Involvement of platelet-activating factor (PAF) in endotoxin-induced intestinal motor disturbances in rats

Intestinal myoelectrical activity was investigated in conscious fasted rats chronically implanted with Nichrome electrodes in the duodeno-jejunum. Motility of the small intestine was characterized by the presence of migrating myoelectric complex (MMC) occurring regularly at 16.2 +/- 5.8 minute intervals. Intravenous administration of endotoxin (E. coli S.0111:B4) at a dose of 50 micrograms/kg increased the interval between MMC to 112.6 +/- 26.8 min, the duration of these effects being dose-related between 10 to 100 micrograms/kg. Such a typical myoelectrical alteration, corresponding to rapidly propagated groups of spike bursts, was mimicked by the IP administration of PAF at doses of 10 to 50 micrograms/kg. Previous administration of BN 52021, a specific PAF antagonist at a dose of 50 mg/kg abolished the motor alterations induced by IP injection of PAF (25 micrograms/kg) and significantly (p less than 0.01) reduced by 61.2% those induced by IV endotoxin (50 micrograms/kg). Indomethacin (10 mg/kg IP) as well as SC 19220 (5 mg/kg IV), a PGE2 antagonist, injected prior to endotoxin (50 micrograms/kg IV) or PAF (25 micrograms/kg IP) also reduced significantly (p less than 0.01) the duration of MMC inhibition. It is concluded that endogenous release of PAF is partly responsible for the intestinal motor alterations induced by endotoxin; these effects, strongly reduced after treatment with BN 52021, are also mediated through the release of prostaglandins.     

Immunofluorescent localization of platelet-activating factor (PAF) in the rat

Summary An immunofluorescent staining method for detecting platelet-activating factor (PAF) is described. This method employs a polyclonal anti-PAF rabbit antibody. When rat brain, heart, lung, liver or kidney tissue was stained using this method, the heart, lung and kidney exhibited PAF-specific staining. Analysis of the amount of PAF in different organs, either by immunofluorescence or by bioassay, showed that kidney tissue contains the greatest amount of PAF.     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

Release, purification, and characterization of platelet-activating factor (PAF)

Platelet-activating factor (PAF) is a phospholipid mediator, released by basophils, macrophages and neutrophils under immunological and non immunological stimuli. It aggregates platelets and liberates their vasoactive contents. We studied the "spontaneous" release of PAF from hog blood leukocytes : optimal conditions were 22 degrees C, pH 9.5 in BSA and Ca2+-containing Tyrode's. This release was inhibited by the Ca2+-chelating agent, EDTA, and by the phospholipase A2 inhibitor, bromophenacyl bromide. Disruption of the cells did not yield PAF, indicating that it is not a "preformed" mediator. A preparative procedure for the extraction and purification of bulk quantities of PAF was developed. Purification was performed by silicic acid columns followed by high pressure liquid chromatography. The active fraction was eluted between sphingomyelin and lysophosphatidylcholine. The PAF purest fractions were still contaminated with these phospholipids as shown by thin layer chromatography and chemical ionization mass spectrometry. PAF activity was not affected by treatment with diazomethane, acetylation or hydrogenation. Our results combined with those obtained from our previous studies of the PAF structure using specific phospholipases indicate that PAF is a glycero-phospholipid devoid of ester function at position 1. This allowed us to establish precise criteria to distinguish PAF from other aggregating agents.     

Rabbit blastocysts accumulate platelet-activating factor (PAF) and lyso-PAF in vitro.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a very potent phospholipid, which has been demonstrated to stimulate smooth muscle and change vascular permeability. PAF has been detected in the rabbit preimplantation uterine endometrium and has been demonstrated to bind specifically to rabbit uterine membranes. To evaluate the possible role of PAF in maternal-embryonic chemical communication, we report here that rabbit blastocysts can accumulate [3H]PAF from their environment. Blastocysts were able to accumulate [3H]PAF as time-, buffer-, age-, and concentration-dependent functions. The accumulation was inhibited by some PAF receptor antagonists, such as U66985, as well as by unlabeled PAF and lyso-PAF, indicating that the accumulation process may be receptor mediated. The data support the current model of PAF as a paracrine factor in preimplantation stages of reproduction.     

Hypotensive actions of some analogues of platelet-activating factor (PAF) with higher potencies than natural PAF

Substituents on the nitrogen atom of the phosphorylcholine moiety of natural C16 platelet-activating factor (PAF) were modified or replaced by more bulky groups, and their hypotensive activities were examined with rats. As a result, it was found that N-methylpiperidine and N-methylpyrrolidine analogues were 3-10 times more potent than natural C16-PAF.     

Simple and rapid measurement of platelet-activating factor (PAF) in whole blood.

A simple assay of platelet-activating factor (PAF) in whole blood was developed, employing acetone extraction and thin layer chromatography (TLC) purification of blood sample. The activity of acetylhydrolase present in blood sample was almost completely suppressed by ice-cold acetone extraction, and other inhibitory substances interfering the activity of PAF were effectively removed from the acetone extract by TLC. Then, the treated samples were subjected to a conventional PAF bioassay using rabbit platelets. The recovery rate of PAF by the above procedure was constant and feasible (46-48%). The lower limit of the present assay was estimated to be 1.0 x 10(-10) M. Employing the present method, it was able to determine the amount of PAF in blood (1.2-6.0 x 10(-10) M) of 6 out of 14 septic patients, while no significant PAF activity was detected in the samples from 6 healthy subjects. These results indicate a potential application of the present method in the clinical assay of PAF in blood.     

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.     

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress

We detected platelet-activating substance in gastrointestinal areas, which was confirmed to be platelet-activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin-layer chromatography; 2) it did not aggregate PAF-desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L-652,731. The level of PAF was determined with a bioassay method based on the release of [3H]serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 +/- 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 +/- 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 +/- 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

Specific binding of platelet-activating factor (PAF) by human peripheral blood mononuclear leukocytes

Binding of platelet-activating factor (PAF) to human peripheral blood mononuclear leukocytes was time-dependent, reversible, and saturable. [3H]PAF binding to the cells was inhibited dose-dependently by unlabeled PAF and PAF receptor antagonists: L-659,989, triazolam, and alprazolam. Scatchard analysis of saturation binding data indicated one class of receptors for PAF with KD = 5.7 nM and Bmax = 18 fmol/10(6) cells (11,100 receptors/cell). PAF (10 nM) increased intracellular free calcium concentration in human lymphocytes and this effect was inhibited by L-659,989 dose-dependently. Our data suggest that human peripheral blood mononuclear leukocytes have specific receptors for PAF.     

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from patients with ischemic cerebrovascular disease

Platelet-activating factor (PAF) is metabolized by a specific enzyme, PAF acetylhydrolase, which may play an important role in the manifestation of the biological activities of PAF in vivo. The activity of PAF acetylhydrolase in plasma of patients with ischemic stroke was higher than that in healthy controls. The incidence of irreversible platelet aggregation in response to PAF, as well as to ADP, was found to be higher in patients than in controls. The patients whose platelets responded with irreversible aggregation to PAF displayed a higher activity of plasma PAF acetylhydrolase than those with only reversible aggregation. In controls, PAF acetylhydrolase activity correlated positively, although weakly, with LDL-cholesterol, which may reflect the major role of LDL in carrying this enzyme. However, since there was no significant difference in plasma levels of lipids and apoproteins between patients and controls (except for apo B) and there was no significant relationship between the enzyme activity and the levels of other lipids and apoproteins, it is unlikely that increased plasma level of PAF acetylhydrolase activity in stroke patients is accounted for by an abnormality of lipoprotein metabolism. Platelet hyperfunction may be associated with augmented generation of PAF, which, in turn, may bring about the induction of the inactivating enzyme, PAF acetylhydrolase.     

Augmentation of DNA synthesis in guinea pig bone marrow cells by platelet-activating factor (PAF)

When guinea pig bone marrow cells were incubated in the presence of 10(-7) to 10(-5) M platelet-activating factor (PAF) for 24 to 72 hours, [3H]thymidine incorporation of cells was time-dependently augmented. The enantiomer of PAF and lysoPAF, a major metabolite of PAF, did not show significant enhancement. A non-metabolizable potent PAF agonist, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-sn-glycero-3-phospholine, enhanced the [3H]thymidine incorporation at 10(-10) to 10(-8) M. This augmentation of DNA synthesis in bone marrow cells was abolished by specific PAF antagonists, CV-6209 or FR-900452. When the conditioned medium of PAF-stimulated bone marrow cells was added to another culture of bone marrow cells, the augmentation of DNA synthesis was also observed. These results suggest that PAF may affect the proliferation of one or some classes of guinea pig bone marrow cells through release of soluble factor(s).     

Role of platelet-activating factor (PAF) in the initiation of the decidual reaction in the rat

Injection of PAF into the left uterine horn induced a dose-dependent decidua-like reaction in the pseudopregnant rat. This reaction was maximal when PAF was injected at Day 5 of pseudopregnancy and was blocked by the specific PAF antagonist, BN 52021. BN 52021 did not interfere with the decidual reaction induced by prostaglandin E-2 or insertion of a cotton thread in the uterine horn. In contrast, a decidua-like reaction was not evoked by the inactive lyso-PAF, demonstrating the specificity of the action of PAF. The decidua-like reaction induced by PAF involves the generation of cyclooxygenase metabolites of arachidonic acid since it was inhibited by indomethacin. The histological alterations induced by PAF were similar to those observed after embryo implantation, strengthening the postulate for a role of the autacoid in the early stages of pregnancy.