The effect of pharmacologic inhibition of phospholipid methylation on human platelet function

Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L- methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza- adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza- aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.     

Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids.

Rat brain synaptosomes contain an enzyme, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), that catalyzes the methylation of phosphatidylethanolamine to form its mono-, di-, and trimethyl (phosphatidylcholine) derivatives. Synaptosomal phosphatidylethanolamine is much richer in polyunsaturated fatty acids (43.4%) than is synaptosomal phosphatidylcholine (4.6%). It thus seemed possible that the phosphatidylcholine derived via the N-methylation of phosphatidylethanolamine might also be especially enriched in polyunsaturated fatty acids. To test this hypothesis, we examined the incorporation of [3H]methyl groups into various molecular species of phosphatidylcholine, by incubating rat synaptosomes for 10, 30, or 90 min in a medium containing S-adenosyl[methyl-3H]methionine. Phosphatidylcholine was extracted and separated from other lipids by TLC, after which its molecular species were isolated by argentation TLC (which distinguishes among the phospholipid molecules by the degree of unsaturation of their fatty acid moieties.) We found that approximately 65% of the [3H]methyl incorporated into phosphatidylcholine during the incubation period was present in the fraction associated with pentaene or hexaene fatty acids; an additional 30% was present in the tetraene fraction, while the remaining phosphatidylcholine radioactivity was distributed between the dienes and monoenes. Similar distributions were observed among synaptosomes incubated for 10 or 30 min; however, after 90 min the phosphatidyl[3H]choline contained proportionately less of the tetraenes. These observations indicate that neuronal phosphatidylcholine molecules formed via N-methylation are especially richer in polyunsaturated fatty acids, and they raise the possibility that these molecules constitute a distinct pool with particular physiologic functions.     

Stimulation of phospholipid methylation and thyroid hormone secretion by thyrotropin

Synthesis of phosphatidylcholine (PC) by S-adenosylmethionine-dependent methylation of phosphatidylethanolamine has previously been associated with receptor-mediated histamine and pituitary hormone secretion. We investigated stimulation of phospholipid methylation by TSH and its possible role in thyroid hormone secretion. Rat hemithyroids were incubated in Krebs-Henseleit-glucose-BSA buffer and the effect of various treatments on the incorporation of [3H-methyl]L-methionine into PC and T4/T3 secretion was studied. TSH treatment elevated thyroid phosphatidylethanolamine methyltransferase activity, the incorporation of [3H-methyl]methionine into PC, and T4/T3 secretion. The increase in PC synthesis was linear up to 6 h in a dose-dependent fashion (half-maximal stimulation at 2.5 micrograms TSH/ml). Stimulation required protein synthesis, because cycloheximide inhibited the increase in PC synthesis by 77%. Inhibitors of phospholipid methylation (100 microM adenosine + 10 microM L-homocysteine thiolactone + 10 microM erythro-9[2-hydroxy-3-nonyl]adenine) significantly decreased TSH-stimulation of phospholipid methylation but not T3/T4 secretion. In conclusion, stimulation of thyroid phospholipid methylation by TSH is not required for stimulated secretion of thyroid hormones.     

Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration

This study was undertaken to isolate phospholipids released from activated platelets and to investigate their biological activities. Freshly washed platelets were activated with freezing/thawing, thrombin, ionophore 23187, and arachidonic acid. Thrombin was incubated with platelet-rich plasma to promote synthesis and release of phospholipids from platelets. Phospholipids in supernatants of activated platelets were extracted with butanol and separated by thin-layer chromatography. Release of phosphatidylserine (PS) and phosphatidic acid (PA) increased when platelets were treated with freezing/thawing, ionophore, and thrombin. The lysophosphatidyl ethanolamine (LPE) appeared not to be induced with freezing/thawing, but increased significantly by thrombin, ionophore, and arachidonic acid. The effects of platelet phospholipids on hemostasis and angiogenesis were studied with platelet aggregation and endothelium chemotaxis. Phospholipids isolated from thrombin-stimulated platelet-rich/platelet-poor plasmas were used as synergistic agonists in platelet aggregation and as chemotactic agents in endothelial cell migration. Several phospholipids increased chemotaxis and platelet aggregation; these were PS, PA, LPE, and sphingosine-1-phosphate. Also, chemotaxis of those phospholipids increased when combined with charcoal-stripped fetal bovine serum, suggesting that cofactors in serum enhanced phospholipid-induced cell migration. These observations suggest that activated platelets release biologically active phospholipids into the blood stream, where they may play an important role in thrombosis and angiogenesis.     

Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration

This study was undertaken to isolate phospholipids released from activated platelets and to investigate their biological activities. Freshly washed platelets were activated with freezing/thawing, thrombin, ionophore 23187, and arachidonic acid. Thrombin was incubated with platelet-rich plasma to promote synthesis and release of phospholipids from platelets. Phospholipids in supernatants of activated platelets were extracted with butanol and separated by thin-layer chromatography. Release of phosphatidylserine (PS) and phosphatidic acid (PA) increased when platelets were treated with freezing/thawing, ionophore, and thrombin. The lysophosphatidyl ethanolamine (LPE) appeared not to be induced with freezing/thawing, but increased significantly by thrombin, ionophore, and arachidonic acid. The effects of platelet phospholipids on hemostasis and angiogenesis were studied with platelet aggregation and endothelium chemotaxis. Phospholipids isolated from thrombin-stimulated platelet-rich/platelet-poor plasmas were used as synergistic agonists in platelet aggregation and as chemotactic agents in endothelial cell migration. Several phospholipids increased chemotaxis and platelet aggregation; these were PS, PA, LPE, and sphingosine–1-phosphate. Also, chemotaxis of those phospholipids increased when combined with charcoal-stripped fetal bovine serum, suggesting that cofactors in serum enhanced phospholipid-induced cell migration. These observations suggest that activated platelets release biologically active phospholipids into the blood stream, where they may play an important role in thrombosis and angiogenesis.     

Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of insulin to plasma membranes stimulated the methylation of endogenous phosphatidylethanolamine, as evidenced by an increase in the levels of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N, N-dimethylethanolamine, and phosphatidylcholine. The effect of insulin was rapid and concentration-dependent, with 100 microunits/ml providing near maximal stimulation. The incorporation of [3H]methyl into phospholipids of control and insulin-stimulated plasma membranes was enhanced by the addition of exogenous methyltransferase substrates phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidyl-N,N-dimethylethanolamine. The stimulatory effect of insulin on adipocyte plasma membrane phospholipid methylation may have a physiological role in insulin action.     

Rat basophilic leukemia cell lines defective in phospholipid methyltransferase enzymes, Ca2+ influx, and histamine release: reconstitution by hybridization.

Variants of the rat basophilic leukemia (RBL) cell line were isolated and screened for phospholipid methyltransferase I and II activities, enzymes that convert phosphatidylethanolamine to phosphatidylcholine. Two variants were found that had decreased phospholipid methyltransferase enzyme levels and were unable to cause an influx of Ca2+ or release histamine in an IgE-mediated reaction. However, these cells were able to release histamine through an ionophore-induced reaction, indicating that the releasing mechanism distal to the Ca2+ channel was intact. One cell line, 1C1.B1, had low specific activity for phospholipid methyltransferase I. A second variant, 2H3.B6, had reduced phospholipid methyltransferase II activity. Although both variants were unable to incorporate label from [methyl-3H]methionine or [3H]serine into phosphatidylcholine, they were able to incorporate [methyl-3H]choline and myo-[2-3H(N)]inositol into phospholipids. Fusion of the two cell lines and isolation on selective media resulted in the growth of eight independent hybrids. All eight had an increased number of chromosomes and normal phospholipid methyltransferase activities. Stimulation of the hybrids with IgE resulted in CA2+ influx and histamine release. These results indicate that phospholipid methylation precedes and is necessary for Ca2+ influx, and they further support the hypothesis that methylation is a necessary early step in the IgE-mediated histamine release reaction in RBL cells.     

Regulation by guanosine 3'':5''-cyclic monophosphate of phospholipid methylation during chemotaxis in Dictyostelium discoideum.

In Dictyostelium discoideum, the chemoattractant cyclic AMP activates the enzyme guanylate cyclase, giving a brief up to 10-fold increase in the intracellular cyclic GMP content. The addition of physiological cyclic GMP concentrations to a homogenate of D. discoideum cells markedly increased the incorporation of the 3H-labeled methyl group from S-adenosyl-L-[methyl-3H]methionine into mono- and dimethylated phosphatidylethanolamine and phosphatidylcholine. Lipid methylation was inhibited by S-adenosyl-L-homocysteine, which inhibits transmethylation. When whole cells prelabeled with L-[methyl-3H]methionine were exposed to cyclic AMP, a rapid transient increase in the amount of [methyl-3H]phosphatidylcholine was observed. The time course of [methyl-3H]phosphatidylcholine formation agrees with its being mediated by the intracellular increase in cyclic GMP originating during chemotactic stimulation. Addition of the 8-Br derivative of cyclic GMP to whole cells also increased the levels of labeled phosphatidylcholine. It is therefore likely that cyclic GMP contributes to chemotaxis by regulating membrane function via phospholipid methylation.     

Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line.

Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used as precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here we used a population of purely cholinergic cells (human neuroblastoma, LA-N-2), incubated in the presence of [methyl-3H]methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. PtdCho, purified by thin-layer chromatography, contained 90% of the label incorporated into lipids, demonstrating that LA-N-2 cells contained phosphatidylethanolamine N-methyltransferase. Three peaks of radioactive material that cochromatographed with authentic Ac-Cho, choline, and phosphocholine were observed when the water-soluble metabolites of the [3H]PtdCho were purified by high-performance liquid chromatography. Their identities were ascertained by subjecting them to enzymatic modifications with acetylcholinesterase, choline oxidase, and alkaline phosphatase, respectively. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine.     

Inhibition of cyclooxygenase-independent platelet aggregation by low vitamin E concentration

Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 microM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 microM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A2 formation was less than 10% in samples incubated with 100 microM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 microM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.     

Phospholipid N-methylation as a Signal Transduction Mechanism in Normal and Failing Hearts

The phospholipid N-methylation pathway comprises of three successive N-terminal methylations of phosphatidylethanolamine where S-adenosyl-L-methionine acts as the physiological donor. Under optimal conditions in cardiac membranes, the catalytic sites I, II, and III of methyltransferase have been identified which are responsible for the synthesis of the major product, phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine, respectively. The characterization of the phosphatidylethanolamineN-methyltransferase system has shown that each of the catalytic sites exhibits different biochemical properties. The phospholipid N-methylation pathway has also been observed to regulate heart function by inducing localized structural, compositional, and functional changes in cardiac membranes under different pathological conditions of chronic nature. This review deals with the phosphatidylethanolamine N-methylation–mediated signal transduction mechanism involving modification of the Ca2+-transporting activities of the sarcolemmal and sarcoplasmic reticular membranes of the cardiomyocyte. In this regard, special attention is given to the status of this pathway and its relevance for the functioning of membrane-related Ca2+-transport systems in heart dysfunction due to different cardiac pathologies, such as diabetes-induced cardiomyopathy, catecholamine-induced cardiomyopathy, genetically linked cardiomyopathy, and adriamycin-induced cardiomyopathy. In addition, changes in phosphatidylethanolamine N-methylation in heart dysfunction due to cardiac hypertrophy, Ca2+-paradox hearts, and ischemic-reperfused hearts have been described. It is suggested that an increase in phosphatidylethanolamine N-methylation activity may play an adaptive role, whereas a depression may contribute towards contractile dysfunction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phosphatidylcholine metabolism is altered in a monocyte-derived macrophage model of Gaucher disease but not in lymphocytes

Gaucher disease is caused by defective activity of acid-beta-glucosidase (GlcCerase), resulting in accumulation of glucosylceramide (GlcCer) mainly in macrophages. We now demonstrate that secondary biochemical pathways regulating levels of phospholipid metabolism are altered in a Gaucher disease macrophage model. Upon treatment of macrophages with the GlcCerase inhibitor, conduritol-B-epoxide, phosphatidylcholine (PC) labeling with the metabolic precursor, [methyl-14C]choline, was elevated after 6 or 12 days in macrophages but not in lymphocytes. These changes correlated with increases in the cytoplasmic/nuclear ratio and with levels of [3H]GlcCer accumulation. Moreover, metabolic labeling with L-[3-3H]serine and L-[methyl-3H]methionine demonstrated that PC synthesis via the methylation of phosphatidylethanolamine is also increased in CBE-treated macrophages. Since PC is a major structural component of biological membranes and the source of various second messengers, we suggest that changes in its metabolism in macrophages may be relevant for understanding Gaucher disease pathology.     

Studies on the mechanism of the inhibition of platelet aggregation and release induced by high levels of arachidonate

We have previously reported that arachidonic acid induced a biphasic pattern of platelet aggregation and the release of both dense and alpha- granule components. Low levels of arachidonate (0.025--0.1 mM) specifically induced aggregation and release, while high concentrations (0.15--0.35 mM) caused a progressive inhibition of these platelet responses in human gel-filtered platelets (GFP). We now report studies of the mechanism(s) responsible for this arachidonate-induced turn-off of platelet function. Electron micrographic studies demonstrated that there was no gross damage to the platelets during the turn-off. Active synthesis of malondialdehyde and thromboxane A2 was seen at the high arachidonate levels, despite the inhibition of aggregation. Furthermore, GFP inhibited by 0.25 mM arachidonate were capable of undergoing aggregation and serotonin release in response to other stimuli, such as collagen or thrombin. Thus, GFP appeared to be metabolically intact and functional during the inhibiton by high arachidonate levels. Thin-layer chromatographic studies revealed that prostaglandin metabolism was not changed at the high arachidonate levels. In addition, indomethacin (20 microM) did not abolish the arachidonate-induced inhibition of platelet function. Therefore, the inhibitory effect of high arachidonate did not depend on its conversion to other prostaglandin products. Platelet cyclic AMP levels increased twofold at the high arachidonate concentrations (1.3 +/- 0.3 pmole/10(8) platelets at peak aggregation, compared with 2.9 +/- 0.4 pmole/10(8) platelets at inhibition by 0.25 mM arachidonate, p less than 0.001). Prostaglandin-D2, a platelet inhibitor known to increase cyclic AMP, generated a similar rise (to 2.4 +/- 0.2 pmole/10(8) platelets). Thus, the magnitude of the arachidonate-induced increase in platelet cyclic AMP levels can account for the inhibition of aggregation and release.     

Phospholipid transfer between plasma and platelets in vitro

Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of 32PO4 into rabbits. At certain time intervals during a 6-hr incubation at 37 degrees C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phospholipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed through-out the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hr, 4% of total platelet phospholipid, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet- induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma. Labeling of both platelet lysolecithin and lecithin could be due to uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchanged of platelet lecithin may be important mechanisms in the modification by plasma lipids of platelet membrane phospholipid fatty acid composition and platelet function.     

Phosphatidylcholine biosynthesis in myocardial ischaemia

In this study the effect of myocardial ischaemia was evaluated on two aspects of phospholipid metabolism: (i) the de novo synthesis of myocardial phospholipids, as indicated by the incorporation of (methyl-3H) choline and (ii) the incorporation of radiolabelled long chain fatty acids into tissue phospholipids. Two models of ischaemia were used namely normothermic ischaemic arrest and hypoxic, low-flow perfusion of the isolated rat heart. The results showed that within 10 min, hypoxic low-flow perfusion significantly inhibited the incorporation rate of (methyl-3H) choline into tissue phospholipids. Since the tissue choline content remained unaltered under these conditions, the results suggested that the de novo synthesis of phosphatidylcholine is very susceptible to ischaemic damage. Inhibition of (methyl-3H) choline incorporation into tissue phospholipids appeared to be due to both a reduction in choline uptake and specific inhibition of the CDP pathway. Perfusion with glucose (10 mM) as substrate completely abolished the ischaemia-induced reduction in (methyl-3H) choline incorporation, indicating that glycolytically produced ATP played an important role in phosphatidylcholine biosynthesis. In contrast to these results, myocardial ischaemia stimulated the incorporation of long-chain saturated and unsaturated fatty acids into tissue phospholipids. In summary, the results obtained showed that myocardial ischaemia profoundly affected phospholipid metabolism which, in turn, might contribute to membrane damage.     

Stimulation of phospholipid N-methylation by isoproterenol in rat hearts

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activities were studied in rat heart sarcolemmal and sarcoplasmic reticular fractions after a single intraperitoneal injection of isoproterenol (0.5-5.0 mg/kg). Three active sites (I, II, and III) for PtdEtn N-methylation were assayed by measurement of [3H]methyl group incorporation from 0.055, 10, and 150 microM S-adenosyl-L-[methyl-3H]methionine into membrane PtdEtn molecules. Total methylation activity for catalytic site I of both sarcolemma and sarcoplasmic reticulum was stimulated within 2 minutes by isoproterenol in a dose-dependent manner. Although the increased methyltransferase activity in sarcoplasmic reticulum was normalized at 10 minutes, the enzyme activity in sarcolemma was normalized at 5 minutes but was again increased at 10-30 minutes after isoproterenol injection. No changes in response to isoproterenol were seen for site II and III N-methylation activities in either membrane. Individual N-methylated phospholipids (phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine), which specifically formed at each site, showed similar behavior. Pretreatment of the animals with a beta-blocking drug, atenolol, for 2 days prevented the isoproterenol-induced changes in hemodynamic parameters and sarcolemmal methylation without affecting the enhanced methylation activities in sarcoplasmic reticulum. In vitro addition of cyclic AMP-dependent protein kinase (catalytic subunit) plus Mg-ATP enhanced methyltransferase activities in sarcolemma and sarcoplasmic reticulum from control hearts by 2.7- and 2.3-fold, respectively; however, under the same in vitro conditions, only about 20% activation was seen in both subcellular membranes isolated from the heart of isoproterenol-injected animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations of phospholipid methyltransferase(s) activity in the rat pituitary: estrous cycle and sex differences

We previously demonstrated a specific stimulatory action of estrogens on phosphatidylethanolamine methylation in rat pituitary membranes. To investigate the physiological relevancy of this effect, the activity of methylating enzyme(s) was evaluated during the rat estrous cycle, a period in which both endogenous ovarian steroid levels and the sensitivity of pituitary membrane receptors fluctuate. Anterior pituitary membranes (P2) were prepared from adult female rats at different stages of the estrous cycle and assayed for phospholipid methylation in the presence of S-adenosyl-[methyl-3H]methionine as a donor of 3H-methyl groups. Methylated phospholipids were separated by TLC. Formation of phosphatidyl-mono- and dimethylethanolamine and that of phosphatidylcholine increased significantly in the morning, reaching maximal values on the afternoon of proestrus; they decreased thereafter during estrus, metestrus, and diestrus. Plasma estradiol concentrations increased in late diestrus and then varied similarly with the fluctuations of phospholipid methyltransferase activity throughout the cycle. In parallel, plasma levels of LH and PRL were significantly elevated during the afternoon of proestrus, but remained low throughout the rest of the cycle. Under the same experimental conditions, phospholipid methylation in membranes prepared from mediobasal-hypothalamic structures was not affected. These data demonstrate that under physiological conditions the increased pituitary methyltransferase activity is associated with the progressive increment of plasma estradiol levels occurring shortly before proestrus and precedes the release of LH and PRL. Ovariectomy significantly decreased methyltransferase activity; however, 17 beta-estradiol treatment of ovariectomized rats for 5 days restored the enzyme activity, which was further augmented after progesterone administration. Attempting to investigate variations of pituitary methyltransferase activity in male rats, we demonstrated that the intact males showed weaker activity than that of females; orchidectomy diminished the phospholipid methylation, but adrenalectomy had no effect.     

Relationship between plasma and platelet phospholipid fatty acid composition in healthy subjects

In order to furtherly clarify the mechanisms regulating the fatty acid composition of platelet phospholipids the relationships between the fatty acid composition of major phospholipid fractions from plasma and platelets were investigated in 30 healthy male subjects. Strict correlations between all but two plasma and platelet fatty acids were observed for phosphatidylcholine (PC) (r = 0.51-0.95, at least P < 0.01), whereas only poor correlations were found for the other fractions. These results suggest that the direct transfer of PC molecules from plasma to platelet membrane is a pivotal mechanism for renovation of platelet PC fatty acids, while other mechanisms appear to play a major role for renovation of other phospholipid fractions of the platelet membrane.     

Modifying Hepatic Phospholipid Synthesis Associates with Biliary Phospholipid Secretion Rate in a Transporter-Independent Manner in Rats

Biliary phospholipid secretion is mediated by a multidrug resistance gene product, and its molecular subselection occurs at the site of secretion to modulates bile metastability. The aim of this study was to determine the effect of modifying hepatic phospholipid synthesis on canalicular phospholipid transporter expression and membrane fluidity. Bile-duct cannulation was performed in male Sprague-Dawley rats pretreated with or without intravenous infusion of dimethylethanolamine, an intermediate phospholipid metabolite along the pathway of phosphatidylcholine synthesis of phosphatidylethanolamine N-methylation (0.01 mg/min/100 g body wt) for 15 hr, followed by sodium taurocholate infusion (50 nmol/min/100 g body wt) with or without sulfobromophthalein (50 nmol/min/100 g body wt). Dimethylethanolamine enhanced biliary phospholipid secretion in association with a decrease in biliary phospholipid hydrophobicity. Dimethylethanolamine also increased canalicular membrane fluidity defined by 1,6-diphenyl-1,3,5-hexatriene fluorescence depolarization, whereas the expression of multidrug resistance gene product and multidrug resistance associated protein was unchanged. In contrast, a disproportionate reduction of biliary phospholipid secretion caused by sulfobromophthalein (uncoupling) was enhanced by under the treatment with dimethylethanolamine. In conclusion, the increase in biliary phospholipid secretion and canalicular membrane fluidity without a drastic change of its canalicular transporter by dimethylethanolamine suggests that such a canalicular membrane fluidity facilitates the transporter activity and/or phospholipid molecular movement from the canalicular outer membrane into the bile. A more drastic reduction in phospholipid secretion under sulfobromophthalein-caused uncoupling indicates the possibility of a preferential distribution of relatively hydrophilic phosphatidylcholine molecules to bile salt micelles since sulfobromophthalein is known to reduce the micellar capacity to extract membrane lipids for biliary secretion.     

Relationship between plasma and platelet phospholipid fatty acid composition in healthy subjects

In order to furtherly clarify the mechanisms regulating the fatty acid composition of platelet phospholipids the relationships between the fatty acid composition of major phospholipid fractions from plasma and platelets were investigated in 30 healthy male subjects. Strict correlations between all but two plasma and platelet fatty acids were observed for phosphatidylcholine (PC) (r = 0.51-0.95, at least P < 0.01), whereas only poor correlations were found for the other fractions. These results suggest that the direct transfer of PC molecules from plasma to platelet membrane is a pivotal mechanism for renovation of platelet PC fatty acids, while other mechanisms appear to play a major role for renovation of other phospholipid fractions of the platelet membrane.