Phosphatidylethanol Formation Research Articles

The effect of incidental ethanol exposures on the formation of blood phosphatidylethanol.

Blood phosphatidylethanol (PEth), a metabolite of ethanol, is emerging as a direct biomarker of choice for characterizing ethanol consumption in clinical, research, and forensic contexts. An accumulating body of evidence, and a recent international consensus conference, supports a cutoff of 20 μg/L of PEth (16:0/18:1) to distinguish abstinence from beverage ethanol consumption. There is a dearth of research, however, on whether exposures to nonbeverage ethanol sources are sufficient to produce PEth concentrations that exceed this cutoff. To explore this possibility, we recruited 30 participants, who indicated past-90-day abstinence from beverage alcohol, to characterize their past-30-day nonbeverage ethanol exposures (including source, frequency, and intensity of exposures) and to undergo PEth testing. Two of the 30 participants (6.7%) produced PEth concentrations ≥20 μg/L. One of these participants (PEth = 26 μg/L) reported multiple ethanol exposure sources, including near-daily intensive exposures to ethanol vapor. The other participant (PEth = 49 μg/L) reported only once-daily use of an ethanol-containing mouthwash; the veracity of his abstinence claim is refuted. The results of this study support a rebuttable presumption that PEth ≥20 μg/L is indicative of beverage ethanol consumption. They suggest, however, that intensive, incidental alcohol exposures have the potential, under unusual circumstances, to result in PEth concentrations that modestly exceed this threshold.

Formation of phosphatidylethanol and ethylglucuronide after low to moderate alcohol consumption in volunteers with a previous three-week alcohol abstinence.

Phosphatidylethanol (PEth) is only formed when ethanol is present in blood. This direct alcohol marker has been widely discussed, including the minimum amount of ethanol being necessary to form as much PEth as to exceed the threshold of 20ng/mL in previously PEth negative subjects. In order to corroborate hitherto existing results, a drinking study including 18 participants after a 3-week alcohol abstinence was performed. They consumed a pre-calculated amount of ethanol to reach a blood alcohol concentration (BAC) of at least 0.6g/kg. Blood was drawn before and periodically seven times after alcohol administration on day 1. Blood and urine were also collected the next morning. Dried blood spots (DBS) were prepared immediately from collected venous blood. BAC was determined by head space gas chromatography and the concentrations of both PEth (16:0/18:1, 16:0/18:2 and five additional homologues) and ethyl glucuronide (EtG) were analysed using liquid chromatography-tandem mass spectrometry. Out of 18, 5 participants had concentrations of PEth 16:0/18:1 above the threshold of 20ng/mL, and 11 out of the 18 subjects had concentrations between 10 and 20ng/mL. In addition, four persons had PEth 16:0/18:2 concentrations above 20ng/mL the following morning. All test subjects tested positive for EtG in DBS (≥ 3ng/mL) and urine (≥100ng/mL) upon 20-21h after alcohol administration. By combining both a lower cutoff of 10ng/mL and the homologue PEth 16:0/18:2, the sensitivity to detect a single alcohol intake after a 3-week abstinence increases to 72.2%.

Activity‐based chemoproteomics reveals targets of phospholipase D‐derived phosphatidyl ethanol lipids

Alcohol consumption leads to formation of phosphatidylethanol (PEth) via the transphosphatidylation activity of phospholipase D (PLD) enzymes. Though this non‐natural phospholipid routinely serves as a biomarker of chronic alcoholism, its pathophysiological roles remain unknown. We use a minimalist diazirine alkyne alcohol as an ethanol surrogate to generate clickable, photoaffinity lipid reporters of PEth localization and lipid–protein interactions via PLD‐mediated transphosphatidylation. We use these tools to visualize phosphatidyl alcohols in a manner compatible with standard permeabilization and immunofluorescence methods. We also use click chemistry tagging, enrichment, and proteomics analysis to define the phosphatidyl alcohol interactome. Our analysis reveals an enrichment of putative interactors at various membrane locations, and we validate one such interaction with the single‐pass transmembrane protein basigin/CD147. Further, we identify binding sites of phosphatidyl alcohols to basigin/CD147 via both proteomics and targeted mutagenesis. This study provides a comprehensive view of the molecular interactions of phosphatidyl alcohols with the cellular proteome and points to future work to connect such interactions to potential pathophysiological roles of PEth.

A Chemoproteomics Approach to Profile Phospholipase D-Derived Phosphatidyl Alcohol Interactions.

Alcohol consumption leads to formation of phosphatidylethanol (PEth) via the transphosphatidylation activity of phospholipase D (PLD) enzymes. Though this non-natural phospholipid routinely serves as a biomarker of chronic alcoholism, its pathophysiological roles remain unknown. We use a minimalist diazirine alkyne alcohol as an ethanol surrogate to generate clickable, photoaffinity lipid reporters of PEth localization and lipid-protein interactions via PLD-mediated transphosphatidylation. We use these tools to visualize phosphatidyl alcohols in a manner compatible with standard permeabilization and immunofluorescence methods. We also use click chemistry tagging, enrichment, and proteomics analysis to define the phosphatidyl alcohol interactome. Our analysis reveals an enrichment of putative interactors at various membrane locations, and we validate one such interaction with the single-pass transmembrane protein basigin/CD147. This study provides a comprehensive view of the molecular interactions of phosphatidyl alcohols with the cellular proteome and points to future work to connect such interactions to potential pathophysiological roles of PEth.

Thrombin Stimulation of Human Endothelial Cell Phospholipase D Activity. Regulation by Phospholipase C, Protein Kinase C, and Cyclic Adenosine 3' 5’ -Monophosphate

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Inter Individual Variation and Factors Regulating the Formation of Phosphatidylethanol.

Alcohol use disorders are a major but often unrecognized health problem. Alcohol markers can therefore be of great value for diagnosis, follow-up, and treatment evaluation. Phosphatidylethanol in blood (B-PEth) is an alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers but has shown a considerable interindividual variation in relation to reported consumption. An in vitro system was used to investigate factors, which may affect the formation rate of PEth or which may give rise to interindividual variation in the rate of formation. In this system, isolated erythrocytes from 31 individuals were incubated in the presence of various concentrations of ethanol (EtOH). The concentration of PEth and phosphatidylcholine (PC), the parent molecule of PEth, was determined by chromatographic methods. Time, EtOH, and PC concentration were major factors determining the amount of PEth formed. The interindividual variation in PEth formation rate, calculated at an EtOH concentration of 50mmol/l, showed a coefficient of variation (CV) from 23 to 31% for the different PEth forms studied (PEth 16:0/18:2, total PEth and PEth 16:0/18:1). The concentration of PC was found to be an important determinant of this variation. The formation rate for PEth 16:0/18:2 was somewhat higher than for PEth 16:0/18:1. The formation of PEth 16:0/18:1 but not PEth 16:0/18:2 showed a positive correlation to the concentration of PEth at baseline (endogenous PEth). Calculation of enzyme kinetics for the reaction resulting in the formation of PEth 16:0/18:1 or PEth 16:0/18:2 showed an apparent Km (Michaelis constant) of approximately 160 to 170mmol/l. Interindividual variation in the formation rate of PEth appears to be a significant but relatively modest source of variation in the relation between B-PEth and reported consumption. Correction for interindividual variation in PC concentrations might substantially reduce the interindividual variability in PEth formation and consequently in B-PEth.

Phospholipase D: its role in metabolism processes and disease development

Phospholipase D (PLD) is one of the key enzymes that catalyzes the hydrolysis of cell membrane phospholipids. In this review current knowledge about six human PLD isoforms, their structure and role in physiological and pathological processes is summarized. Comparative analysis of PLD isoforms structure is presented. The mechanism of the hydrolysis and transphosphatidylation performed by PLD is described. The PLD1 and PLD2 role in the pathogenesis of some cancer, infectious, thrombotic and neurodegenerative diseases is analyzed. The prospects of PLD isoform-selective inhibitors development are shown in the context of the clinical usage and the already-existing inhibitors are characterized. Moreover, the formation of phosphatidylethanol (PEth), the alcohol abuse biomarker, as the result of PLD-catalyzed phospholipid transphosphatidylation is considered.

Formation of phosphatidylethanol from endogenous phosphatidylcholines in animal tissues from pig, calf, and goat

In the presence of alcohol, phosphatidylcholine (PC) is transformed to the direct alcohol biomarker phosphatidylethanol (PEth). This reaction is catalyzed by the enzyme phospholipase D (PLD) and dependent on substrate availability. As recent methods have solely focused on the determination of PEth, information about the PC composition was generally missing. To address this issue and monitor PC (16:0/18:1 and 16:0/18:2) and PEth (16:0/18:1 and 16:0/18:2) simultaneously, a reversed phase LC–MS/MS method based on a C8 core–shell column, coupled to a Sciex 5500 QTrap instrument was developed. By application of polarity switching, at first, PC was measured in ESI positive SRM mode, while PEth was determined at a later stage in ESI negative SRM mode. The PEth method was validated for human blood samples to show its robustness and subsequently applied for the investigation of systematic in vitro PEth formation in animal tissue samples (brain, kidney, liver, and blood) from a pig, a calf, and a goat. Homogenized tissue was incubated at 37°C with varying ethanol concentrations from 1 to 7g/kg (determined by HS-GC-FID) for 5h, whereby a sample was taken every 30min. For all tissue samples, an increase in PEth was measurable. PEth concentrations formed in blood remained below the LLOQ, in agreement with literature. Data analysis of Michaelis–Menten kinetics and PC within the tissue provided a detailed insight about PEth formation, as the occurrence of PEth species can be linked to the observed PC composition. The results of this study show that PEth formation rates vary from tissue to tissue and among different species. Furthermore, new recommendations for PEth analysis are presented.

The Formation, Elimination, Interpretation, and Future Research Needs of Phosphatidylethanol for Research Studies and Clinical Practice.

1 The Formation, Elimination, Interpretation and Future Research Needs of Phosphatidylethanol 2 (PEth) for Research Studies and Clinical Practice 4 Judith A. Hahn, PhD 5 University of California, San Francisco 7 Raymond F. Anton, MD 8 Medical University of South Carolina 10 Martin A. Javors, PhD 11 University of Texas Health Science Center, San Antonio 13 Address correspondence to: 14 Judith A. Hahn, PhD 15 University of California, San Francisco 16 550 16 th St. 3 rd floor 17 San Francisco, CA 94143 18 Phone: 415-476-5815 19 e-mail: judy.hahn@ucsf.edu 21 Funding: NIAAA K24AA022586 (Hahn), NIAAA U01AA020776 (Hahn), NIAAA K05AA017435 22 (Anton), R01AA022361 (Javors)

Age-Related Changes in the Phospholipase D-Dependent Signal Pathway of Insulin in the Rat Neocortex

Insulin participates in stabilization of normal functioning of the CNS; phospholipase D (PLD) is an integral part of the signal cascade of this hormone. With age, the lipid spectrum of the cells is modified, which implicates dysfunction of different signal pathways, including that of insulin. In our study, we found that the contents of ceramides and free fatty acids in the neocortex of old (24 months) rats are appreciably higher than that in 3-month-old animals. Under such conditions, the ability of insulin to activate PLD sharply drops. Incubation of neocortical tissues obtained from young animals in the presence of exogenous С2 ceramide or palmitic acid (a precursor of sphingolipids) leads to an increase in the content of endogenous ceramides. This process is accompanied by suppression of the insulinstimulated formation of phosphatidylethanol (PET, a product of reaction of transphosphatidylation of ethanol by PLD). Thus, the obtained data indicate that the activity of a PLD-dependent link of the signal cascade of insulin in the neocortex is significantly weakened with aging; ceramide plays an important role in this process.

Polyphosphoinositide Metabolism and Golgi Complex Morphology in Hippocampal Neurons in Primary Culture is Altered by Chronic Ethanol Exposure

Ethanol affects not only the cytoskeletal organization and activity, but also intracellular trafficking in neurons in the primary culture. Polyphosphoinositide (PPIn) are essential regulators of many important cell functions, including those mentioned, cytoskeleton integrity and intracellular vesicle trafficking. Since information about the effect of chronic ethanol exposure on PPIn metabolism in neurons is scarce, this study analysed the effect of this treatment on three of these phospholipids. Phosphatidylinositol (PtdIns) levels as well as the activity and/or levels of enzymes involved in their metabolism were analysed in neurons chronically exposed to ethanol. The levels of phospholipases C and D, and phosphatidylethanol formation were also assessed. The consequence of the possible alterations in the levels of PtdIns on the Golgi complex (GC) was also analysed. We show that phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate levels, both involved in the control of intracellular trafficking and cytoskeleton organization, decrease in ethanol-exposed hippocampal neurons. In contrast, several kinases that participate in the metabolism of these phospholipids, and the level and/or activity of phospholipases C and D, increase in cells after ethanol exposure. Ethanol also promotes phosphatidylethanol formation in neurons, which can result in the suppression of phosphatidic acid synthesis and, therefore, in PPIn biosynthesis. This treatment also lowers the phosphatidylinositol 4-phosphate levels, the main PPIn in the GC, with alterations in their morphology and in the levels of some of the proteins involved in structure maintenance. The deregulation of the metabolism of PtdIns may underlie the ethanol-induced alterations on different neuronal processes, including intracellular trafficking and cytoskeletal integrity.

Formation of Phosphatidylethanol and Its Subsequent Elimination During an Extensive Drinking Experiment Over 5 Days

For almost 30 years, phosphatidylethanol (PEth) has been known as a direct marker of alcohol consumption. This marker stands for consumption in high amounts and for a longer time period, but it has been also detected after 1 high single intake of ethanol (EtOH). The aim of this study was to obtain further information about the formation and elimination of PEth 16:0/18:1 by simulating extensive drinking. After 3 weeks of alcohol abstinence, 11 test persons drank an amount of EtOH leading to an estimated blood ethanol concentration of 1 g/kg on each of 5 successive days. After the drinking episode, they stayed abstinent for 16 days with regular blood sampling. PEth 16:0/18:1 analysis was performed using liquid chromatography-tandem mass spectrometry (high-performance liquid chromatography 1100 system and QTrap 2000 triple quadrupole linear ion trap mass spectrometer. Values of blood alcohol were obtained using a standardized method with headspace gas chromatography flame ionization detector. Maximum measured concentrations of EtOH were 0.99 to 1.83 g/kg (mean 1.32 g/kg). These values were reached 1 to 3 hours after the start of drinking (mean 1.9 hours). For comparison, 10 of 11 volunteers had detectable PEth 16:0/18:1 values 1 hour after the start of drinking, ranging from 45 to 138 ng/ml PEth 16:0/18:1. Over the following days, concentrations of PEth 16:0/18:1 increased continuously and reached the maximum concentrations of 74 to 237 ng/ml between days 3 and 6. This drinking experiment led to measurable PEth concentrations. However, PEth 16:0/18:1 concentrations stayed rather low compared with those of alcohol abusers from previous studies.

Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils.

The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.

Phospholipase D in human platelets: Presence of isoenzymes and participation of autocrine stimulation during thrombin activation

Phospholipase D (PLD), which hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline, is present in human platelets. Thrombin and other agonists have been shown to activate PLD but the precise mechanisms of activation and PLDs role in platelet activation remains unclear. We measured thrombin-stimulated PLD activity in platelets as formation of phosphatidylethanol. Since no specific PLD inhibitors exist, we investigated possible roles for PLD in platelets by correlating PLD activity with platelet responses such as thrombin-mediated secretion and F-actin formation (part of platelet shape change). Extracellular Ca2+ potentiated thrombin-stimulated PLD, but did not stimulate PLD in the absence of thrombin. Thrombin-induced PLD activity was enhanced by secreted ADP and binding of fibrinogen to its receptors. In contrast to others, we also found a basal PLD activity. Comparison of time courses and dose responses of platelets with PLD showed many points of correlation between PLD activation and lysosomal secretion and F-actin formation. The finding of different PLD activities suggested that different PLD isoenzymes exist in platelets as reported for other cells. Here we present evidence for the presence of both PLD1 and PLD2 in platelets by use of specific antibodies with immunoblotting and immunohistochemistry. Both isoforms were randomly localized in resting platelets, but became rapidly translocated to the proximity of the plasma membrane upon thrombin stimulation, thus indicating a role for PLD in platelet activation.

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 μM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.

Role of phospholipase D signaling in ethanol-induced inhibition of carbachol-stimulated DNA synthesis of 1321N1 astrocytoma cells.

Inhibition of astrocyte proliferation has been suggested to be an important event in the developmental neurotoxicity associated with ethanol. We have previously shown that the acetylcholine analog carbachol induces astroglial cell proliferation through activation of muscarinic M3 receptors, and that ethanol strongly inhibits this effect by inhibiting activation of protein kinase C (PKC) zeta and its down-stream effector 70-kDa ribosomal S6 kinase (p70S6K). In this study, we investigated whether inhibition by ethanol of this signal transduction pathway in 1321N1 human astrocytoma cells may be due, at least in part, to inhibition of the formation of the PKC zeta activator phosphatidic acid (PA), which is formed by hydrolysis of phosphatidylcholine by phospholipase D (PLD). 1-Butanol, which is a substrate for PLD and inhibits PA formation, inhibited carbachol-induced cell proliferation and the underlying intracellular signaling, whereas its analog tert-butanol, which is a poor substrate for PLD, was much less effective. In addition, exogenous PAs were able to increase DNA synthesis and to activate PKC zeta and p70S6K. Furthermore, in carbachol-stimulated cells, ethanol increased the formation of phosphatidylethanol and inhibited the formation of PA. Taken together, these results indicate that PLD activation plays an important role in carbachol-induced astroglial cell proliferation by generating the second messenger PA, which activates PKC zeta. Moreover, the effect of ethanol on carbachol-induced proliferation appears to be mediated, at least in part, by its ability to interact with PLD leading to a decreased synthesis of PA.

Involvement of phosphatidylcholine hydrolysis by phospholipase C in prostaglandin F2alpha-induced 1,2-diacylglycerol formation in osteoblast-like MC3T3-E1 cells.

We previously demonstrated that a prostaglandin F2alpha (PGF2alpha)-induced, sustained increase in 1,2-diacylglycerol (DAG) production was important for proliferation in osteoblast-like MC3T3-E1 cells. The 1,2-DAG formation is mediated by various enzymes, such as phos-phoinositide (PI)-specific phospholipase C (PLC), phospholipase D (PLD), and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). In the present study, to elucidate the mechanism of the 1,2-DAG formation, we have examined the PGF2alpha-induced production of [(3)H]phosphorylcholine, a product of PC-PLC activity, in [(3)H]choline-labeled MC3T3-E1 cells. The PGF2alpha-induced [(3)H]phosphorylcholine production was inhibited by genistein, a potent protein tyrosine kinase inhibitor, and increased by vanadate, a potent protein tyrosine phosphatase inhibitor. However, there were no effects after treatment with protein kinase C (PKC) inhibitors, the guanosine triphosphate (GTP) binding protein activator, NaF/AlCl(3), a Ca(2+)-ionophore, or the potent activator of PKC, phorbol 12-myristate 13-acetate (PMA), suggesting that a tyrosine kinase(s) was involved in the PGF2alpha-induced [(3)H]phosphorylcholine formation. Furthermore, a PGF2alpha analogue, 16-(3-trifluoromethylphenoxy)-Omega-tetranor-trans-Delta(2) PGF2alpha methyl ester (ONO-995), stimulated the proliferation of MC3T3-E1 cells to a level similar to that seen with PGF2alpha, and also caused phosphorylcholine and 1,2-DAG generation. However, neither an increase in intracellular free calcium ion ([Ca(2+)]i) levels by PI-PLC, nor phosphatidylethanol formation (and choline production) by PC-PLD were observed. From these results, we conclude that PGF2alpha-induced 1,2-DAG accumulation was mediated mainly via tyrosine kinase(s)-dependent PC hydrolysis by PLC activity in osteoblast-like MC3T3-E1 cells.

Increase in diacylglycerol production by liver and liver cell nuclei at old age

Early experiments have shown that sustained elevation of diacylglycerol (DAG) level in liver cell plasma membranes of aged rats is an essential prerequisite for disturbances in agonist responsiveness of hepatocytes. However, the mechanisms responsible for the age-related rise in DAG content in liver cells have not been clearly identified. The aim of the present study was to determine if phospholipid degradation by endogenous phospholipases precedes the DAG accumulation in liver cells and cell nuclei. The DAG formation in liver slices, hepatocytes and liver cell nuclei has been studied in 90-and 720-day-old rats. The experiments were performed in either the [ 14C]CH 3COOH-labeled rat liver or liver slices, or hepatocytes pre-labeled by [ 14C] linoleic or [ 14C] oleic or/and [ 3H] arachidonic acid. The metabolism of [ 14C-linoleil]phosphatidylcholine (PC) and [ 14C-methyl] PC was investigated in isolated liver cell nuclei. The [ 14C]DAG production in pre-labeled liver slices and in hepatocytes increased significantly and [ 14C]phosphatidylethanol formation decreased with age. DAG formed in liver of old animals showed labeling ratios ( 14C/ 3H) close to those of PC, pointing to PC as the primary DAG source in senescent liver. The liver slices of old rats, pre-labeled in situ by [ 14C]CH 3COOH, demonstrated the enhanced ability to produce DAG and sphingomyelin (SM) in a time-dependent manner while the PC level decreased in liver of old rats. The production of [ 14C]DAG, [ 14C]SM and [ 14C]phosphocholine in the nuclei of old rats was significantly higher than that in adult animals. These results suggest that PLC and SM synthase activities play a key role in a regulation of DAG basal levels present in the liver cells and in the nuclei of old rats.

Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst by cirsimaritin involves inhibition of phospholipase D signaling in rat neutrophils.

In this study, the cellular localization of the inhibitory effect of a natural flavonoid cirsimaritin against formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils was investigated. Cirsimaritin concentration-dependently inhibited the superoxide anion (O(*-)(2))generation and O(2) consumption (IC(50) 11.5+/-2.2 micro M and 17.0+/-3.9 micro M, respectively) of neutrophils. Cirsimaritin did not reduce, but slightly enhanced the O(*-)(2) generation in phorbol 12-myristate 13-acetate (PMA)-activated or arachidonic acid-stimulated NADPH oxidase preparation as well as during the autoxidation of dihydroxyfumaric acid. Cirsimaritin did not elevate cellular cAMP levels, and only partially inhibited the fMLP-induced [Ca(2+)](i) changes in the presence or absence of extracellular Ca(2+). The phosphorylation of protein tyrosine, extracellular signal-regulated protein kinase and Akt caused by fMLP were attenuated by cirsimaritin in a concentration-dependent manner. In contrast, cirsimaritin had no effect on the phosphorylation of p38 mitogen-activated protein kinase. Cirsimaritin produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol, in the presence of ethanol, from fMLP-stimulated neutrophils (IC(50) 15.1+/-6.5 micro M and 15.6+/-3.4 micro M, respectively), but did not affect the phosphatidylethanol formation in response to PMA. Under the similar concentration range, cirsimaritin attenuated the membrane translocation of ARF and Rho A. However, the GTPgammaS-stimulated membrane-associated ARF and Rho in cell lysate were unaffected by cirsimaritin. Collectively, these results indicate that the inhibition of fMLP-induced respiratory burst by cirsimaritin in rat neutrophils is likely mainly through the blockade of phospholipase D signaling pathway.

Formation of phosphatidylethanol in vitro in red blood cells from healthy volunteers and chronic alcoholics

Phosphatidylethanol (PEth) is an abnormal phospholipid, formed only in the presence of ethanol via a transphosphatidylation reaction of phospholipase D (PLD). PEth in blood is a promising new marker of alcohol abuse. Blood PEth is found almost exclusively in red cells. This study was performed to investigate a possible PEth formation in human red cells from alcoholics and healthy individuals, at physiologically relevant ethanol concentrations. Blood was drawn from six healthy volunteers (controls) and six chronic inpatient alcoholics. Hematological analyses were performed, and red blood cells were separated and incubated in plasma with ethanol to study PEth formation. Lipids were extracted and PEth analyzed with high pressure liquid chromatography and evaporative light-scattering detection. Incubation of red cells in 50 mM ethanol yielded detectable PEth after 12 hours. Formation of PEth was concentration dependent at 10 to 50 mM ethanol. In vitro formation of PEth was significantly higher (P <.001) in red cells from alcoholics (5.2 ± 1.1 μmol/l) compared to controls (2.4 ± 0.6 μmol/l) (mean ± SD). A significant correlation (P <.01) was observed between initial mean corpuscular volume and accumulated PEth. This study demonstrates that PEth is formed in human red cells at physiologically relevant ethanol concentrations. Alcoholics accumulate about twice as much PEth than controls. The accumulation rate of PEth is slower in red cells compared to rates reported for other tissues. (J Lab Clin Med 2002;140:79-83)