Metabolism Of Platelet-activating Factor Research Articles

Albuterol Isomers Modulate Platelet-Activating Factor Synthesis and Receptor Signaling in Human Bronchial Smooth Muscle Cells

Background: Racemic albuterol is a 50:50 mixture of the (R)- and (S)-enantiomers of albuterol. Its clinical efficacy resides in the (R)-enantiomer (levalbuterol). Studies have shown that (S)-albuterol induces human bronchial smooth muscle cell (HBSMC) proliferation via a pathway linked to platelet-activating factor (PAF), but the underlying mechanism by which (S)-albuterol augments PAF effects is not clear. In this study, we compared effect of levalbuterol and (S)-albuterol on PAF receptor (PAFr)-mediated signaling and PAF metabolism by HBSMCs after incubation with the albuterol isomers. Methods: PAF binding and inositol phosphate (IP₃) release were studied on adherent cultured cells. PAFr protein expression was measured by Western blotting, PAF synthesis and catabolism were measured in membrane and cytosolic proteins of cells incubated with albuterol isomers. Results: Compared to control conditions, (S)-albuterol increased PAF binding by 70% after 30 min of preincubation and by 150% after 24 h of preincubation. Levalbuterol had no effect on PAF binding under both conditions. (S)-albuterol also augmented PAF stimulation of IP₃ release, while levalbuterol and the racemic mixture had no effect. WEB 2170, a PAFr antagonist, inhibited the ability of (S)-albuterol to increase PAF binding or stimulate IP₃ release. (S)-albuterol stimulated PAFr protein expression. With PAF metabolism, (S)-albuterol treatment augmented PAF synthesis, but significantly inhibited PAF catabolism. Conclusions: Our data suggest that one mechanism by which (S)-albuterol stimulates HBSMC proliferation involves upregulation of PAFr-mediated effects including increased PAF synthesis and decreased PAF catabolism.

Effects of HAART on Platelet-Activating Factor Metabolism in Naive HIV-Infected Patients I: Study of the Tenofovir-DF/Emtricitabine/Efavirenz HAART Regimen

Platelet-activating factor (PAF) is implicated in human immunodeficiency virus (HIV)-related manifestations. Increased PAF synthesis has been recently detected in HIV-infected patients. In this study, we examined in naive HIV-infected patients the in vivo effects of a highly active antiretroviral therapy (HAART) regimen, containing tenofovir-DF/emtricitabine/efavirenz, on PAF metabolism. The specific activities of PAF basic biosynthetic enzymes, PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (lyso-PAF-AT), but also the ones of PAF-basic catabolic enzymes, PAF acetylhydrolase (PAF-AH) in leukocytes and platelets, and lipoprotein-associated-phospholipase-A(2) (LpPLA(2)) in plasma, were measured in blood samples of eight asymptomatic naive male HIV-infected patients just before and after 1, 3, and 6 months of treatment. CD4 cell counts, viral load, and several biochemical markers were also measured in the same blood samples of these patients. The repeated measures ANOVA and the Pearson r criterion were used to study statistical differences and correlations-partial correlations, while linear mixed models were conducted in order to estimate association(s) between time-dependent changes in these factors. Before treatment, the activities of PAF-CPT in leukocytes and LpPLA(2) in plasma were found to be inversely correlated with CD4 cell counts and positively correlated with the viral load. After 6 months of treatment, the activities of basic PAF-biosynthetic enzymes, PAF-CPT and lyso-PAF-AT, were both reduced in leukocytes. At 6 months, PAF-AH activity was also reduced in these cells, while LpPLA(2) remained stable. The reduction of PAF-CPT occurred even from the first month, while there is a time-dependent correlation between the increase of CD4 and the decrease of both viral load and PAF-CPT of leukocytes during treatment. Apart from its classical antiretroviral activities the tenofovir-DF/emtricitabine/efavirenz regimen also exhibited favorable effects on PAF metabolism and therefore may also display beneficial effects in some HIV-related conditions, such as cardiovascular disease (CVD), in which PAF is implicated.

Platelet-Activating Factor and Its Basic Metabolic Enzymes in Blood of Naive HIV-Infected Patients

Platelet-activating factor (PAF), a mediator of proatherosclerotic inflammatory processes, is also implicated in endothelial dysfunction during human immunodeficiency virus (HIV) infection. We examined PAF metabolism in blood of naive male patients, 8 with early HIV infection (group A) and 17 just before treatment initiation (group B), versus 18 healthy age-matched males (group C). Statistical analysis was performed with 1-way analysis of variance (ANOVA) criterion and Pearson r test. Higher PAF biosynthesis in patients' leukocytes versus group C was accompanied by an increase in lipoprotein-associated phospholipase A2 (Lp-PLA2) activity that degrades PAF. Moreover, PAF synthesis was higher and Lp-PLA2 activity was lower in group B compared to group A. Lipoprotein-associated phospholipase A2 was positively correlated with viral load and negatively correlated with CD4 cell counts in group B. The activities of PAF-basic biosynthetic enzymes in patients' leukocytes were also negatively correlated with CD4 cell counts. The observed continuous increase in PAF biosynthesis during HIV infection progress seems to amplify the risk of AIDS manifestations and/or cardiovascular complications in HIV-infected patients, while a subsequent increase in Lp-PLA2 activity seems to be a host response.

In Vivo Effects of a Ginkgo Biloba Extract on Platelet Activating Factor Metabolism in Two Asymptomatic Hiv-Infected Patients

Ginkgo biloba products seem to protect from several pathological conditions, including HIV manifestations, where Platelet Activating Factor (PAF) is implicated. In the present study, we examined for the first time the in vivo effects of a standardized formulation of Ginkgo biloba extract (150 mg daily, per os) on PAF metabolism in blood cells and plasma of two male, asymptomatic HIV-infected patients, not receiving antiretroviral treatment, during a 9-month period. These patients differed at baseline in terms of duration of HIV infection, viral load levels, CD4 cell counts and Highly Active Antiretroviral Therapy (HAART) experience. In the first patient with early HIV infection, after an initial transient increase, a return of both plasma viral load and PAF biosynthetic enzyme activities in leukocytes to their baseline levels was observed during Ginkgo biloba use. As a result PAF degradation also remained low in this patient. The second patient with late but not advanced HIV infection, had higher levels of viral load and a lower CD4 cell count at baseline. The use of 150 mg of a Ginkgo biloba extract was probably insufficient to induce PAF degradation and/or to suppress the induction of PAF biosynthesis observed. At the same time, the initial high levels of viral load were further increased and CD4 cell counts were finally decreased during the study. The observed differences in PAF metabolism during Ginkgo use seem to be related to the initial heterogeneity of these patients. It appears that in some HIV-infected patients inhibition of the PAF/PAF-receptor system, along with a decrease/down-regulation of PAF-biosynthesis, illustrates a new potential role for Ginkgo biloba compounds in the treatment of HIV infection and its manifestations. However, more tests on a larger number of patients are needed in order to support these preliminary observations.

In Vitro and In Vivo Effects of Statins on Platelet-Activating Factor and Its Metabolism

Platelet activating factor (PAF) is implicated in cardiovascular disease (CVD). Statins are widely used in these situations. Therefore, we assessed their effect on the biological activities and metabolism of PAF. Several statins, including simvastatin, exhibited an inhibitory effect against PAF, comparable with that of PAF-inhibitors. Simvastatin also suppressed in vivo PAF-biosynthesis via the de novo pathway, in leukocytes of 6 simvastatin-treated volunteers. Total cholesterol and low-density lipoprotein cholesterol were also significantly decreased, whereas high-density lipoprotein cholesterol, triacylglycerol, EC(50), and lag time were unaffected in these participants. Simvastatin with an intact lactone ring also inhibited PAF-activities, while incubation of human mesangial cells with it also resulted in decreased de novo PAF-biosynthesis. This suggests that these simvastatin-dependent effects are independent of its lactone ring. These new actions of statins should be further studied in PAF-implicated pathological conditions such as CVD, cancer, and renal disease.

Effects of highly active antiretroviral therapy (HAART) on platelet activating factor (PAF) metabolism in HIV-infected patients: in vivo results

Purpose of the study PAF, a potent inflammatory mediator, seems to play a role in the pathogenesis of several AIDS manifestations. PAFantagonists have been studied in this context with promising results. We have previously described the in vitro interaction between HAART and PAF-induced platelet aggregation, as well as between HAART and the specific activities of the basic PAF metabolic enzymes.

Mo-W14:1 Novel approaches and new biomarkers for CHD risk assessment

Platelet Activating factor (PAF) is a potent inflammatory mediator that is involved in the initiation and the prolongation of atherosclerosis. The purpose of the study was to investigate the effect of wine consumption on the activity of PAF metabolic enzymes and on IL-6 levels as a cytokine inflammatory marker. Healthy men participated in 4 daily trials and consumed a standardized meal along with Robola wine (trial R), or Cabernet Sauvignon (trial CS), or ethanol solution (trial E), or water (trial W).A significant trial effect was found in the activity of lyso-PAF acetyltransferase (Lyso-PAF AT) (ptrial = 0.01). In specific, R trial decreased enzyme activity compared to E trial (p = 0.03) while a trend for differentiation was observed between CS trial and E one (p = 0.06) as well as between R trial and W one (p = 0.07). Concerning PAF-cholinephosphotransferase (PAF-CPT) activity, a significant trial effect was found (ptrial < 0.00). Specifically, both R (p = 0.002) and CS (p = 0.001) trials decreased enzyme activity compared to E trial. Concerning lipoprotein-associated phospholipase A2 (LpPLA2) no time either trial effect was observed. Concerning IL-6 levels a significant time effect was found (ptime < 0.00) while no trial effect was revealed.In conclusion, the protective effect of wine consumption could partly be explained through the modulation of PAF metabolism by wine micro-constituents that lead to lower PAF levels.

In vivo antiatherogenic properties of olive oil and its constituent lipid classes in hyperlipidemic rabbits

Background and aim The consumption of olive oil has been associated with lower incidence of cardiovascular disease in the Mediterranean countries. This may be due in part to the action of platelet-activating factor (PAF) antagonists which we have previously demonstrated to be present in olive oil. In order to assess the in vivo effects of olive oil lipids and PAF in the development of atherosclerosis, the effects of diet supplementation with olive oil (OO), olive oil polar lipid extract (OOPLE) and olive oil neutral lipid extract (OONLE) were studied in rabbits fed a cholesterol-enriched diet. Methods and results Rabbits were fed for 45 days with atherogenic diet (Group A) supplemented with OO (Group B), OOPLE (Group C) or OONLE (Group D). Lipoprotein profiles, plasma in vitro oxidation, blood PAF levels, PAF-induced platelet aggregation and PAF-acetylhydrolase (PAF-AH) activity, were measured on day 0 and 45. Atherosclerotic lesions formed in the aortic wall and wall elasticity were assessed on day 45. Changes in lipid profile were in accordance with previous studies. Blood PAF levels were higher in group A and decreased in group D on day 45. In rabbits fed an atherogenic diet (Group A) blood PAF and PAF-AH increased, atherosclerotic lesions formed and the elasticity of vessel walls declined. In animals fed olive oil (Group B) or OOPLE (Group C) blood PAF-AH increased, platelet aggregation was attenuated, less oxidation occurred in plasma, lesion thickness was reduced and vessel walls retained elasticity. Most of these beneficial changes were not seen in animals fed OONLE (Group D) although blood PAF and plasma oxidation were lower. Conclusions The antiatherogenic effects of OO result from OOPLE. The beneficial effect of these factors is linked to PAF metabolism and proaggregant activity.

Cardiac surgery for a patient with familial lecithin: Cholesterol acyltransferase deficiency

A 62-year-old female with familial lecithin: cholesterol acyltransferase (LCAT) deficiency was subjected to cardiac surgery consisting of mitral valve repair, tricuspid valve annuloplasty, and left atrium plication, under cardiopulmonary bypass. Familial LCAT deficiency is a very rare disease with clinical features including corneal opacity, hemolytic anemia, and renal failure. Our patient manifests partial LCAT activity and no renal failure. Patients with LCAT deficiency have fragile red blood cells, renal dysfunction, and disturbed metabolism of platelet-activating factor. To date, no record of cardiac surgery on patients with LCAT deficiency has been reported. Thus, we present the result of this experience to share what we learned about this complicated and rare disease.

Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 ≤ 5 nM for C 8–C 18 analogs and 0.1–0.6 nM for C 13 and C 14 compounds; human plasma IC50 ≤ 2 nM for C 13–C 18 analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C 13–C 18 compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C 13 n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.

Delineation of the role of platelet-activating factor in the immunoglobulin G2 antibody response.

Localized aggressive periodontitis (LAgP) is a chronic inflammatory disease characterized by severe destruction of periodontal tissues surrounding the first molars and incisors. LAgP subjects produce large amounts of immunoglobulin G2 (IgG2) antibody against oral pathogens, and this response is inversely correlated with the severity of disease. We previously demonstrated that platelet-activating factor (PAF) is required for optimal IgG2 responses. The present investigation was designed to determine the mechanism of IgG2 induction by PAF. Exogenous PAF acetylhydrolase suppressed approximately 80% of pokeweed mitogen-stimulated IgG2 production, confirming that PAF is essential for optimal responses. PAF-activated leukocytes produced gamma interferon (IFN-gamma), a Th1 cytokine that has been associated with IgG2 responses in previous studies. The monocyte-derived cytokines interleukin-12 (IL-12) and IL-18 are upstream of IFN-gamma production, and IgG2 production was suppressed by neutralizing antibodies against these proteins. In addition, PAF induced monocyte-derived dendritic cells (DC) but not macrophages (MPhi) to secrete IL-12 and IL-18. This observation was interesting because monocyte differentiation in LAgP subjects is skewed to the DC phenotype. Although other investigators have implicated IFN-gamma in IgG2 production, its precise role in this response is controversial. Our studies suggest that IFN-gamma induces isotype switching to IgG2 but only in concert with the Th2 cytokine IL-4. Thus, it appears that the unique PAF metabolism of LAgP monocytes or DC promotes Th1 responses that are essential for optimal IgG2 antibody production. As IgG2 antibodies opsonize oral bacteria and promote their clearance and destruction, these alterations in PAF metabolism may be essential for limiting disease severity in LAgP patients.

Lycopene in association with α-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with α-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H 2O 2-induced oxidative stress. The results indicated that in each case H 2O 2-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with α-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that α-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.

Activation of protein kinase C inhibits the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages.

Background and Aims: Platelet activating factor (PAF), a potent phospholipid mediator, has been implicated in a number of reproductive processes through ovulation to parturition. To clarify the regulatory mechanism of PAF metabolism in the decidua, we have investigated the effect of activation of protein kinase C (PKC) on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by human decidual macrophages. Methods: Decidual macrophage populations were isolated from human decidua by using enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The cells were treated with a PKC activator (TPA), H-7, dibutyryl cyclic adenosine monophosphate (AMP), Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl)-ester (BAPTA/AM) and/or nifedipine. The activity of PAF-AH secreted in the culture medium was assayed. Results: The PKC activator, TPA, inhibited the PAF-AH secretion by decidual cells in a dose-dependent manner. The TPA also decreased the enzyme secretion by flow cytometrically purified macrophages. The inhibitory effect of TPA was blocked by a PKC inhibitor, H-7. Protein kinase A (PKA) activation by dibutyryl cyclic AMP was without effect on the enzyme secretion. Calcium channel blockers, BAPTA/AM and nifedipine had no effect on the PAF-AH secretion. Conclusion: It is suggested that the TPA-induced inhibition of PAF-AH secretion may be mediated, in part, by a PKC-dependent signal transduction, and that activation of PKC may result in the increase in the local concentration of PAF in the decidua because of its inhibitory effect on the PAF-AH secretion by decidual macrophages. (Reprod Med Biol 2003; 2: 121-126).

Metabolism of platelet activating factor by intrapulmonary vascular smooth muscle cells. Effect of oxygen on phospholipase A 2 protein expression and activities of acetyl-CoA acetyltransferase and cholinephosphotransferase

We have demonstrated that platelet activating factor (PAF) plays an important physiological role in the maintenance of high pulmonary vascular tone in fetal lambs, a role attributable to increased PAF receptor binding (J. Appl. Physiol. 85 (1998) 1079; Am J. Physiol. 278 (2000) H1168). In this study, we examined the possibility that increased PAF synthesis via de novo and remodeling pathways as well as decreased PAF catabolism in hypoxic state of fetal lungs may account for the PAF action in vivo. We investigated effect of oxygen tension on PAF synthesis by ovine fetal intrapulmonary venous (PV) and arterial (PA) smooth muscle cells pulsed with [ 3 H ]choline (de novo), or [ 3 H ]acetate (remodeling), while PAF catabolism was studied by assay of acetylhydrolase (PAF-Ah) activity. Hypoxia stimulated PAF synthesis by choline incorporation (pmol/10 6 cells) in both PVSMC (1.14±0.13 vs 0.53±0.05) and PASMC (0.39±0.12 vs 0.22±0.04). Hypoxia stimulated PAF synthesis via remodeling pathway only in PVSMC (408±32 vs 225±17) which was 5-fold greater than in PASMC (77±15 vs 105±24), however, with A23187 in remodeling pathway, PAF synthesis increased 5-fold compared to baseline conditions and then synthesis in hypoxia was greater than in normoxia in both cell types. Phospholipase A 2 protein expression was significantly higher in hypoxia in both cells and was ∼2-fold higher in PVSMC. PAF-Ah activity (nmol lyso-PAF/min/mg protein) was greater in hypoxia vs normoxia in PVSMC (0.81±0.24 vs 0.44±0.088), but in PASMC activity was less in hypoxia vs normoxia (1.68±0.24 vs 3.93±0.44). Compared to PVSMC PAF-Ah activity in PASMC was 4-fold higher in hypoxia. Our data demonstrate that (1) PAF synthesis in intrapulmonary SMC of fetal lambs occurs by both de novo studied by choline incorporation and remodeling pathways, the latter being predominant. (2) There is heterogeneity in PAF synthetic and catabolic activities in lung vasculature of fetal lambs. We conclude that increased PAF synthesis in veins by the two synthetic pathways coupled with decreased catabolism will result in a higher venous PAF levels in the hypoxic environment of fetal lungs. We speculate that in vivo, a high PAF level in veins will make more PAF available for binding to its receptors so as to sustain the desired high venous tone in the fetal pulmonary circulation.

Receptor-dependent Metabolism of Platelet-activating Factor in Murine Macrophages

Degradation of platelet-activating factor (PAF) was examined by incubating PAF with macrophages from PAF receptor-deficient mice. The degradation rate was halved as compared with wild-type mice. The reduction of the rate was comparable with the presence of a PAF antagonist WEB 2086 in wild-type cells. PAF was internalized rapidly (t(12) approximately 1 min) into wild-type macrophages. The PAF internalization was inhibited by the treatment of 0.45 m sucrose but was not affected by phorbol 12-myristate 13-acetate, suggesting that PAF internalizes into macrophages with its receptor in a clathrin-dependent manner. Internalized PAF was degraded into lyso-PAF with a half-life of 20 min. Treatment of concanavalin A inhibited the conversion of PAF into lyso-PAF, suggesting that uptake of PAF enhances PAF degradation. Lyso-PAF was subsequently metabolized into 1-alkyl-2-acyl-phosphatidylcholine. In addition, release of PAF acetylhydrolase from macrophages was enhanced when wild-type macrophages were stimulated with PAF but not from macrophages of PAF receptor-deficient mice. Thus, the PAF stimulation of macrophages leads to its degradation through both intracellular and extracellular mechanisms.

1-O-alkylglycerols improve boar sperm motility and fertility.

1-O-alkylglycerols are naturally occurring ether lipids with potent biological activities. They may interfere with lipidic signaling, and they amplify platelet-activating factor (PAF) biosynthesis in a monocyte cell line. The PAF is produced by mammalian sperm and is an important activator of sperm motility. The aim of this study was to evaluate the effect of in vitro treatment of boar spermatozoa with natural 1-O-alkylglycerols (10 microM) on 1) boar sperm motility; 2) production of PAF and its metabolite, lyso-PAF, by spermatozoa; and 3) fertility in artificial inseminations of breeding sows. Using a computer-assisted spermatozoa analyzer, we found that 1-O-alkylglycerols increased percentage motility as well as velocity parameters after 24 h. These effects were partially or totally reversed by the PAF receptor-antagonist SR 27417. After [3H]-1-O-alkylglycerol incubation with boar spermatozoa, we identified [3H]lyso-PAF by high-performance liquid chromatography. Production of PAF and lyso-PAF was measured with a biological assay using [3H]serotonin release from rabbit platelets. 1-O-alkylglycerols significantly increased lyso-PAF production but had no effect on PAF production. The effect of 1-O-alkylglycerols on fertilization was also evaluated in industrial breedings: 1-O-alkylglycerol-treated or untreated semen dilutions were alternately used for artificial inseminations of sows on 12 farms. 1-O-alkylglycerol treatment increased the number of farrows but had no effect on the mean size of the litters. This study demonstrates that 1-O-alkylglycerol treatment of boar spermatozoa in vitro improves their motility and fertility, and it suggests that this effect is related to PAF metabolism and function in boar spermatozoa.

Maturational Changes in Ovine Pulmonary Metabolism of Platelet-Activating Factor: Implications for Postnatal Adaptation

We recently reported that PAF acetylhydrolase (PAF-Ah) mRNA level and PAF-Ah activity in lamb lungs are up-regulated in the immediate newborn period, thereby facilitating the fall in postnatal PAF levels as well as a fall in pulmonary vascular resistance (B. O. Ibe, F. C. Sardar, and J. U. Raj, Mol Genet Metab 69:46–55, 2000). We have studied hypoxia effects on PAF synthesis and PAF-Ah activity in fetal lamb pulmonary arterial smooth muscle cells (FPASMC) and endothelial cells (FPAEC). We also studied PAF synthesis by platelets, and PAF-Ah activity in plasma of perinatal lambs at different ages. PAF synthesis (means ± SEM, pmol/106 cells) by SMC in baseline was 168 ± 27 and increased 3-fold on stimulation with A23187. Hypoxia augmented A23187-stimulated PAF synthesis by 30%. In FPAEC, baseline synthesis was 0.54 ± 0.062 and increased 3-fold to 1.72 ± .34. Hypoxia had no effect on PAF synthesis by EC. FPASMC produced over 300-fold more PAF than FPAEC. PAF synthesis by platelets was 47.02 ± 7.1, 63.4 ± 6.6, 71.5 ± 9.9, and 62.2 ± 5.2 for fetal, and newborn lambs <2 h, <1 day, and 6–12 days, old, respectively. PAF synthesis by platelets of <1 day-old lambs was different from that of fetal lambs. PAF-Ah activity (nmol lyso-PAF/min/mg protein) by FPASMC in normoxia was 3.41 ± 0.38 which was 50% higher than the rate in hypoxia. Activity in FPAEC was 1.75 ± 0.37 which was not different from hypoxia. PAF-Ah activity in fetal lamb plasma was 47.83 ± 6.87 which was different from 155.32 ± 12.10, the activity in plasma of newborn <1 day old. Activity in the other perinatal lambs did not differ from fetal or newborn <1 d. Our data suggest that lower pulmonary vascular PAF synthesis in normoxia together with higher PAF-Ah activity during immediate postnatal period is necessary to ensure rapid catabolism of PAF in vivo so as to facilitate postnatal adaptation of the pulmonary and systemic circulations.

The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.

Dietary polyunsaturated fatty acid (PUFA) supplementation has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in a recent randomized, controlled trial. These compounds are known to modulate the inflammatory cascade and to influence intestinal health in a variety of ways. Although the pathophysiology of NEC is not well understood, recent evidence suggests that platelet-activating factor (PAF) is a key endogenous mediator of intestinal necrosis in animals. Using a neonatal rat model of NEC that includes the key risk factors of asphyxia and formula feeding, we investigated the role of dietary PUFA supplementation on the incidence and pathophysiology of NEC. Our findings suggest that PUFA reduce the incidence of NEC by modulating PAF metabolism and endotoxin translocation.

Lipid acetylation reactions and the metabolism of platelet-activating factor.

In this review properties of lipid acetyltransferase enzymes are outlined. The three activities of interest are lyso PAF acetyltransferase (acetyl CoA: 1-alkyl-sn-glycero-3-phosphocholine acetyltransferase), AGP acetyltransferase (acetyl CoA: 1-alkyl sn-glycero-3-phosphate acetyltransferase) and a transacetylase activity that can transfer acetyl groups from PAF to lipid acceptors in the formation of 1-alkenyl-2-acetyl-sn-glycero-3-phosphoethanolamine and N-acetyl sphingosine (C2 ceramide). This review focuses on the role of acetyltransferases and transacetylases within the metabolism of platelet-activating factor and specifically addresses characteristics of the enzymes, including subcellular localization, substrate selectivity, and enzymatic regulation.

The regulation of CoA-independent transacylation reactions in neuronal nuclei by lysophospholipid, free fatty acid, and lysophospholipase: the control of nuclear lyso platelet-activating factor metabolism.

CoA-independent transacylase activities generating alkylacylglycerophosphocholine (AAGPC) from alkylglycerophosphocholine (1-alkyl GPC) were considerably enriched in neuronal nuclei isolated from rabbit cerebral cortex. Specific nuclear transacylation activities were 13 times the corresponding microsomal values. Several lysophospholipids, notably 1-acyl glycerophosphocholine (1-acyl GPC), 1-alkenyl GPC and 1-alkenyl GPE (1-alkenyl glycerophosphoethanolamine) inhibited the transacylation of 1-alkyl GPC. The inhibitory effects of 1-acyl GPC were seen in the presence of MAFP (methyl arachidonoylfluorophosphonate) or free oleate, compounds that inhibit neuronal nuclear lysophospholipase. When neuronal nuclei were preincubated with 1-alkyl GPC, the radioactive AAGPC product served as donor in transacylation reactions, to generate 1-alkyl GPC. In these nuclear reactions, 1-palmitoyl GPE and 1-palmitoyl GPC appeared to be poor acceptor substrates, when compared with corresponding 1-alkyl and 1-alkenyl analogues. The presence of free oleate or MAFP in the reactions containing 1-acyl GPC boosted the release of 1-alkyl GPC from AAGPC. These observations are of particular relevance to brain ischemia in which lysophospholipid, free fatty acid, and platelet-activating factor (PAF) levels rise dramatically. PAF can be made by the nuclear acetylation of 1-alkyl GPC, which is formed by nuclear transacylation mechanisms. Yet transacylase also removes 1-alkyl GPC, and thus this enzyme activity can regulate 1-alkyl GPC availability. Our observations indicate that lysophospholipids promote the formation of 1-alkyl GPC from nuclear AAGPC via transacylation, while free fatty acid likely prolongs the lifetime of 1-acyl lysophospholipids substrates by lysophospholipase inhibition. Similarly, once 1-alkyl GPC is formed, other lysophospholipids effectively compete with this 1-alkyl analogue and reduce its conversion back to AAGPC by transacylation. Free oleate, in this case, sustains 1-acyl lysophospholipid inhibitors of 1-alkyl GPC transacylation. Thus the cycle of transacylation may favour 1-alkyl GPC formation during ischemia, increasing levels of 1-alkyl GPC for nuclear acetylation reactions and PAF formation. The nuclear generation of PAF is of considerable importance as PAF can play regulatory roles in transcription events associated with inflammation.