Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

CIRBP promotes ferroptosis by interacting with ELAVL1 and activating ferritinophagy during renal ischaemia-reperfusion injury

Renal ischaemia-reperfusion (IR) is a major cause of acute kidney injury (AKI). Cold-inducible RNA-binding protein (CIRBP) may contribute to AKI because its deficiency protects against renal IR injury in a mechanism believed to involve ferroptosis. We aimed to investigate whether ferroptosis is associated with CIRBP-mediated renal damage. The differential expression of CIRBP was examined in tubular epithelial (HK2) cells during hypoxia-reoxygenation (HR) or in response to erastin, an inducer of ferroptosis. CIRBP expression was increased in response to HR or erastin in HK2 cells but the silencing of CIRBP inhibited HR and erastin-induced ferroptosis together with ferritinophagy. We discovered an interaction between CIRBP and ELAVL1 using STRING software, which was verified through co-immunoprecipitation and fluorescence colocalization assays. We found that ELAVL1 is a critical regulator in the activation of ferritinophagy and the promotion of ferroptosis. HR or erastin also induced the expression of ELAVL1. An autophagy inhibitor (hydroxychloroquine) or si-ELAVL1 transfection reversed CIRBP-enhanced ferritinophagy activation and ferroptosis in HK2 cells under HR. Injection of anti-CIRBP antibody into a mouse model of IR inhibited ferroptosis and decreased renal IR injury in vivo. In summary, our results provide evidence that ferritinophagy-mediated ferroptosis could be responsible for CIRBP-enhanced renal IR injury.     

Enzymes of platelet activating factor synthesis in brain

In this review, evidence is summarized for the production of PAF in brain, in response to stimulation associated with pathology. As well, there is a growing literature on the duality of actions of this lipid autocoid upon nervous tissue, indicated by extracellular and intracellular actions and binding sites for PAF in brain. The metabolic routes to PAF can be divided into the de novo and remodelling pathways of synthesis. The de novo route consists of 1-alkyl glycerophosphate acetyltransferase, and the subsequent actions of distinct phosphohydrolase and cholinephosphotransferase activities. This acetyltransferase can be activated by phosphorylation, and inhibited by MgATP and fatty acyl CoA thioesters, inhibitions which have particular relevance to brain ischemia. There is also evidence that the cholinephosphotransferase is controlled by phosphorylation, and regulated by levels of CDP-choline. The remodelling pathway to PAF relies upon the actions of phospholipase A₂ or CoA-independent transacylases to generate the l-alkyl glycerophosphorylcholine, as substrate for a distinct acetyltransferase. Following stimulation, rising intracellular calcium may trigger arachidonate selective cytosolic phospholipase activity which leads to increased PAF synthesis. The l-alkyl glycerophosphocholine acetyltransferase activity is quite small in brain in comparison with the de novo acetyltransferase activity, and is also controlled by phosphorylation. Evidence has been presented for the actions of both pathways in brain, in response to biologically relevant stimulation pertinent to the disease state.Special issue dedicated to Dr. Leon S. Wolfe.     

Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

We treated four anesthetized dogs (Canis familiaris) with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous 111In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone (3 mg/kg) administered 5 min prior to ischemia and continuously (1 mg/kg/hr) throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude (% of baseline) when compared to controls (27-36% vs 9-14%, p less than 0.05). We estimated platelet accumulation as 111In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered 111In-labeled platelet accumulation when compared to controls (6158 +/- 2386 vs 9979 +/- 3852, mean +/- SEM). Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved.     

Platelet activating factor inhibits irritant-induced leukocyte migration into mouse peritoneal cavity

The effect of platelet-activating factor (PAF) on the leucocytes accumulation into mouse peritoneal cavity, that induced by high-molecular irritants carrageenan and polyacrylic acid pentaerytritol (carbopol) was studied. It was found that injection of PAF into peritoneal cavity one hour before i.p. injection of the irritants did not change their effects. On the contrary the injection of PAF simultaneously with the irritants or 3 and 6 hours after these injections inhibited the leucocyte accumulation into the peritoneal cavity and/or these injections increased the leucocyte adhesion to the peritoneal covering. It was supposed that finding effects are mediated by the change of leucocytes adhesion under action of PAF.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

Platelet activating factor induces cytoskeletal reorganization through Rho family pathway in THP-1 macrophages

In the process of atherosclerosis, platelet activating factor (PAF) promotes the infiltration of inflammatory cells into atherosclerotic plaque by modulating their cytoskeleton. Here, we examined whether Rho family proteins are involved in PAF-induced cytoskeletal reorganization in THP-1 macrophages. PAF stimulation rapidly induced cell elongation, accompanied by filopodia formation. The inhibition of Rho family proteins by the overexpression of Rho-GDI attenuated the PAF-mediated morphological changes. Both RhoA and Cdc42 were activated in response to PAF. Inhibition of RhoA or Cdc42 by dominant negative mutants abrogated morphological changes induced by PAF. Collectively, PAF regulates cytoarchitecture through Rho family proteins in macrophages.     

Effects of aminoguanidine against renal ischaemia-reperfusion injury in rats

Aminoguanidine is an inhibitor of nitric oxide synthase (NOS), with high selectivity for the inducible isoform (iNOS). In addition to being an inhibitor of NOS, aminoguanidine also exhibits antioxidant activity. Recent studies suggest that aminoguanidine reduces ischaemia-reperfusion (I/R)-induced damage. However, the role of aminoguanidine, in renal injury associated with I/R remains unknown. This study was designed to investigate the effects of aminoguanidine on renal I/R injury. There were three groups of eight rats each. I/R was induced by occlusion of the left renal vessels for 60 min, followed by 24 h reperfusion in rats. Malondialdehyde (MDA) levels, a stable metabolite of the free radical-mediated lipid peroxidation cascade, were found to be significantly higher in the I/R group (30.3 +/- 0.1 nmol g(-1) tissue) than in the control group (10 +/- 0.05 nmol g(-1)). Aminoguanidine (100 mg kg(-1)) administration to rats significantly reduced the MDA values. We also demonstrated that I/R leads to structural change but aminoguanidine did not reverse this change. Aminoguanidine, according to the biochemical finding is protective but histopathological findings did not reveal protection against I/R injury in kidney. The effects of aminoguanidine on I/R-induced damage remain a subject for future investigations.     

Platelet activating factor intravenous infusion in rats stimulates vascular lysosomal hydrolase secretion independent of blood neutrophils

Intravenous infusion of platelet activating factor into rats at doses of 5-20 nmoles/kg results in a rapid and dose-dependent increase in the plasma level of several lysosomal hydrolases, notably glucosaminidase. This hydrolase secretion occurs concomitantly with the increased vascular permeability but subsequent to the neutropenia and hypotensive responses to platelet activating factor. The glucosaminidase release in vivo does not exhibit any lasting desensitization, does not require cytochalasin B, and is quantitatively the same in rats made neutropenic with anti-neutrophil serum, and thus is different from the published in vitro degranulating effect of this lipid with neutrophils. Therefore, lysosomal hydrolase secretion may be an important pathophysiologic response to very low blood levels of platelet activating factor.     

Platelet activating factor receptor-deficient mice present delayed interferon-gamma upregulation and high susceptibility to Leishmania amazonensis infection

We investigated the role of the platelet activation factor (PAF) receptor (PAFR) in the outcome of infection with Leishmania amazonensis. PAFR deficient (PAFR(-/-)) mice were infected with L. amazonensis and the course of infection was followed. We found that PAFR(-/-) mice in the C57BL/6 background were more susceptible to infection with L. amazonensis than the wild-type controls, as seen both by lesion size and parasite number at the site of infection. Interferon (IFN)-gamma production was delayed in PAFR(-/-) mice, and lower levels of Ccl5 were found in lesions. Expression of nitric oxide synthase-2 mRNA was found impaired in PAFR(-/-) associated with higher levels of arginase-1 mRNA. Moreover, higher levels of antibodies were produced in response to L. amazonensis by PAFR(-/-) mice. We conclude that signaling through the PAFR is essential for the ability of the murine host to control L. amazonensis infection by driving an adequate immune response.     

Mechanisms in bradykinin stimulated arachidonate release and synthesis of prostaglandin and platelet activating factor

Regulatory mechanisms in bradykinin (BK) activated release of arachidonate (ARA) and synthesis of prostaglandin (PG) and platelet activating factor (PAF) were studied in bovine pulmonary artery endothelial cells (BPAEC). A role for GTP binding protein (G-protein) in the binding of BK to the cells was determined. Guanosine 5-O- (thiotriphosphate), (GTPtauS), lowered the binding affinity for BK and increased the Kd for the binding from 0.45 to 1.99 nM. The Bmax remained unaltered at 2.25 x 10(-11) mole. Exposure of the cells to aluminium fluoride also reduced the affinity for BK. Bradykinin-induced release of ARA proved pertussis toxin (PTX) sensitive, with a maximum sensitivity at 10 ug/ml PTX. GTPtauS at 100 muM increased the release of arachidonate. The effect of GTPtauS and BK was additive at suboptimal doses of BK up to 0.5 nM but never exceeded the levels of maximal BK stimulation at 50 nM. PTX also inhibited the release of ARA induced by the calcium ionophore, A23187. Phorbol 12-myristate 13-acetate or more commonly known as tetradecanoyl phorbol acetate (TPA) itself had little effect on release by the intact cells. However, at 100 nM it augmented the BK activated release. This was downregulated by overnight exposure to TPA and correlated with down-regulation of protein kinase C (PKC) activity. The down-regulation only affected the augmentation of ARA release by TPA but not the original BK activated release. TPA displayed a similar, but more potent amplification of PAF synthesis in response to both BK or the calcium ionophore A23187. These results taken together point to the participation of G-protein in the binding of BK to BPAEC and its activation of ARA release. Possibly two types of G-protein are involved, one associated with the receptor, the other activated by Ca(2+) and perhaps associated with phospholipase A(2) (PLA(2)). Our results further suggest that a separate route of activation, probably also PLA(2) related, takes place through a PKC catalysed phosphorylation.