Invited review: Plant polyphenols and rumen microbiota responsible for fatty acid biohydrogenation, fiber digestion, and methane emission: Experimental evidence and methodological approaches.

The interest of the scientific community in the effects of plant polyphenols on animal nutrition is increasing. These compounds, in fact, are ubiquitous in the plant kingdom, especially in some spontaneous plants exploited as feeding resources alternative to cultivated crops and in several agro-industry by-products. Polyphenols interact with rumen microbiota, affecting carbohydrate fermentation, protein degradation, and lipid metabolism. Some of these aspects have been largely reviewed, especially for tannins; however, less information is available about the direct effect of polyphenols on the composition of rumen microbiota. In the present paper, we review the most recent literature about the effect of plant polyphenols on rumen microbiota responsible for unsaturated fatty acid biohydrogenation, fiber digestion, and methane production, taking into consideration the advances in microbiota analysis achieved in the last 10 yr. Key aspects, such as sample collection, sample storage, DNA extraction, and the main phylogenetic markers used in the reconstruction of microbial community structure, are examined. Furthermore, a summary of the new high-throughput methods based on next generation sequencing is reviewed. Several effects can be associated with dietary polyphenols. Polyphenols are able to depress or modulate the biohydrogenation of unsaturated fatty acids by a perturbation of ruminal microbiota composition. In particular, condensed tannins have an inhibitory effect on biohydrogenation, whereas hydrolyzable tannins seem to have a modulatory effect on biohydrogenation. With regard to fiber digestion, data from literature are quite consistent about a general depressive effect of polyphenols on gram-positive fibrolytic bacteria and ciliate protozoa, resulting in a reduction of volatile fatty acid production (mostly acetate molar production). Methane production is also usually reduced when tannins are included in the diet of ruminants, probably as a consequence of the inhibition of fiber digestion. However, some evidence suggests that hydrolyzable tannins may reduce methane emission by directly interacting with rumen microbiota without affecting fiber digestion.

Effect of different dietary tannin extracts on lamb growth performances and meat oxidative stability: comparison between mimosa, chestnut and tara.

Little information is available on the effects of different sources of tannins on ruminant product quality. Nowadays several tannin-rich extracts, produced from different plants, are available and contain tannins belonging to different chemical groups, but most of these have not been used so far as feed supplements. The present study aimed at comparing the effects of feeding three tannin extracts (one containing condensed tannins and two containing hydrolysable tannins) to lambs on growth performances and meat oxidative stability. Comisana male lambs were divided into four groups (n=9 each) and were fed for 75 days: a concentrate-based diet (CON), or CON supplemented with 4% tannin extracts from either mimosa ( MI; Acacia mearnsii, De Wild; condensed tannins), chestnut (CH; Castanea sativa, Mill; hydrolysable ellagitannins) or tara (TA; Cesalpinia spinosa, (Molina) Kuntze; hydrolysable gallotannins). Only CH reduced growth rate, final weight, carcass weight and feed intake (P0.05). The TA diet increased (P<0.001) the concentration of γ-tocopherol in muscle and tended to increase that of α-tocopherol (P=0.058). Oxidative stability of raw and cooked meat, or of meat homogenates incubated with pro-oxidants, was not affected by the extracts. These results, compared with those reported in the literature, highlight that some effects of tannins cannot be easily generalized, but may strictly depend on their specific characteristics and on conditions inherent to the basal diet and the metabolic status of the animals.

Variations in stable isotope ratios in lamb blood fractions following dietary changes: a preliminary study.

RATIONALE: In meat production it is common practice to use finishing diets based on concentrates, even for those animals previously raised on pasture. No studies have investigated the variations of stable isotope ratios in lamb plasma and erythrocytes, following a switch from pasture to a concentrate-based diet during the last days before slaughter. For meat traceability it is important to verify how and whether these parameters are affected in blood fractions. METHODS: Blood of ten male Italian Merino lambs, whose diet was switched from pasture to concentrate, was sampled 8 times (days 1, 2, 3, 4, 5, 6, 7 and 14) in the last 14 days before slaughter. The variations in the δ(13)C, δ(15)N, δ(18)O and δ(34)S values of blood plasma and erythrocytes were investigated. The stable isotope ratios of the samples were measured using a stable isotope mass spectrometer coupled with an elemental analyser (C, N, S) and a CO2/H2O equilibration system (O(plasma)). RESULTS: The δ(13) C(plasma), δ(18)O(plasma) and δ(34)S(plasma) values were shown to be different 7 days after the abrupt variation in the diet. The comparison between erythrocytes and plasma stable isotope ratios could be suitable for verifying whether the animal was actually pasture-raised and could merit a higher price. The erythrocytes isotopic signature was not affected by very short finishing periods in previously grazed animals and maintained the pasture-raised fingerprint, while the analysis of plasma could detect very short finishing periods with concentrate and hay. CONCLUSIONS: The present study has demonstrated that the combination of blood plasma and erythrocytes stable isotope ratios of carbon, nitrogen, oxygen and sulphur can be used to infer the dietary background of lambs and thus offers a tool for the authentication of the animal production system.

Volatiles in raw and cooked meat from lambs fed olive cake and linseed.

This study was conducted to determine the effects of feeding olive cake and linseed to lambs on the volatile organic compounds (VOCs) in raw and cooked meat. Four groups of eight male Appenninica lambs each were fed: conventional cereal-based concentrates (diet C), concentrates containing 20% on a dry matter (DM) basis of rolled linseed (diet L), concentrates containing 35% DM of stoned olive cake (diet OC), or concentrates containing both rolled linseed (10% DM) and stoned olive cake (17% DM; diet OCL). The longissimus dorsi muscle of each lamb was sampled at slaughter and was subjected to VOC profiling through the use of SPME-GC-MS. In the raw meat, the concentration of 3-methylpentanoic acid was higher in treatment C as compared with treatments L, OC and OCL (P<0.01). Moreover the level of nonanoic acid was greater in treatments C and OC than in treatment L (P<0.05). With respect to alcohols, in raw meat the amount of 2-phenoxyethanol in treatment OCL was lower than in treatments C (P<0.01) and OC (P<0.05), while in cooked meat the amount of 1-pentanol was higher in treatment C than in treatment OC (P<0.05). Apart from these compounds, none of the lipid oxidation-derived volatiles was significantly affected by the dietary treatment. Therefore, the results suggest that the replacement of cereal concentrates with linseed and/or olive cake did not cause appreciable changes in the production of volatile organic compounds in lamb meat.

Antioxidant effects of ryegrass phenolics in lamb liver and plasma.

Sixteen lambs were divided into two groups and fed two different diets. Eight lambs were stall-fed with a concentrate-based diet (C), and the remaining eight lambs were allowed to graze on Lolium perenne (G). The antioxidant status was measured in the liver and plasma samples before and after solid-phase extraction (SPE) to probe the antioxidant effects that grass phenolic compounds may have conferred onto the animal tissues. The liver and plasma samples from grass-fed lambs displayed a greater antioxidant capacity than the tissues from C lamb group, but only if samples had not been passed through SPE cartridges. Finally, the feed and animal tissues, which had been purified by SPE, were analysed by liquid chromatography combined with mass spectrometry (LC���MS) to identify phenolic compounds present in L. perenne and to evaluate the results from the antioxidant assays. It would appear that the improvement of the antioxidant capacity of lamb liver and plasma from lambs fed ryegrass was not related to the direct transfer of phenolic compounds from grass to the animal tissues.

Stable isotope ratios of blood components and muscle to trace dietary changes in lambs.

Multielemental stable isotope ratio (SIR) analysis was used in lamb plasma, erythrocytes and muscle to detect the switch from a pasture- to a concentrate-based diet, with the aim of verifying the possibility to trace the change of feeding in animal tissues. During 89 days of experimental feeding, lambs were subjected to four dietary treatments: pasture (P), pasture followed by concentrate in the stall for either 14 days (P-S14) or 37 days (P-S37) or concentrate in the stall (S). Pasture and concentrate diets comprised C3 plants only and had different values of 13C/12C, 18O/16O, 2H/1H and 34S/32S ratios. Muscle 13C/12C and 34S/32S and plasma 13C/12C and 18O/16O ratios in P, P-S14 and P-S37 lambs were significantly different. A multivariate analytical approach revealed that 13C/12C and 18O/16O ratios in plasma were the most powerful variables for the discrimination among the dietary treatments.

Effect of Quillaja saponaria dietary administration on colour, oxidative stability and volatile profile of muscle longissimus dorsi of Barbarine lamb.

Eighteen Barbarine lambs were assigned during 77 days to three dietary treatments (n=6): control, oat hay ad libitum and 400 g of concentrate; QS60 and the QS90 control diet supplemented with 60 mg and 90 mg Quillaja saponaria (QS) bark extract/kg dry matter, respectively. The analysis of pre-frozen longissimus dorsi muscle showed that the QS90 treatment reduced meat redness (a*) and saturation (C*) measured after 2h of blooming. It also reduced the rate of decrease in a* values (P=0.02) during 14 days of refrigerated storage. Supplementation with QS extended meat colour stability by reducing (P<0.05) the rate of increase in hue angle (H*) values. Neither the rate of metmyoglobin accumulation at the meat surface nor lipid peroxidation over storage duration differed between treatments. The overall meat volatile compound profile was similar between the groups. We conclude that supplementing QS affects meat colour development at the meat surface and extends its stability without producing detrimental effects on meat volatile compounds.

The restriction of grazing duration does not compromise lamb meat colour and oxidative stability.

Over 72 days, 33 lambs were fed: concentrates in stall (S), grass at pasture for 8 hours (8 h), or grass at pasture for 4 hours in the afternoon (4h-PM). The 4h-PM treatment did not affect the carcass yield compared to the 8h treatment. Meat colour development after blooming was unaffected by the treatments. The 4 h-PM treatment increased the proportion of polyunsaturated fatty acids (PUFA; P<0.0005) and of the highly peroxidizable fatty acids (HP-PUFA; P<0.001) in meat compared to the 8h treatment. The S treatment increased lipid oxidation (higher TBARS values) and impaired colour stability (higher H* values) of meat over storage compared to the 8h and 4 h-PM treatments (P<0.0005 and P=0.003, respectively). No difference in meat oxidative stability was found between the 8h and the 4h-PM treatments. In conclusion, growing lambs can tolerate a restriction of grazing duration without detrimental effects on performances and meat oxidative stability.

Vitamin E and polyunsaturated fatty acids in bovine muscle and the oxidative stability of beef from cattle receiving grass or concentrate-based rations.

The present study was designed to assess the balance between antioxidant and prooxidant components and the oxidative stability of beef from cattle fed exclusively grazed pasture (PAS) or a barley-based concentrate offered indoors (CONC) for 11 mo, or fed grass silage indoors for a 5-mo winter period, followed for the remaining 6-mo summer period by grazed pasture (SiP) or by grazed pasture plus concentrate at 50% of the dietary DM (SiPC). Muscle prooxidant and antioxidant components were determined by measuring fatty acids and α-tocopherol concentration of LM, respectively. Lipid oxidation and color stability were monitored in ground LM, packaged in a high-oxygen modified atmosphere, over 11 d of refrigerated storage. Vitamin E concentration decreased (P < 0.0005) with an increasing proportion of concentrate in the diet (2.59, 2.45, 1.76, and 1.15 µg/g for PAS, SiP, SiPC, and CONC, respectively). A greater proportion of PUFA was found in LM from cattle in the PAS, SiP, and SiPC groups compared with animals in the CONC group (9.62, 11.04, 8.96, and 6.94%, respectively; P < 0.0005). A greater concentration of highly peroxidizable PUFA was found in LM from heifers in the PAS, SiP, and SiPC groups compared with those in the CONC group (0.84, 0.85, 0.87, and 0.65 mg/g of muscle, respectively; P = 0.02). Dietary treatment affected lipid oxidation (P < 0.0005), with greater 2-thiobarbituric acid reactive substance values in beef from heifers in the SiPC group than in beef from those in the PAS, SiP, and CONC groups. Dietary treatment affected myoglobin oxidation (P = 0.002) during storage, with greater metmyoglobin accumulation in beef from animals receiving concentrate (CONC and SiPC treatments) than in beef from cattle in the PAS and SiP groups. Consequently, feeding concentrate impaired meat color stability over the storage duration, with greater H* (hue angle) values (P < 0.0005) in meat from heifers in the SiPC and CONC groups compared with meat from those in the PAS and SiP groups. The results of the present study confirm a positive effect of grass-based feeding systems on meat color stability compared with concentrate-based dietary strategies. It appears that vitamin E in muscle alone does not explain the resistance of meat to oxidative deterioration because a clear interaction with highly peroxidizable PUFA exists.

Effect of dietary saponins from Quillaja saponaria L. on fatty acid composition and cholesterol content in muscle Longissimus dorsi of lambs.

The purpose of this study was to evaluate the effects of increasing levels of saponins from Quillaja saponaria on fatty acid (FA) composition and cholesterol content in muscle Longissimus dorsi of lambs. A total of 24 Barbarine lambs were assigned to four dietary treatments: control diet (C) consisting of oat hay ad libitum and 400 g of concentrate (80% barley, 17.5% soybean meal and 2.5% vitamin and mineral supplement); C diet plus 30 ppm of Q. saponaria L. (QS30); C diet plus 60 ppm of Quillaja (QS60); C diet plus 90 ppm of Quillaja (QS90). Saponin supplementation reduced the concentration of C14:1 cis-9 (P = 0.001) and of its desaturation index (P = 0.002). None of the FA intermediates of ruminal biohydrogenation (BH) was affected by Quillaja saponin supplementation (P > 0.05). The concentration of C20:4n-6 was higher in the meat of animals receiving 60 ppm of Quillaja than C and QS30 groups. Supplementing 60 ppm of Quillaja reduced the ratio between α-linolenic and linoleic acids compared with the C group (P = 0.023). We did not find any significant effect of Quillaja saponins on muscle cholesterol level. Further investigations are necessary to assess the metabolic fate of saponins in the rumen and to understand whether there is an effect of saponin on Δ9-desaturase enzyme activity, ruminal BH and cholesterol metabolism in ruminants. Supplementing up to 90 ppm of Quillaja saponins did not produce detrimental effects on the overall meat FA profile.

Altered redox status of coenzyme Q9 reflects mitochondrial electron transport chain deficiencies in Caenorhabditis elegans.

Mitochondrial disorders are often associated with primary or secondary CoQ10 decrease. In clinical practice, Coenzyme Q10 (CoQ10) levels are measured to diagnose deficiencies and to direct and monitor supplemental therapy. CoQ10 is reduced by complex I or II and oxidized by complex III in the mitochondrial respiratory chain. Therefore, the ratio between the reduced (ubiquinol) and oxidized (ubiquinone) CoQ10 may provide clinically significant information in patients with mitochondrial electron transport chain (ETC) defects. Here, we exploit mutants of Caenorhabditis elegans (C. elegans) with defined defects of the ETC to demonstrate an altered redox ratio in Coenzyme Q9 (CoQ9), the native quinone in these organisms. The percentage of reduced CoQ9 is decreased in complex I (gas-1) and complex II (mev-1) deficient animals, consistent with the diminished activity of these complexes that normally reduce CoQ9. As anticipated, reduced CoQ9 is increased in the complex III deficient mutant (isp-1), since the oxidase activity of the complex is severely defective. These data provide proof of principle of our hypothesis that an altered redox status of CoQ may be present in respiratory complex deficiencies. The assessment of CoQ10 redox status in patients with mitochondrial disorders may be a simple and useful tool to uncover and monitor specific respiratory complex defects.

Metabolic fate of fatty acids involved in ruminal biohydrogenation in sheep fed concentrate or herbage with or without tannins.

A 2 x 2 factorial experiment was carried out to evaluate the effect of herbage or concentrate feeding and dietary tannin supplementation on fatty acid metabolism and composition in sheep ruminal fluid, plasma, and intramuscular fat. Twenty-eight male lambs were divided into 2 equal groups at 45 d of age and kept in individual pens. One group was given exclusively fresh herbage (vetch), and the other group was fed a concentrate-based diet. Within each treatment, one-half of the lambs received supplementation of quebracho powder, providing 4.0% of dietary DM as tannins. Before slaughter, blood samples were collected. The animals were slaughtered at 105 d of age, and ruminal contents and LM were collected. Blood plasma, ruminal fluid, and LM fatty acid composition was determined by gas chromatography. Tannin supplementation reduced (P < 0.05) the concentration of stearic acid (-49%) and increased the concentration of vaccenic acid (+97%) in ruminal fluid from concentrate-fed lambs. Within concentrate- and herbage-based diets, tannin supplementation reduced the accumulation of SFA in blood (P < 0.05) compared with lambs fed the tannin-free diets. When tannins were included in the concentrate, the LM contained 2-fold greater concentrations of rumenic acid compared with the LM of the lambs fed the tannin-free concentrate (0.96 vs. 0.46% of total extracted fatty acids, respectively; P < 0.05). The concentration of PUFA was greater (P < 0.05) and SFA (P < 0.01) less in the LM from lambs fed the tannin-containing diets as compared with the animals receiving the tannin-free diets. These results confirm, in vivo, that tannins reduce ruminal biohydrogenation, as previously reported in vitro. This implies that tannin supplementation could be a useful strategy to increase the rumenic acid and PUFA content and to reduce the SFA in ruminant meats. However, the correct dietary concentration of tannins should be carefully chosen to avoid negative effects on DMI and animal performance.

Lipid and colour stability of meat from lambs fed fresh herbage or concentrate.

Fourteen male Comisana lambs were divided into two groups at 45days of age and were individually penned for 105days. Over this period, seven lambs were fed a concentrate-based diet (C), whereas the remaining animals received vetch (Vicia sativa; H) harvested daily and given fresh to the animals. Lipid oxidation was measured in both minced cooked meat (semimembranosus muscle, SM) over 4days of aerobic refrigerated storage and on minced raw meat stored over 14days in a high oxygen atmosphere. Colour descriptors, haem pigment concentration, and metmyoglobin percentages were also determined during storage duration on the minced raw meat. Lipid oxidation increased over time in cooked and raw meat (P<0.0005), but lower TBARS values were found in both cooked and minced meat from lambs fed vetch compared to those given concentrates (P=0.001; P=0.006, respectively). Higher a* values, lower b* values and lower hue angle values were observed in meat from H-fed animals as compared to meat from C-fed lambs (P=0.006; P=0.02; P=0.005, respectively). Metmyoglobin formation increased over time (P<0.0005), but the H diet resulted in lower metmyoglobin percentages than the C diet (P=0.006). Haem pigment concentration decreased over the 14days of storage (P<0.0005). We conclude that, under conditions that promote oxidative stress in meat, a herbage-based diet can improve the oxidative stability of meat compared to a concentrate-based diet.

Meat odour and flavour and indoles concentration in ruminal fluid and adipose tissue of lambs fed green herbage or concentrates with or without tannins.

A 2 × 2 factorial experiment was carried out to evaluate the effect of herbage or concentrate feeding system and tannin addition to diet on skatole and indole in ruminal fluid and adipose tissue and meat sensory properties. Twenty-eight male lambs aged 45 days were randomly assigned to one of two feeding systems (vetch green herbage or concentrates, n = 14) and within feeding system to one supplement (quebracho tannins added to the diet or none). Animals were kept in singular pens and slaughtered at the age of 105 days. Indole (P < 0.05) and skatole (P < 0.01) concentrations in ruminal fluid were higher in lambs fed herbage compared to those given concentrates. Skatole in ruminal fluid tended to be present at lower concentrations in animals that received the tannin supplementation (P = 0.07). Indole was also higher in the caudal fat of animals fed green vetch compared to those fed concentrate (P = 0.04). Skatole concentration was lower in the fat of lambs fed concentrates compared to those given herbage (P = 0.05) and was lower in the fat of animals supplemented with tannins compared to the animals not supplemented (P = 0.01). Sheep meat odour was lower in meat from animals supplemented with tannins compared to those not supplemented (P < 0.01). It is concluded that tannins are more effective in reducing skatole formation in ruminants when they are associated with concentrate diets than green herbages.

Dietary tannins improve lamb meat colour stability.

Fourteen male Comisana lambs were divided into two groups at 45days of age: lambs fed a concentrate diet (C), or lambs fed the same concentrate with the addition of quebracho (Schinopsis lorentzii) tannins (T). Sheep were slaughtered at 105days of age. Lipid oxidation, colour coordinates, haem pigment concentration, and metmyoglobin percentages were measured on minced semimembranosus muscle (SM) over 14days of refrigerated storage in a high oxygen modified atmosphere. Tannin supplementation increased (P<0.01) a(∗) values and reduced (P<0.01) b(∗) values of the SM when compared to C. Lower hue angles (P<0.001) and metmyoglobin formation (P=0.07) were observed in lamb from T-fed compared to C-fed sheep during the 14-days storage period. Furthermore, feeding T resulted in greater (P<0.001) haem pigment concentrations in the SM during refrigerated storage; however, diet had no (P=0.28) effect on lipid oxidation. Therefore, including quebracho tannins in sheep diets can improve meat colour stability of fresh lamb during extended refrigerated storage.

Intramuscular fatty acid composition of lambs given a tanniniferous diet with or without polyethylene glycol supplementation.

The aim of this trial was to investigate the effects that dietary tannins have on lamb intramuscular fatty acids. Twenty-seven lambs were divided into three homogeneous groups: control group, receiving commercial concentrate based on maize; tannin group, fed a diet based on carob pulp (45% as fed basis); PEG group, receiving the same diet as the latter with addition of 42g/kg of polyethylene glycol (PEG, a binding agent that eliminates the effects of condensed tannins). The duration of the trial was 45 d. Intramuscular fatty acids were measured in the longissimus dorsi muscle. The isomer cis-9 trans-11 of linoleic acid (conjugated linoleic acid or CLA) and linolenic acid were higher in the longissimus muscle fat from animals fed the control diet compared to the other groups (P<0.0005); these fatty acids were higher in the fat from animals fed the carob diet supplemented with PEG compared to those fed the same diet without PEG (P<0.05). trans-Vaccenic acid (C18:1 trans-11) was found at higher concentration in fat from control and PEG lambs compared to tannin lambs (P<0.01); the CLA/C18:1 trans-11 ratio was lower in lambs fed control and PEG diets than in tannin-fed animals (P<0.05).

Ruminant fat volatiles as affected by diet. A review.

Volatile compounds in meat have been widely studied for their favourable or undesirable effects on meat flavour, or for their potential use in tracing the animal feeding system. To date, the chemical mechanisms causing the appearance of volatile compounds in meat have been largely understood. Several variables are involved in the accumulation of volatiles in animal tissues and among them animal diet plays a key role. The purpose of the present review is to highlight the effects of different dietary regimes (concentrate, green grass and fat-enriched diets) on the appearance of fat volatile compounds in ruminant meat. Grain-based diets induce greater accumulations in meat of branched-chain fatty acids, some aldehydes, and lactones while meat fat from grazing animals contains high levels of phenols, terpenes, indoles and sulphur compounds. Fat-enriched diets exert their effect mainly on those volatiles which originate from polyunsaturated fatty acids. Cooking procedures have been considered for their contribution to fat volatiles in meat by reactions induced by high temperatures.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3.

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Characterization of sphingomyelinase activity released by thrombin-stimulated platelets.

In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells.

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.