Platelet Activating Factor In Neutrophils Research Articles

The allosterically modulated FFAR2 is transactivated by signals generated by other neutrophil GPCRs.

Positive allosteric modulators for free fatty acid receptor 2 (FFAR2/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFAR2 was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFAR2 from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFAR2 in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFAR2s, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFAR2 activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFAR2 activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFAR2 activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFAR2 modulator.

The Subcellular Localization of the Receptor for Platelet-Activating Factor in Neutrophils Affects Signaling and Activation Characteristics

The localization in neutrophils, of the receptor for platelet-activating factor (PAFR), has been determined using subcellular fractionation and a receptor mobilization protocol. We show that the PAFR is expressed primarily in the plasma membrane. Although activation of neutrophils by PAF induces responses typical also of agonists that bind the formyl peptide receptors (FPR), known to be stored in mobilizable organelles, some quantitative as well as qualitative differences were observed when neutrophils were activated through these receptors. PAF is equipotent to fMLF (high affinity agonist for FPR1) to cleave off L-selectin and to induce granule/vesicle secretion but is more potent than fMLF to induce a rise in intracellular Ca2+. Similar to fMLF, PAF induced also a robust release of reactive oxygen species, but with higher EC50 value and was less sensitive to a PI3K inhibitor compared to the fMLF response. Despite the lack of a granule localized storage pool of receptors, the PAF-induced superoxide production could be primed; receptor mobilization was, thus, not required for priming of the PAF response. The desensitized PAFR could not be reactivated, suggesting that distinct signaling pathways are utilized for termination of the responses triggered through FPR1 and PAFR.

Plasma Lipids and Plasma and Urinary Acetyl Hydrolase Activity in Normal and Hypertensive Pregnancies

Objective: In a previous study we showed neutrophil platelet-activating factor (PAF) to be reduced in preeclampsia, while plasma lyso-PAF was increased. The aim of the current study was to examine (PAF) acetyl hydrolase activity (the enzyme that degrades PAF) in plasma and urine of new groups of preeclamptic, hypertensive, and normal pregnant women, to see if alterations in the activity of this enzyme might explain the changes in neutrophil PAF and plasma lyso-PAF previously seen in severe preeclampsia.Methods: Plasma and urinary acetyl hydrolase activity was measured in 22 women with proteinuric preeclampsia and 22 age- and gestational-age-matched normal pregnant women. To determine whether alterations in acetyl hydrolase activity in preeclampsia were due to hypertension per se, a comparison was made to a group of 10 age- and gestational-age-matched pregnant women with uncomplicated essential hypertension. The pregnant women were studied prior to delivery and at 6 weeks and 6 months postpartum, and were ...

Neutrophil Platelet-Activating Factor in Normal and Hypertensive Pregnancy and in Pregnancy-Induced Hypertension

1. Platelet-activating factor is a phospholipid with potent vasodilator and platelet-activating properties. To test the hypothesis that a generalized change in cellular platelet-activating factor metabolism may be involved in the systemic vasodilatation of normal pregnancy or pregnancy-induced hypertension, we studied platelet-activating factor and eicosanoid synthesis in isolated leucocytes obtained from pregnant women before and after delivery compared with age-matched non-pregnant control subjects. Parallel observations were carried out in age- and gestation-matched women with uncomplicated hypertension in pregnancy and in women with pregnancy-induced hypertension and a further set of normotensive pregnant control subjects. 2. Leucocyte counts were higher in all pregnant groups compared with non-pregnant control subjects. Neutrophil production of platelet-activating factor and metabolites of prostacyclin, prostaglandin E2 and thromboxane in response to calcium ionophore stimulation were all lower in pregnant women compared with non-pregnant control subjects, but returned to similar levels 6 weeks post partum. There was no significant difference between essential hypertensive and normotensive groups. When women with pregnancy-induced hypertension were a priori sub-divided into those with or without proteinuria, subjects with proteinuria showed significantly lower levels of neutrophil platelet-activating factor synthesis. 3. Plasma levels of the platelet-activating factor metabolite (lyso-platelet-activating factor) were also lower in pregnancy, suggesting alterations in the activity of enzymes controlling synthesis or degradation of this phospholipid in pregnancy. In pregnancy-induced hypertension the levels of plasma lyso-platelet-activating factor were higher than in normal pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Calaguala and an active principle, adenosine, on platelet activating factor.

Calaguala, an extract from the fern Polypodium decumanum, has been used to treat psoriasis and related immunological disorders. In an effort to explain Calaguala's medicinal effects the inhibitory activity of the extract in two platelet activating factor (PAF) related models has been investigated. In the first model, PAF was used to induce release of the proteolytic enzyme elastase in human neutrophils. Calaguala inhibited this effect with an IC50 of 0.1 mg/ml. The known PAF antagonist ginkgolide BN 52021 was used as a positive control and had an IC50 of 0.034 mg/ml. In the second model the inhibition of biosynthesis of PAF in neutrophils using lyso-PAF and labeled acetyl-CoA was studied. Also in this assay Calaguala showed a dose-dependent activity, the IC50 being 0.2 mg/ml. Since recent findings have indicated that PAF might be involved in the pathogenesis of psoriasis, it is possible that the activity shown by Calaguala in these PAF assays may contribute to the clinical efficacy of the extract. The PAF induced exocytosis assay was further used to guide the fractionation of the crude extract. From the acetone supernatant the nucleoside adenosine was isolated as an active principle. Pure adenosine dose-dependently inhibited the exocytosis induced by PAF (IC50 = 0.024 micrograms/ml) but was inactive in the biosynthesis assay. Adenosine is most probably one of the bioactive compounds of Calaguala responsible for its therapeutic properties.

Metabolism of platelet-activating factor in neutrophils isolated from Pichinde virus-infected guinea pigs.

Metabolism of platelet-activating factor (PAF) was studied in cultured polymorphonuclear neutrophils (PMNs) obtained from Pichinde virus-infected strain 13 guinea pigs. Neutrophils obtained from control and infected guinea pigs on postinoculation days 10 and 14 were incubated with [3H]lyso-PAF, [3H]PAF, or [3H]acetate. After incubation for 1 h at 37 degrees C, formation of [3H]acyl-PAF from either [3H]lyso-PAF or [3H]PAF increased significantly in PMNs from infected guinea pigs compared to control PMNs. Furthermore, total radioactivity from [3H]lyso-PAF or [3H]PAF was higher in PMNs from infected animals than in those from controls. However, compared to PAF production by control PMNs, production of PAF by infected PMNs was unchanged. These results suggest that PMNs may not be the major source of increased blood PAF levels during Pichinde viral disease.

Metabolic fate of platelet-activating factor in neutrophils.

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-acetyl-GPC) incubated with rabbit polymorphonuclear leukocytes was metabolized to 1-O-alkyl-2-acyl-GPC containing long chain groups at the 2 position. Within 5 min, 60% of the total label added to the cell suspension was found in the 1-O-[3H]alkyl-2-acyl-GPC. At earlier time points, 1-O-[3H]alkyl-2-lyso-GPC was present. No metabolites were detected that would indicate the ether bond had been cleaved or the polar head group had been altered by the cells. At subaggregating and subdegranulating concentrations of labeled 1-O-[3H]alkyl-2-acyl-GPC, the same pattern of product formation was observed. Furthermore, when 1-O-[3H]alkyl-2-lyso-GPC was added to the cells, it was taken up and acylated in the same manner as that from 1-O-alkyl-2-acetyl-GPC. The nature of the long chain acyl residues incorporated into the 2 position was then examined by argentation chromatography and high performance liquid chromatography. Argentation chromatography of 1-O-[3H]alkyl-2-acyl-3-acetylglycerols obtained from 1-O-[3H]alkyl-2-acyl-GPC after acetolysis indicated that mono-, di-, and tetraenoic fatty acids were the predominant molecular species being incorporated into the 2 position of the molecule (18, 55, and 18%, respectively). Furthermore, high performance liquid chromatographic analysis using synthetic 1-O-alkyl-2-acyl-GPC standards indicated that three fatty acids, linoleic, arachidonic, and oleic (50, 15, and 12%, respectively), were the major chains being incorporated into the 1-O-hexadecyl-linked species. Analysis of 1-O-[3H]alkyl-2-acyl-GPC derived from exogenously added 1-O-[3H]alkyl-2-lyso-GPC revealed the same distribution of acyl groups linked to the 2 position. The findings are consistent with a pathway in which two enzymatic activities are responsible for the metabolism of exogenous platelet-activating factor in the rabbit neutrophils: one that hydrolyzes the acetyl residue (acetylhydrolase) and another that transfers a fatty acyl chain to the 1-O-alkyl-2-lyso-GPC formed (possibly acyl-CoA:1-O-alkyl-2-lyso-GPC acyltransferase). These events appear to play an important role in inactivating this potentially lethal phospholipid.