Identification of platelet-activating factor receptors in P388D1 murine macrophages

Platelet-activating factor (PAF) binding and metabolism by eight murine and human cell lines was analyzed. Only the murine P388D1 macrophage line had specific, high affinity PAF binding sites. PAF binding reached saturation within 10 min at room temperature and was irreversible. Minimal PAF metabolism was observed at the time binding saturation was achieved. Scatchard analysis of PAF binding revealed a single class of PAF receptors (7872 +/- 1310/cell) which had a dissociation constant of 0.08 +/- 0.01 nM (mean +/- SEM, eta = 6). The dissociation constant was confirmed independently by quantifying the kinetics of initial specific PAF binding. PAF binding was stereospecific, required an sn-2 acetyl substituent, and was inhibited by structurally diverse PAF antagonists including kadsurenone, BN 52021, triazolam, and CV3988. The fact that the receptors are functionally active was shown by the observation that 1 to 100 pM PAF increased free intracellular calcium in P388D1 cells in a dose-related manner. These studies demonstrate that P388D1 macrophages have functional PAF receptors whose affinity and structural specificities are similar to PAF receptors in other cells. The availability of a stable cell line that binds but does not metabolize PAF will greatly facilitate studies of the PAF receptor.     

The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.     

Calcium is necessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G-proteins

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizable bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]i and the release of PAF but not the Na+/H+ antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

Platelet-activating factor binding to human platelet membranes

Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor.     

Anti-apoptotic actions of the platelet-activating factor acetylhydrolase I alpha2 catalytic subunit

Platelet-activating factor (PAF) is an important mediator of cell loss following diverse pathophysiological challenges, but the manner in which PAF transduces death is not clear. Both PAF receptor-dependent and -independent pathways are implicated. In this study, we show that extracellular PAF can be internalized through PAF receptor-independent mechanisms and can initiate caspase-3-dependent apoptosis when cytosolic concentrations are elevated by approximately 15 pM/cell for 60 min. Reducing cytosolic PAF to less than 10 pM/cell terminates apoptotic signaling. By pharmacological inhibition of PAF acetylhydrolase I and II (PAF-AH) activity and down-regulation of PAF-AH I catalytic subunits by RNA interference, we show that the PAF receptor-independent death pathway is regulated by PAF-AH I and, to a lesser extent, by PAF-AH II. Moreover, the anti-apoptotic actions of PAF-AH I are subunit-specific. PAF-AH I alpha1 regulates intracellular PAF concentrations under normal physiological conditions, but expression is not sufficient to reduce an acute rise in intracellular PAF levels. PAF-AH I alpha2 expression is induced when cells are deprived of serum or exposed to apoptogenic PAF concentrations limiting the duration of pathological cytosolic PAF accumulation. To block PAF receptor-independent death pathway, we screened a panel of PAF antagonists (CV-3988, CV-6209, BN 52021, and FR 49175). BN 52021 and FR 49175 accelerated PAF hydrolysis and inhibited PAF-mediated caspase 3 activation. Both antagonists act indirectly to promote PAF-AH I alpha2 homodimer activity by reducing PAF-AH I alpha1 expression. These findings identify PAF-AH I alpha2 as a potent anti-apoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.     

Presence of platelet-activating factor in rhesus (Macaca mulatta) spermatozoa.

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-phosphocholine; PAF] is a unique signaling phospholipid which has been implicated in a number of biological activities (e.g., reproduction). PAF has been detected in the spermatozoa from a number of laboratory and domestic species, including, but not limited to, rabbit, bovine, and the mouse. The concentration of PAF is inversely related to human (Homo sapien) spermatozoal quality. Additionally, PAF levels are significantly higher in Bolivian squirrel monkey (Saimiri sciureus) spermatozoa obtained during the breeding season than spermatozoa obtained during the nonbreeding season. There are no reports on the presence of PAF in rhesus (Macaca mulatta) spermatozoa. Therefore, the primary objective of this study was to detect the presence of PAF in rhesus spermatozoa. A second objective was to determine if PAF spermatozoa levels differ between animals housed individually (single-caged) versus free-ranging (open corrals). Semen were collected from mature rhesus via electro-ejaculation. Spermatozoa were washed free of ejaculatory plug and quick frozen in PBS. Endogenous lipids were extracted from thawed spermatozoa and ejaculatory plugs then assayed for the presence of PAF by [125I]-radioimmunoassay. PAF was not detected in any ejaculatory plugs. PAF levels were significantly higher (P < 0.01) in spermatozoa obtained from free-ranging males (mean: 1.16 pmol/10(6) spermatozoa) than males housed individually in single cage units (mean: 0.53 pmol/10(6) spermatozoa). PAF was present in rhesus spermatozoa. Additionally, PAF levels were higher in spermatozoa obtained from corral-housed animals. Additional studies are warranted to elucidate the role of PAF in spermatozoa function.     

Platelet-activating factor in human endometrium

Platelet-activating factor (PAF) is a phospholipid actively produced by human endometrium and deeply involved in the processes of ovoimplantation and labor. We recently found that PAF represents a new autocrine growth factor for a human adenocarcinoma cell line, HEC-1A. Indeed, biologically active PAF is synthesized by HEC-1A cells, under progesterone control. In HEC-1A cells, PAF regulates intracellular calcium concentration ([Ca²⁺]), DNA synthesis and expression of early oncogenes. All these effects are blocked by the receptor antagonist L659,989. However, while nanomolar concentrations of PAF mobilize [Ca²⁺], only micromolar concentrations affect cell growth, suggesting heterogeneity of PAF receptors or signaling. Two distinct populations of PAF receptors are present in HEC-1A cells, which bind PAF in nanomolar and micromolar concentrations, respectively. Since HEC-1A cells are producing elevated concentrations of PAF and micromolar concentrations of the PAF antagonist L659,989 inhibit cell proliferation, an autocrine role for PAF is suggested in HEC-1A cells.     

Human neutrophil platelet-activating factor: molecular heterogeneity in unstimulated and ionophore-stimulated cells

The molecular heterogeneity of platelet-activating factor (PAF) in resting and ionophore (A23187) -stimulated human neutrophils was measured by a very sensitive gas chromatography-negative ion chemical ionization mass spectrometric method. The molecular species compositions of PAF, which are due to variations in the 1-O-alkyl chain length, were significantly different between resting and ionophore-stimulated polymorphonuclear leukocytes. The major species of PAF produced by unstimulated polymorphonuclear leukocytes were 16:0, 17:0, 18:1 and 18:0, representing 55, 14, 8 and 10%, respectively, of the total PAF; 16:0 was the predominant PAF (74%) in A23187-stimulated polymorphonuclear leukocytes. The PAF molecular species from unstimulated polymorphonuclear leukocytes was similar to compositions from those of the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, whereas those from the ionophore-stimulated polymorphonuclear leukocytes differed from the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, thus indicating a very high degree of substrate selectivity for PAF synthesis. Although the physiological implications of the variations in PAF composition are not known, these studies indicate that the PAF produced by resting polymorphonuclear leukocytes are significantly different from those produced in response to ionophore.     

The release of platelet-activating factor from human endothelial cells in culture

The release of platelet-activating factor (PAF) from stimulated human endothelial cells (HEC) cultured from normal term, umbilical cord veins is described. HEC in primary cultures released PAF after challenge with A23187, rabbit anti-human factor VIII (RaHu/FVIII), angiotensin II, and vasopressin. HEC subcultures maintained the ability to release PAF in the presence of A23187 and RaHu/FVIII, whereas the release of PAF in response to angiotensin II and vasopressin was not constant and was reduced. Control cultured, smooth muscle cells derived from umbilical cord veins, previously depleted of endothelial cells, did not release PAF under the above-mentioned stimulation. Plastic-adherent or cultured monocytes released PAF with A23187, but not with RaHu/FVIII, angiotensin II, and vasopressin. The release of PAF from HEC in primary cultures required the presence of extracellular cations and the activation of membrane phospholipase A2. PAF release induced by A23187, RaHu/FVIII, angiotensin II, and vasopressin was unaffected by indomethacin, an inhibitor of cyclooxygenase, which, however, favored the release of PAF from HEC stimulated with thrombin, a stimulus that did not affect HEC in the absence of indomethacin. PGI2 inhibited PAF release from stimulated HEC. The relevance of an acetylation process in the biosynthesis of PAF and HEC was supported by the following evidence: 1) the increase in PAF yield in the presence of sodium acetate and, particularly, of acetyl-CoA; 2) the incorporation of [14C]acetate into PAF molecules; 3) the loss of radioactivity and of biologic activity after treatment with phospholipase A2. These results indicate that HEC in culture are able to release PAF and that metabolic pathways similar to those described for leukocytes are involved.     

Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

Synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) in exocrine glands and its control by secretagogues

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet-activating factor (PAF] stimulate exocytosis in isolated lobules from guinea pig parotid glands or pancreas by an acetylcholine-like mechanism (S?ling, H. D., Eibl, H. J., and Fest, W. (1984) Eur. J. Biochem. 144, 65-72). We show here that both tissues are able to synthetize PAF themselves. Isolated guinea pig parotid gland acini incorporate labeled acetate into the 2-position of PAF. Stimulation with A23187 or carbamoylcholine lead to a significant stimulation of this process. The newly synthetized PAF is partially released into the medium. Addition of lyso-PAF to the incubation medium does not significantly affect the rate of incorporation of labeled acetate into PAF in the absence or presence of carbamoylcholine. Isolated pancreatic lobules are also able to incorporate labeled acetate into PAF, and cholecystokinin and caerulein lead to a strong stimulation of this process. Incorporation of radioactive lyso-PAF into PAF, but not into 1-O-alkyl-2-long chain acyl-sn-glycero-3-phosphocholine was also significantly stimulated by carbamoylcholine in isolated parotid acini. Under these conditions, the time-dependent stimulation of amylase release paralled that of lyso-PAF incorporation into PAF. The same holds for the concentration dependency of the carbachol effect on these two parameters. In isolated pancreatic lobules, caerulein also stimulated the incorporation of lyso-PAF into PAF. Pulse-chase experiments with radioactive lyso-PAF indicate that stimulation of incorporation of radioactive lyso-PAF into PAF represents increased net synthesis of PAF rather than increased PAF-turnover. Using the platelet aggregation test, substantial amounts (0.79 nmol/g) of PAF could be determined in isolated acini from guinea pig parotid glands.     

Platelet-activating factor and cardiac diseases: therapeutic potential for PAF inhibitors

Platelet-activating factor (PAF) is a potent phospholipid mediator released from inflammatory cells in response to diverse immunologic and non-immunologic stimuli. Animal studies have implicated PAF as a major mediator involved in coronary artery constriction, modulation of myocardial contractility and the generation of arrhythmias which may bear on cardiac disorders such as ischemia, infarction and sudden cardiac death. PAF effects are induced by direct actions of PAF on cardiac tissue to modify chronotropic and inotropic activity, or indirectly via the release of eicosanoids such as thromboxane A₂ (TXA,), leukotrienes (LT) or cytokines (TNFx). The development of selective, high affinity PAF receptor antagonists has permitted investigations on the role of PAF in experimental animal models of cardiac injury. In vivo and in vitro studies strongly suggest that PAF receptor antagonists might convey therapeutic benefits in ischemic conditions and certain arrhythmias. In addition, PAF antagonists might have a cardiac allograft-preservation effect. Although clinical studies with PAF receptor antagonists in patients with cardiac diseases have not yet been reported, the experimental results to date suggest that PAF receptor antagonist might be useful in some specific cardiac disorders in humans.     

The blocking action of platelet-activating factor (PAF) on the calcium currents of the atrial fibers in the frog and guinea pig

The effects of platelet-activating factor (PAF) on the myocardial cell membrane Ca-current (ICa) and Ca-action potential (Ca-AP) were investigated. In double sucrose-gap voltage-clamped frog atrial trabeculae PAF (2 X 10(-7) M) reduced ICa-amplitude to 40-50%; at the same time the IK-amplitude was increased to the same value. These changes of ICa and IK amplitudes were protected by simultaneous action of PAF and PAF antagonist BN 52021 (4 X 10(-6) M). In the partially depolarized (K+0 = 15-20 mM) of the guinea pig myocardial auricles PAF decreased Ca-AP amplitude and Vmax of its upstroke and shortened the Ca-AP duration (intracellular microelectrodes) like the isometric tension responses. These effects were prevented by PAF antagonist U-66985. Histamine was also able to protect from the PAF-induced changes of Ca-AP and tension responses. Our data demonstrated both by direct and by indirect methods of ICa registration in myocardia membrane that PAF induces reversed blocking of ICa. Because the blocking effects of PAF on frog and guinea pig myocardium are identical, these results imply that the mechanisms of PAF action on cold- and warm-blooded animals are similar in principle. The coupling of ICa and IK changes confirm our earlier supposition that PAF-induced Ca-AP shorting can be explained by IK augmentation.