Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes.

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.

Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets.

Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt soluble (HSS) and detergent-soluble (DS) fractions. These accounted, respectively, for about 30%, 7% and 60% of total AChE activity. Applications of hydrophobic chromatography on phenyl-agarose to three solubilized fractions revealed that hydrophilic forms were almost exclusively located in the LSS fraction ( approximately 27% of total AChE), whereas most amphiphilic forms were present in DS extracts ( approximately 59% of total AChE), the remaining forms being distributed among aqueous soluble fractions. Enzyme molecular forms in the solubilized extracts were identified by centrifugation in 5-20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate between hydrophilic or amphiphilic species. A predominance of hydrophilic dimeric forms ( approximately 22%), with small amounts of hydrophilic monomers (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fractions had a single peak in the sedimentation profiles with sedimentation coefficients of about 6S in gradients with Triton X-100; these were slightly shifted in the presence of Brij 97. After treatment with dithiothreitol, this molecular form solubilized in DS was converted to another molecular form with a lower sedimentation coefficient. Our results show that amphiphilic globular dimers are the dominant molecular form in sheep platelet AChE, suggesting a partial conversion of this membrane-bound form into soluble dimers and monomers, mainly with a hydrophilic character, through the action of either endogenous proteases and phospholipases or residual endogenous reducing agents.

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms.

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine.

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.

Phospholipid and fatty acid composition in stored sheep erythrocytes of different densities.

The mammalian erythrocyte is an ideal model for studies of membrane aging under conditions of storage. The present study describes the variations in the membrane lipid composition of three density groups (light, 1.110 < d < 1.125; intermediate, 1.125 < d < 1.130, and dense, 1.130 < d < 1.140) of sheep erythrocytes separated by centrifugation in a discontinuous Ficoll density gradient after storage at 4 degrees C in a nutrient medium for up to 6 days. The only changes in phospholipid composition took place in the erythrocyte light fraction where sphingomyelin (SM) and phosphatidic acid increased (p < 0.05), whereas phosphatidylethanolamine (PE) decreased (p < 0.05). Moreover, polyunsaturated fatty acids (20:4 and 22:6) decreased during storage, whereas lipid fluorescence increased (p < 0.01) after 24 hours of storage in all the fractions separated. These observations suggest a lipid peroxidation process in all three erythrocyte groups during storage.

Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity.

Incubation of human or sheep platelet crude membranes with xanthine oxidase/hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosphatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity in a time-dependent manner, this inhibition being significant within 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20-45 min after the start of the treatment. The inhibition of PTPase activity in general showed good a correlation with the production of thiobarbituric acid-reactive substances (TBARS). The results with several antioxidants suggest that the inhibition of PTPase activity is related to the generation of alkoxyl and/or peroxyl radicals. Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treatment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. However, the amount of protein thiols was reduced only by treatment with trans-2-nonenal. Fluorescence product formation was always higher with trans-2-nonenal, these products being mainly located in the protein fraction. The results with aldehydes suggest that secondary degraded products of lipid hydroperoxides affect PTPase activity. Kinetic studies of PTPase activity indicated that with all treatments enzyme inhibition is mainly due to a decrease in the Vmax value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/ hypoxanthine in the presence of Fe2+/ADP is a very complex, time-dependent process, and that it is probably related, at least after long periods of peroxidation, to changes in protein thiols and amino groups. We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing importance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular.

Changes in the fatty acid composition of stored erythrocytes from sheep of different ages.

This study examined the profile in sheep erythrocyte fatty acids from animals of different ages during storage at 4 degrees C in a nutritive medium for up to 6 days. The changes found in the fatty acyl profile were a decrease (P < 0.01) in the percentage of arachidonic acid and an increase (P < 0.01) in the percentage of minor fatty acids (representing < 2% in each case; 20:2, 22:0 and 22:1) with respect to fresh erythrocytes in all age groups. However, the saturated/unsaturated ratios and unsaturated index started almost constant in all cases. The changes observed occurred after 24-48 h of storage, with significant increases (P < 0.01) in the fluorescence detected in the lipid extracts from stored erythrocytes during this period. The above findings suggest peroxidative damage and changes in the erythrocyte lipid membrane during storage.

Changes in the phospholipid and fatty acid composition in normal erythrocytes from sheep of different ages. Aminophospholipid organization in the membrane bilayer.

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.

Vesiculation and changes in fluidity and lipid composition of platelet membranes after storage of sheep platelets in plasma or Seto solution.

To relate the improvement of platelet storage in synthetic media with possible structural changes, we conducted serial studies on the membranes of platelets and microparticles shed during platelet storage for up to 5 days at 4 degrees C either in plasma or in Seto solution. Spontaneous microparticle formation proceeded linearly for up to 2 days in both storage media, although the processes seemed to be different because microparticles from Seto solution had a higher lipid/protein ratio than those released in plasma. Microparticles were heterogeneous structures showing beta-N-acetylhexosaminidase, glucose-6-phosphatase and succinate dehydrogenase activities. After 2-5 days of storage, microparticles contained 60% of total cellular acetylcholinesterase (AChE), were doubly enriched in cholesterol. and showed identical phospholipid profiles but with a decrease in the lipid unsaturation index with respect to fresh platelets. Fluorescence anisotropy studies pointed to a remarkable increase in the deep lipid core fluidity of microparticles during storage of platelets in plasma. With respect to platelets, only those stored in plasma showed significant changes in lipid contents, with a 3-fold decrease in the phospholipid to protein ratio, a decrease in phosphatidylethanolamine (PE) levels and a parallel increase in phosphatidylcholine (PC) percentages in their phospholipid profile, together with a significant reduction in the lipid unsaturation index after 1 day of storage. The fluidity of the negatively charged surface of the platelet membranes decreased in platelets stored for 5 days in both media, whereas the fluidity of the membrane deep core was only increased in platelets stored in plasma. These findings suggest that Seto solution permits better storage of platelets for 5 days than plasma and support the notion that lipid peroxidation could play an important role in the structural changes observed.

The effect of GTP on the aluminum fluoride- and forskolin-activated adenylyl cyclase from human embryonic kidney 293 cells.

GTP has been shown to inhibit AlF4(-)-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4(-)-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4(-)- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4(-)-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4(-)-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4- or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4-. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4(-)-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4-.

Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization.

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55 degrees C. The Km values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cations and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least alpha-mannose and N-acetylglucosamine residues in the glycan chain.

Characterization of phosphotyrosine phosphatase activity in sheep platelets: amphiphilic and hydrophilic forms.

Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K(m) values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl2 and only weakly inhibited by Zn2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.

Fatty acid composition of subcellular particles from sheep platelets and topological distribution of phosphatidylethanolamine fatty acids in the plasma membrane.

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18:1; 32-41%), linoleic (18:2, 10-17%), stearic (18:0; 13-15%), palmitic (16:0; 11-15%) and arachidonic (20:4; 8-12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22:0), 18:0 and 16:0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18:1n-9. Our findings suggest that fatty acids are asymmetrically distributed between the choline versus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzene-sulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18:2 and significantly less 18:1 and 20:5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturated versus saturated PE acyl chains.

Effects of dietary oil related to the toxic oil syndrome on the lipids of guinea pig liver microsomes.

The potential effects of oil specimens related to cases of toxic oil syndrome (TOS) on the liver microsomal lipid composition from guinea pigs were investigated. For four weeks, animals were fed diets supplemented with either "case oil" (oil related to cases of TOS) or "control oil" (oil unrelated to cases of TOS), either previously heated or not. Results were compared with those from guinea pigs fed the same diet with no oil. The administration of case oil produced changes in liver microsomal lipid composition. Statistically significant differences were also found between heated case and heated control oils. The cholesterol/phospholipid molar ratios and the major phospholipid class distribution were unaffected under these diet conditions. However, increases in the relative contents of linoleic and arachidonic acids and, simultaneously, a reduction in palmitic and palmitoleic acid levels were observed by diet effects. Heated oil administration decreased the saturated/unsaturated ratios in all cases. Our data suggest that changes observed in the fatty acid composition are attributable to the free fatty acid contents of administered oils. The toxic constituents of case oil seem to be able to alter the liver microsomal lipid composition.

Phospholipid fatty acid and lipid peroxidation in liver microsomes from guinea pigs fed oil related to the toxic oil syndrome.

The potential effects of oil specimens both related and unrelated to cases of Toxic Oil Syndrome (TOS) on the phospholipid fatty acid composition, some antioxidant enzyme activities, and lipid peroxidation in guinea pig liver microsomes were investigated. For 4 weeks, animals were fed diets supplemented with either oil related to cases of TOS or control oil, previously heated or not. In all cases, the fat diet produced the incorporation of approximately 7% of linoleic acid exclusively in the phosphatidylethanolamine (PE) of liver microsomes. A pronounced increase in lipid peroxidation products, measured as malondialdehyde (MDA) and 4-hydroxyalkenals, was detected in animals fed nonheated control oil. Heated oil diets produced significant increases in superoxide dismutase and glutathione peroxidase activities with concomitant decreases in the lipid peroxidation status. Heated oils also increased the oleic/stearic acid ratio in the phosphatidylserine plus phosphatidylinositol (PS + PI) fraction. This ratio was also increased in the same fraction from animals fed non heated case oil. The study shows that case oil produces a decrease in the lipid peroxidation products with minimal alterations in phospholipid fatty acid composition of liver microsomes, which is dependent rather on the composition of dietary fat than on toxic effects.

Changes in forebrain, cerebellum and brain stem phospholipid patterns from mothers and newborn rats after chronic pentazocine treatment during the gestation period.

Pregnant female Wistar rats were injected with pentazocine intramuscularly during the first nineteen days of the gestation period. The initial dose (1.8 mg/kg body weight) was increased daily by 1.2 mg of pentazocine, finishing with 23.4 mg/kg body weight. Chemical analysis of the forebrain, cerebellum and brain stem from mothers and their newborns points to statistically non-significant changes in total protein, phospholipid and cholesterol contents after pentazocine treatment. The phospholipid pattern only changed significantly in the brain stem of the mother rats treated with pentazocine, showing an increase (p less than 0.05) in inositol phosphoglycerides (2.5-fold) and phosphatidylglycerol (3.5-fold) percentages, while ethanolamine- and serine-phosphoglycerides decreased (p less than 0.05) by about 0.9 and 0.6-fold, respectively. The cholesterol profile found in the forebrains of the mother rats and their newborns did not show statistically significant differences after treatment.

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation.

Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34(+) cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22(phox)-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.