyunsaturated fatty acids were present only in the phosphatidylinositol fractions and the polyphosphorylated compounds appeared to be significantly different from the corresponding monophosphorylated ones. The possible significance of the different fatty acid distribution in the three phosphoinositide classes is discussed.

The fatty acid pattern of phosphatidylinositol and other inositol phospholipids is reported to be predominantly 1-stearoyl, 2-arachidonyl. However, literature does not report data about the effect of a modification of this fatty acid composition on the production and acidic pattern of the diacylglycerol (DAG) formed during phosphoinositide hydrolysis. Culturing cardiomyocytes in a docosahexaenoic acid supplemented medium, we obtained an homogeneous cell population whose phospholipid fatty acid pattern was strongly different from control cells, and which produced, after α1-adrenergic stimulation with phenylephrine, an higher amount of DAG. This DAG was different from control DAG in fatty acid composition, too. This structurally different DAG could be responsible for a different activation pattern of protein kinase C.

The delta-6-desaturase (D6D) activity was evaluated in microsomes from liver fragments of cholecystectomized subjects without any liver pathology and from explanted liver of patients affected by cirrhosis of different etiologies. We observed a significant decrease in D6D activity, evaluated by a radiochemical technique using 1-[14C]-linoleic acid as substrate, in cirrhotic patients with no correlation with the etiology of the cirrhosis. The D6D activity within the pathological group was quite similar. No alteration in the 20:4 18:2 ratio obtained by gas chromatographic analysis of fatty acid methyl esters of microsomal membranes was found. Liver disease seems to be the main cause of the decreased enzyme activity independent of its etiology.

The possibility of enhancing glutathione peroxidase (GPx) activity and cytosolic total antioxidant activity (TAA) in normoxia and hypoxia/reoxygenation (H/R) by the supplementation of different concentrations of sodium selenite (SS) or selenomethionine (SM) was investigated in cultured rat cardiomyocytes. To assess the entity of oxidative stress due to H/R, levels of conjugated dienes containing lipids were determined. In normoxia, GPx activity and TAA increased in parallel with the increase in SS and SM supplementation. H/R did not influence GPx activity but lowered TAA; both SS and SM supplementations were effective in increasing GPx activity, the most effective concentration being 1 microM. At this SS and SM concentration, TAA returned to a normoxic value. Conjugated diene production, increased by H/R, was reduced by SS and SM supplementation, the 1 microM concentration appearing to be the most effective one. According to these data Se supplementation represents another possibility to counteract oxidative damage in the myocardium.

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with alpha-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT1 in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by alpha TC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.

Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.

Some thiazolylbenzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Compounds 2d and 2e show a very good activity on K562 cells, whereas compounds 2a and 2b are active on SW620 cells. The importance of the methoxy group on the quinone moiety is confirmed and the function at 4-position of the thiazole ring plays a determining role for the activity.

Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation. During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells. To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis. Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells. Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells. Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis. Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis. So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging.

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o − ), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8–10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8–10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G i/o protein-coupled receptor.

In long term (21 days) primary cultures of neonatal rat cardiomyocytes, utilized as a model of in vitro senescence, we investigated the dual effect of the time length in culture and of the supplementation with n-6:n-3 fatty acid mixtures on linoleic (LA) and α-linolenic acid (ALA) metabolism. Cardiomyocytes were divided into groups receiving: (1) control medium; (2) control medium plus n-3 fatty acids; (3) and (4) control medium plus n-6 and n-3 fatty acids in the ratio 1:2 or 2:1, respectively. In control cells, senescence caused a reduction in the conversion of LA and ALA, and the decrease in their metabolites was bypassed by the different supplementations. The fatty acid composition of cardiomyocyte lipids was therefore affected by both senescence and supplementation, as evidenced by the n-6:n-3 fatty acid ratio and the unsaturation index (U.I.) in cellular lipids. The final result of ageing in culture and of fatty acid supplementations was in all the groups of cells but one (n-6:n-3, 2:1) an unbalance in the n-6:n-3 fatty acid ratio. All the supplementations were able to counteract the decrease in the U.I. observed with senescence, but only the n-6:n-3 (2:1) was able to do so by increasing the cellular content of the fatty acids which are precursors of anti-aggregation eicosanoids without altering the n-6:n-3 fatty acid ratio.

After 10 months of alcohol abstinence a malnourished alcoholic patient improved his nutritional status. The analysis of peripheral blood lymphocyte response to mitogenic stimulation with the antibody anti-CD3 and of the fatty acid composition of the (poly)-phosphoinositide fraction derived from lymphocytes revealed: 1) a similar [3H]-thymidine uptake as in control (non-drinker) subjects; 2) a similar relative molar content of the main fatty acids in the (poly)-phosphoinositides as in control subjects. Alcohol abstinence can normalize both the parameters, which are greatly altered during alcohol abuse. This suggests a link between nutritional status and lymphocyte responsiveness via phosphoinositide fatty acid composition.

The metabolites of linoleic (LA) and alpha-linolenic (ALA) acids are involved in coronary heart disease. Both n-6 and n-3 essential fatty acids (EFAs) are likely to be important in prevention of atherosclerosis since the common risk factors are associated with their reduced 6-desaturation. We previously demonstrated the ability of heart tissue to desaturate LA. In this study we examined the ability of cultured cardiomyocytes to metabolize both LA and ALA in vivo, in the absence and in the presence of gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) alone or combined together. In control conditions, about 25% or LA and about 90% of ALA were converted in PUFAs. GLA supplementation had no influence on LA conversion to more unsaturated fatty acids, while the addition of n-3 fatty acids, alone or combined together, significantly decreased the formation of interconversion products from LA. Using the combination of n-6 and n-3 PUFAs, GLA seemed to counterbalance partially the inhibitory effect of EPA and DHA on LA desaturation/elongation. The conversion of ALA to more unsaturated metabolites was greatly affected by GLA supplementation. Each supplemented fatty acid was incorporated to a significant extent into cardiomyocyte lipids, as revealed by gas chromatographic analysis. The n-6/n-3 fatty acid ratio was greatly influenced by the different supplementations; the ratio in GLA+EPA+DHA supplemented cardiomyocytes was the most similar to that recorded in control cardiomyocytes. Since important risk factors for coronary disease may be associated with reduced 6-desaturation of the parent EFAs, administration of n-6 or n-3 EFA metabolites alone could cause undesirable effects. Since they appear to have different and synergistic roles, only combined treatment with both n-6 and n-3 metabolites is likely to achieve optimum results.

BACKGROUND & AIMS: A direct comparison of jejunal and ileal absorption rates of bile acids has not been reported. The aim of this study was to compare the relative transport rates of different bile acids in the jejunum and ileum. METHODS: Jejunal and ileal rabbit intestinal segments were separately perfused with bile acid solutions, and dose- response curves were obtained for taurocholate, ursodeoxycholate, chenodeoxycholate, deoxycholate, and their glycoconjugates. Membrane fluidity and bile acid transport were assessed in brush border membrane vesicles. RESULTS: Taurocholate showed active transport in the ileum and no transport in the jejunum. Unconjugated bile acids showed passive diffusion in the two tracts, whereas glycoconjugated bile acids showed both components of transport in the ileum and passive diffusion in the jejunum (lower in the latter). A higher membrane fluidity and lower cholesterol-to-phospholipid ratio were found in the jejunum. Ursodeoxycholate reduced bile acid uptake into membrane vesicles from both ileum and jejunum. CONCLUSIONS: Active transport is limited to the ileum. Passive diffusion is higher through a less fluid membrane with a higher cholesterol-to-phospholipid ratio in the ileum than in the jejunum. Ursodeoxycholate inhibition may be at the level of a facilitated, sodium-independent diffusion in the jejunum. (Gastroenterology 1996 Feb;110(2):459-68)

We have investigated the incorporation of cholesterol oxidation products into cardiomyocyte lipids and related this to changes in cell proliferation, evaluated by measuring cellular protein content. Primary cultures of neonatal rat ventricular cells were supplemented with scalar concentrations of several cholesterol oxidation products (cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5 alpha-cholestane-3 beta, 5, 6 beta-triol, 5-cholesten-3 beta, 4 beta-diol, 5-cholesten-3 beta-ol-7-one, and 5-cholesten-3-one). Although all the cholesterol oxidation products were incorporated into the cardiomyocyte lipids when added to the medium at a concentration higher than 0.5 microM, the extent of the incorporation of the different cholesterol oxidation products differed, depending on the concentration in the culture medium and on the chemical structure of the compound. The effects of the cholesterol oxidation products on the cellular protein content were also different: 5 alpha-cholestane-3 beta, 5, 6 beta-triol was shown to be the most potent inhibitor of cell proliferation, followed by cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5-cholesten-3 beta, 4 beta-diol and 5-cholesten-3 beta-ol-7-one. 5-Cholesten-3-one did not affect the cellular protein content. The ability of cholesterol oxidation products to inhibit cell proliferation, and their capacity to increase the permeability of the plasma membrane to calcium, could be deleterious for cardiac cells.

The immunological alterations observed in chronic alcoholic patients may be due to alterations of signal transduction across the lymphocyte membrane. Upon binding of mitogens or antigens to specific plasma membrane receptors, the activation of phospholipase C leads to the hydrolysis of inositol phospholipids, producing inositol phosphates and diacylglycerol. One of the early events in lymphocyte activation is an increase of intracellular calcium concentration, due to both an influx from extracellular fluid and a release from intracellular stores mediated by inositol phosphates. In this study we verified whether the diminished mobilization of intracellular calcium, previously observed in alcoholics, is caused by alteration in phosphoinositide turnover. We evaluated total inositol phosphate production in peripheral blood lymphocytes after anti-CD3 stimulation, comparing control subjects and alcoholic patients. Lymphocyte activation generated inositol phosphates in both controls and alcoholics, but to a different extent, inositol phosphate production being significantly higher in controls than in alcoholics. This reduction in inositol phosphate production could be accounted either to an inhibition of PLC activity or to a modified affinity of phospholipase C for its own substrates, i.e., phosphoinositides, which fatty acid composition has been previously demonstrated to be greatly different in alcoholics in comparison to healthy subjects.

The fatty acid composition of microsomal membranes from Morris hepatomas 9618A, slow growing, and 3924A, fast growing, confirm the higher content in oleic acid and the loss of PUFAs of the tumours with respect to controls. The specific activities of delta-9-desaturase indicate alternative metabolic pathways for the increased production of oleic acid in the two hepatomas. The delta-6-desaturase activity is much lower in tumours than in controls. However the loss of PUFAs found in tumours seems to be mostly due to a low content in linoleic acid.

In order to evaluate the effect of one of the main oxysterols derived from cholesterol oxidation, cholesterol-5 alpha,6 alpha-epoxide (epox), on cardiac cells, we have supplemented the culture medium of neonatal rat cardiomyocytes with scalar concentrations of epox (0.1-100 microM). While 0.1 microM epox supplementation was ineffective, epox supplementation in the range 1-100 microM determined a reduction in cellular protein level, without affecting cell viability, and a dose-dependent epox incorporation into cardiomyocyte lipids. Furthermore, in the same concentration range of epox supplementation, a gas chromatographic peak unambiguously identified by gas chromatography-mass spectrometry as cholestane-3 beta,5 alpha,6 beta-triol, an hydrolytic metabolite of epox, was detected. The mechanism of cytotoxicity of epox to cardiomyocytes could be due to the insertion of epox itself into cellular lipids, and to its metabolization to the more toxic triol.

A double blind placebo-controlled study of two doses of gamma-linolenic acid, provided by evening primrose oil (EPO, Epogam, Searle, U.K.), in children with atopic dermatitis was performed: 1) to examine the effect of gamma-linolenic acid administration on the clinical status of children with atopic dermatitis and abnormalities of IgE-mediated immune responses compared to those without such IgE abnormalities; 2) to investigate the effect of gamma-linolenic acid on red cell fatty acid composition and 3) to assess whether treatment with gamma-linolenic acid induced changes in red cell membrane microviscosity. A significant improvement in the overall severity of the clinical condition was seen in children treated with gamma-linolenic acid, independent of whether the children had manifestations of IgE-mediated allergy. Furthermore, gamma-linolenic acid treatment increased the percentage content of n-6 fatty acids in erythrocyte cell membrane; this increase was more marked in the membranes of children treated with high doses of EPO. In the high dose group a significant increase in dihomogamma-linolenic acid (DGLA) occurred. This may be of particular relevance because of the potential importance of DGLA as a precursor of antiinflammatory prostanoids. Red cell membrane microviscosity did not change in any group after treatment with EPO, even in high doses, despite a significant increase in the proportion of long chain polyunsaturated fatty acids.

Using cultures of beating neonatal rat cardiomyocytes we have studied the fatty acid composition of the diacylglycerol produced after different stimulation times with an alpha 1-agonist (phenylephrine) and we have related it to the previously reported time course of the activation of particulate protein kinase C, in control cells and in cells grown in a medium supplemented with docosahexaenoic acid. Gas chromatography of the diacylglycerol produced after stimulation revealed significant differences between control cells and cells treated with docosahexanoic acid. In the cells treated with docosahexanoic acid, the more persistent activation of the membrane-bound protein kinase C might be sustained by an enrichment of diacylglycerol with docosahexanoic acid. The modification of the fatty acid composition of diacylglycerol can cause an alteration in the response of the cells to alpha 1-adrenoceptor stimulation.

The interaction of the T-cell receptor complex with the ligands is associated with early molecular events involved in the process of signal transduction implicating phosphoinositide breakdown. In elderly people, abnormalities in membrane signal transduction pathways are the basis of the immune deficiency associated with aging. Peripheral blood lymphocytes from aging humans and young subjects were stimulated with anti-CD3 monoclonal antibody and the phosphoinositide fractions were analyzed in order to determine the fatty acid composition in resting and stimulated conditions. In aging humans, in resting conditions, the all three phosphoinositide fractions appeared more saturated than the corresponding fractions in young subjects. Following anti-CD3 stimulation a decrease in arachidonic acid relative molar content was detected in both young and old subjects, but the arachidonic acid content in resting conditions greatly differed between the two groups, suggesting a different modulation of the microenvironment of the T-cell receptor complex in elderly people, so determining alterations in the early activation steps of lymphocytes.

We have studied the fatty acid composition of the diacylglycerol produced after different stimulation times with an alpha 1-agonist (phenylephrine) in cultures of beating neonatal rat cardiomyocytes, and we have related the acidic pattern to the time course of the translocation of protein kinase C from cytosol to the membrane, both in control cells and in cells grown in a medium supplemented with docosahexaenoic acid. Gas chromatography of the diacylglycerol produced after stimulation revealed significant differences between control cells and cells grown in the docosahexaenoic acid supplemented medium. In the control cells, in the early stimulation times, the higher protein kinase C activity was due to a higher relative molar content of arachidonic acid in the diacylglycerol; in the docosahexaenoic acid treated cells the lower but more persistent activation of the membrane-bound protein kinase C might be sustained by an enrichment of diacylglycerol with docosahexaenoic acid. The modification of the fatty acid composition of diacylglycerol can cause an alteration in the response of the cells to alpha 1-adrenoceptor stimulation.

Using cultures of beating cardiomyocytes from neonatal rats, we have studied the fatty acid composition of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and the effect of supplementing the culture medium with docosahexaenoic acid on the fatty acid composition of the three phosphoinositides. Docosahexaenoic acid was incorporated into the phosphatidylinositol fraction of the supplemented cells, but not into the phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate fractions. At complete confluence, the cardiomyocytes were stimulated with an alpha 1-agonist (phenylephrine). This altered the acidic pattern of the phosphoinositides in both control and supplemented cells. The differences observed between the polyphosphorylated classes and the phosphatidylinositol fraction suggest the existence of different mechanisms of selection of fatty acids in the biosynthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate.

Kinetic parameters of the delta-6-desaturation reaction were determined using both cis-linoleic and alpha-linolenic acid as substrates in liver microsomes of rats of different ages. The Km value for delta-6-desaturation of linoleic acid increased proportionally to the animal age, while the Vm did not change until 25 months of age. The Km values for alpha-linolenic acid were similar in young and senescent rats; on the contrary there was a significant aging influence on the Vm values. The affinity of the enzyme for the (n-3) series substrate was not so influenced by aging as the affinity for the (n-6) series substrate. This loss of affinity may be a key factor in aging through altering the polyunsaturated fatty acid content and distribution into cellular phospholipids.

Adenosine has been shown to have a number of actions on the cardiovascular system [l-4]. It relaxes smooth muscles resulting in dilation of coronary arteries and exercises a local control of blood flow in relation to myocardial oxygen needs [5,6]. It is not known how adenosine causes vasodilation and how it exercises its effects on vascular smooth muscle. However, adenosine has been shown to augment the intracellular CAMP level in the ventricular myocardium [6]; moreover, in a number of tissues it has been possible to demonstrate direct stimulation of adenylate cyclase by adenosine [7]. On the other hand, CAMP and adenosine appear to be interrelated also at the level of recently discovered CAMP-adenosine binding factors [S-lo], among which the binding activity of the mouse liver protein, that depends on a process of ‘activation’ which has already been described in some detail [lo]. Therefore we searched directly for a protein capable of binding CAMP and adenosine in the heart, a tissue in which adenosine is thought to act as a physiological regulator. Here we report the purification by affinity chromatography to apparent homogeneity of a CAMPadenosine binding protein from the soluble fraction of rat heart. Recently, data have also been reported suggesting that the CAMP-adenosine binding protein from human placenta [lo] and mouse liver [ 111 is associated with SAH hydrolase activity. The protein from rat heart, purified by affinity chromatography, was devoid of SAH hydrolase activity.

Erythrocyte membrane fluidity was evaluated in chronic alcoholic patients without any liver alteration, assuming different daily ethanol amounts, and in normal subjects and related to ghost fatty acid and total lipid composition obtained by high resolution gas chromatography. Erythrocyte membrane fluidity was significantly increased in a dose dependent manner in chronic alcoholic patients respect to normal subjects. This real fluidizing effect of ethanol "in vivo" was attributed mainly to a significant increase in the polyunsaturated fatty acids amount in patient ghosts in comparison with control subjects. On the other hand the cholesterol/phospholipid ratio was not significantly affected by chronic ethanol assumption.

Some smooth muscle relaxant drugs devoid of anticholinergic action have been tested for their interaction with calmodulin, calmodulin-stimulated cyclic nucleotide phosphodiesterase activity, and uterine membrane binding sites for nitrendipine and adenosine. The myolytic activity of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems. Trimebutine maleate does not bind either to calmodulin or to nitrendipine and adenosine receptors. Tiropramide has no effect on calmodulin-dependent systems and on Ca2+ channels but it shows a competition for the A2-type adenosine receptors.

The aim of this study is to assess "in vitro" the influence of polyunsaturated phosphatidylcholine on the erythrocyte membrane fluidity in alcoholics (greater than 180 g/die) and control subjects (less than 50 g/die), investigated by the evaluation of fluorescent polarization of DPH. In both groups a significant increase of membrane microviscosity was observed after incubation with 0.04 microM and 0.08 microM of PC compared to baseline values.

The activity of delta-6-desaturase (D6D) in liver microsomes and fatty acid composition of microsomal lipids of rats of different ages were studied. The D6D activity was similar in suckling rats and in weaning rats. However, the enzyme showed a significantly decreased activity in oldest animals, and a linear correlation was found between the D6D activity and the animal age. The fatty acid composition data on total lipids of liver microsomes were consistent with the age-dependent changes in fatty acid desaturase activity. The major changes occurred in the linoleate and arachidonate fractions; the 20:4/18:2 ratio in liver microsomes decreased together with D6D activity during aging. The loss of D6D activity may be a key factor in aging through altering lipid membrane composition.

Total lipids and fatty acid composition were determined in liver plasma and mitochondrial membranes from control and dietary obese rats after 4 weeks of the experimental period. The lipid composition of liver plasma and mitochondrial membranes showed an increase of triacylglycerols in obese rats. The liver plasma membranes showed a decrease of saturated/unsaturated fatty acid ratio and an increase of (n-6) polyunsaturated fatty acids, whereas the (n-3) polyunsaturated acids were decreased. Contrary to what occurs with plasma membranes, few modifications were observed in mitochondrial membranes. Changes of the fatty acid composition of the phospholipid bilayer are of potentially great importance in structural and functional parameters of membrane. Fluidity of liver plasma membranes of dietary obese rats was highly increased, while the mitochondrial ones remained unchanged. These results can be well explained by the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+) ATPase activity (a membrane bound enzyme) was found in plasma membranes of dietary obese rats. Mitochondrial enzymatic activities and oxidative phosphorylation showed few changes except a small, but significant decrease of state 3 respiratory rate. In this study we also determined the fatty acid composition of all the foods offered to animals and their daily intakes in order to discuss their possible influence on changes in structural and functional membrane parameters.