Docosahexaenoic acid but not eicosapentaenoic acid withstands dietary cholesterol-induced decreases in platelet membrane fluidity

To determine the differenetial effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight^.day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.     

Dietary supplementation with docosahexaenoic acid,but not with eicosapentaenoic acid,reduces host resistance to fungal infection in mice

The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.     

Binding of coagulation factor XI to washed human platelets

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.     

Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.     

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1β and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase （LAAO）, NA-LAAO, was purified from the venom ofNaja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA- LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAA O greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyros＇mephosphorylationofanumber ofplatelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase C γ2. Unlike convulxin, Fc receptor γchain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO- activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography ofplatelet proteins on an NA-LAAO- Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.     

Dietary docosahexaenoic acid but not eicosapentaenoic acid suppresses lipopolysaccharide-induced interleukin-1 beta mRNA induction in mouse spleen leukocytes

Mice were fed a diet supplemented either with beef tallow (BT), BT plus ethyl eicosapentaenoate (EPA) or BT plus ethyl docosahexaenoate (DHA) for 9 weeks. EPA and DHA supplementation increased the content of the respective fatty acid in spleen leukocyte lipids, which was associated with the reduction in the arachidonate content. IL-1beta mRNA induction upon lipopolysaccharide (LPS) stimulation in spleen leukocytes in the DHA diet group was significantly lower than in the BT diet group, but the EPA diet was without any significant effect. The amount of prostaglandin E2 (PGE2) released from LPS-stimulated spleen leukocytes was significantly lower in both the EPA and DHA groups than in the BT group. Thus, dietary EPA and DHA inhibited arachidonate metabolism similarly but had different effects on IL-1beta mRNA induction in mouse spleen leukocytes.     

15-Hydroperoxyeicosapentaenoic acid, but not eicosapentaenoic acid, shifts arachidonic acid away from cyclooxygenase pathway into acyl-CoA synthetase pathway in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of eicosapentaenoic acid (EPA) and 15-hydroperoxyeicosapentaenoic acid (15-HPEPE) on the PG and AA-CoA formations from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. EPA reduced the PG and AA-CoA formations from both 60 and 5 microM AA. In contrast, 15-HPEPE decreased the PG formation without affecting the AA-CoA formation from 60 microM AA, and increased the AA-CoA formation at the expense of PG formation when 5 microM AA was used as substrate concentration. The experiments utilizing Fe(2+) and an electron spin resonance (ESR) revealed that 15-HPEPE elicits these effects in the form of hydroperoxy adduct. These results suggest that 15-HPEPE, but not EPA, has the potential to shift AA away from COX pathway into ACS pathway at low substrate concentration (close to the physiological concentration of AA).     

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.     

Docosahexaenoic acid, but not eicosapentaenoic acid, lowers ambulatory blood pressure and shortens interval QT in spontaneously hypertensive rats in vivo

This study was designed to evaluate the effects of individual dietary long-chain n-3 polyunsaturated fatty acids (LCPUFA) on hypertension and cardiac consecutive disorders in spontaneously hypertensive rats (SHR) as compared to Wistar-Kyoto rats (WKY). Rats were fed for 2 months an eicosapentaenoic (EPA)- or docosahexaenoic acid (DHA)-rich diet (240 mg/day) or an n-3 PUFA-free diet. Male SHR (n=6), implanted with cardiovascular telemetry devices, were housed in individual cages for continuous measurements of cardiovascular parameters (blood pressure (BP) and heart rate (HR)) during either activity or rest periods, ECG were recorded during the quiet period. The n-6 PUFA upstream of arachidonic acid was affected in SHR tissues. The cardiac phospholipid fatty acid profile was significantly affected by dietary DHA supply, and EPA in a very lower extent, since DHA only was incorporated in the membranes instead of n-6 PUFAs. Endothelium n-6 PUFA content increased in all SHR groups. Compared to WKY, linoleic acid content decreased in both studied tissues. Cardiac noradrenalin decreased while the adrenal catecholamine stores decreased in SHR as compared to WKY. Both n-3 PUFA supply induced a decrease of adrenal catecholamine stores. Nevertheless after 6 weeks, DHA but not EPA induced a lowering-blood pressure effect and shortened the QT interval in SHR, most probably through its tissue enrichment and a specific effect on adrenergic function. Dietary DHA supply retards blood pressure development and has cardioprotective effect. These findings, showing the cardioprotective effects of DHA in living animals, were obtained in SHR, but may relate to essential hypertension in humans.     

Effect of thrombin on the radioactive nucleotides of human washed platelets

Radioactive ATP and ADP were found in platelets after incubation of human platelet-rich plasma with either [8-(14)C]adenosine or [8-(14)C]ADP. Treatment of the labelled and washed platelets with thrombin indicated that, though considerable amounts of ATP and ADP were released to the supernatant, radioactive ATP and ADP remained predominantly in the cellular fraction. Breakdown of radioactive ATP took place to form mainly IMP and hypoxanthine, the latter compound appearing in the supernatant. The results indicate the presence of at least two pools of nucleotide in platelets. Evidence is given that the two pools contain approximately the same amounts of ATP plus ADP, and that the ratio of ATP to ADP in the pool released to the supernatant by the action of thrombin is about 0.7-0.8.     

Receptors for high molecular weight kininogen on stimulated washed human platelets

Binding of human high molecular weight kininogen to washed human platelets was studied by measuring platelet-associated radiolabeled ligand in pellets of centrifuged platelets. High molecular weight kininogen was bound to stimulated platelets in the presence of ZnCl2 in a specific and saturable manner. Calcium ions potentiated ligand binding but did not substitute for zinc ions. Optimal binding of high molecular weight kininogen occurred near the plasma concentrations of both zinc and calcium ions. Scatchard analysis yielded 24 200 binding sites for high molecular weight kininogen with an apparent dissociation constant of 20 nM. These studies show that stimulated human platelets can bind many high molecular weight kininogen molecules with high affinity and suggest that the platelet surface may potentially serve as an important site for localizing the initial reactions of the plasma kinin-forming and intrinsic coagulation pathways.     

Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca(2+)](i) and pH(i) were determined by loading the cells with the fluorescent dyes, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2',7'-bis(2-carboxyethyl)-5-(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [(3)H]-thymidine. The results indicated that (1) LPS (10 microg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca(2+)](i) and [(3)H]-thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [(3)H]-thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca(2+)](i) elevation.     

Monosodium glutamate but not linoleic acid differentially activates gustatory neurons in the rat geniculate ganglion

To date, only one study has examined responses to monosodium glutamate (MSG) from gustatory neurons in the rat geniculate ganglion and none to free fatty acids. Accordingly, we recorded single-cell responses from geniculate ganglion gustatory neurons in anesthetized male rats to MSG and linoleic acid (LA), as well as to sucrose, NaCl, citric acid, and quinine hydrochloride. None of the 52 neurons responded to any LA concentration. In contrast, both narrowly tuned groups of gustatory neurons (sucrose specialists and NaCl specialists) responded to MSG, as did 2 of the broadly tuned groups (NaCl generalist(I) and acid generalists). NaCl-generalist(II) neurons responded only to the highest MSG concentration and only at low rates. No neuron type responded best to MSG; rather, responses to 0.1 M MSG were significantly less than those to NaCl for Na(+) -sensitive neurons and to sucrose for sucrose specialists. Interestingly, most Na(+) -sensitive neurons responded to 0.3 M MSG at levels comparable with those to 0.1 M NaCl, whereas sucrose specialists responded to 0.1 M MSG despite being unresponsive to NaCl. These results suggest that the stimulatory effect of MSG involves activation of sweet- or salt-sensitive receptors. We propose that glutamate underlies the MSG response of sucrose specialists, whereas Na(+) -sensitive neurons respond to the sodium cation. For the latter neuron groups, the large glutamate anion may reduce the driving force for sodium through epithelial channels on taste cell membranes. The observed concentration-dependent responses are consistent with this idea, as are cross-adaptation studies using 0.1 M concentrations of MSG and NaCl in subsets of these Na(+) -sensitive neurons.     

Cathepsin G activates protease-activated receptor-4 in human platelets

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Interferon alpha but not interleukin 12 activates STAT4 signaling in human vascular endothelial cells

STAT4 signaling, activated by either interleukin 12 (IL12) or interferon alpha (IFNalpha), promotes T(H)1 responses in CD4(+) T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of T(H)1 or T(H)2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNalpha but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rbeta1 and IL12Rbeta2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNalpha responses in STAT4-overexpressing EC; and analysis of IFNalpha responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNalpha but not IL12, and that it may modulate the pro-inflammatory behavior of EC.