Showing papers in "Journal of lipid mediators in 1989"

PAF/cytokine Auto-Generated Feedback Networks in Microvascular Immune Injury: Consequences in Shock, Ischemia and Graft Rejection

Abstract: The catastrophe theory evolved by Thom and Zeeman proposes a mathematical definition for the abrupt or 'catastrophic' changes that can suddenly occur in normally well-ordered and smooth-running systems. We have integrated this theory with our own PAF/cytokine feedback network hypothesis to explain the control and dysfunction of the inflammatory response. This process involves the activation of cells and factors such as proteases, and is coordinated by mediators such as PAF, cytokines and growth factors, minute amounts of which can prime cells to respond in an enhanced manner to subsequent agonistic stimuli. PAF and certain cytokines also possess the unique property of being able to induce the release of each other and their own generation in vivo. This 'singularity' may enable a self-generating feedback network to become established. The priming ability of these mediators indicates the extreme sensitivity of the inflammatory process and importance of a homeostatic equilibrium between the vectors involved in the priming and feedback processes and internal suppressive mechanisms. In pathological conditions, one can consider the phenomenon of PAF and cytokine autogeneration as a 'fold' in the feedback network and an expression of the singularity characteristic of the catastrophe hypothesis. This may lead to systemic toxicity and microcirculatory collapse, a characteristic feature of shock, sepsis, asthma, ischemia and graft rejection. A combination of drugs antagonizing the various feedback components may inhibit this catastrophic process and thus provide more successful therapy of these conditions.

Assessment of the role of platelet-activating factor in an animal model of inflammatory bowel disease.

Abstract: Using a rat model of chronic colitis, the role of PAF-acether as a mediator of intestinal inflammation was assessed. Rats were treated with a specific PAF-acether antagonist, BN52021, during the first 4 days after induction of colitis, or during the period of 4-7 days after induction of colitis. The effects of treatment with BN52021 were compared to those of treatment with 5-aminosalicylic acid. BN52021 and 5-aminosalicyclic acid were without significant effect on colonic damage score when administered intracolonically on days 1-4 after induction of colitis. However, when given on days 4-7 after induction of colitis, both drugs significantly accelerated the healing of ulcers and reduced the incidence of adhesions and diarrhea. Intraperitoneal administration of BN52021 also resulted in a significant reduction of colonic damage scores, while administration of 5-aminosalicyclic acid via this route was without significant effect. Using an in vitro superfusion system, the effects of PAF-acether on contractility of segments of ascending colon were assessed. Tissue segments from normal rats contracted to doses of PAF-acether as low as 0.5 pg. However, non-inflamed segments of ascending colon from rats in which colitis was induced 2 weeks earlier were relatively insensitive to PAF-acether. The results of the present study demonstrate that PAF-acether is unlikely to play an important role in the acute inflammatory response in this model, but may be important in the prolongation of inflammation and ulceration in this model. The studies on contractility of ascending colon suggest that changes in tissue sensitivity to PAF-acether may occur as a consequence of inflammation.

RP 55778, a PAF receptor antagonist, prevents and reverses LPS-induced hemoconcentration and TNF release.

Abstract: Platelet-activating factor (PAF) and tumor necrosis factor (TNF) are present in the plasma of animals injected with endotoxin (LPS). Furthermore, when exogenously administered to animals, PAF and TNF induce similar pathological effects. Thus, in order to explore a possible link between these two factors, the effects of a PAF receptor antagonist, RP 55778, and a glucocorticoid, dexamethasone, were studied on LPS-induced hemoconcentration in rats and on the release of TNF induced by exposing isolated murine macrophages to LPS. RP55778 administered either before or after LPS inhibited these endotoxin effects whereas dexamethasone was effective only when given prior to the LPS challenge. Additionally, in murine macrophages the strong TNF mRNA signal induced by LPS was abolished by RP 55778 and dexamethasone treatment. These results indicate that PAF and TNF can mediate the functional manifestations associated with endotoxemia and only RP 55778 appears to show potential for activity against an already established LPS response.

Receptor antagonism of leukotriene B4 myotropic activity by the 2,6 disubstituted pyridine analog U-75302: characterization on lung parenchyma strips.

Abstract: Leukotriene B4 constricts guinea pig lung parenchyma strips in a concentration-dependent manner. The LTB4 structural analog U-75302, 6-(6-[3-hydroxy-1E,5 Z-undecadienyl]-2-pyridinyl)-1,5-hexanediol, was a partial agonist in this system with a potency 300-1000 times less than LTB4. U-75302 constricted lung parenchyma strips only at concentrations greater than 0.3 microM. At concentrations lacking agonist activity U-75302 was an effective antagonist, displacing the LTB4 dose-response curve. Half-maximal responses required 0.10 microM LTB4 in the presence of 0.3 microM U-75302 and 0.01-0.02 microM LTB4 in its absence. The maximal force of contraction was unaffected at this concentration. Concurrent with antagonism of the myotropic response, U-75302 inhibited the LTB4-dependent release of thromboxane B2 from lung parenchyma. This effect was attributable to receptor antagonism, not enzymatic inhibition of phospholipase, cyclooxygenase, or thromboxane synthase. For instance, 0.3 microM U-75302 did not inhibit thromboxane B2 formation by lung parenchyma stimulated with calcium ionophore A23187 and it did not inhibit thromboxane B2 formation by human platelets stimulated with arachidonic acid. U-75302 selectively antagonized the activity of LTB4 and not other myotropic agonists including the thromboxane A2 mimetic U-46619, LTC4, LTD4, AGEPC, PGF2 alpha, and histamine. Receptor antagonists of leukotriene B4 may have multiple beneficial effects on asthmatic or respiratory disorders. These include (i) direct antagonism of LTB4 myotropic actions; (ii) antagonism of LTB4-dependent mediator release; and (iii) antagonism of LTB4 chemotactic action associated with leukocyte infiltration during anaphylactic late phase reactions.

Platelet-activating factor production occurs through stimulation of cholinergic and dopaminergic receptors in the chick retina.

Abstract: In previous work we showed that platelet-activating factor (PAF) was produced upon stimulation of the chick retina with acetylcholine (ACh) and dopamine (DA), via a dithiothreitol-insensitive cholinephosphotransferase (DTT-CPT). Other neurotransmitters were ineffective. Since ACh and DA stimulated PAF production at different stages of development, we advanced the hypothesis that selected cholinergic and dopaminergic receptors were involved. In this paper we demonstrate that only muscarinic or D2 (and not D1 and nicotinic) receptor stimulation induced PAF production in the chick retina either before or after hatching. Moreover, our data show that PAF production was completely prevented by blockage of these receptors by specific antagonists. These data further substantiate the hypothesis that PAF synthesis could be physiologically associated with synapse stimulation.

Phospholipase A2 activity in platelets. Immuno-purification and localization of the enzyme in rat platelets.

Abstract: A comparative study on phospholipase A2 activity in platelet lysates from various species was carried out using identical assay conditions with phosphatidylethanolamine as substrate. Platelet phospholipase A2, both when expressed as activity per ml blood and as specific activity in KCl extracts, was low in human, cow, pig and goat. Moderate activities, in increasing order, were found in sheep, horse and rabbit, while rats showed by far the highest activity. In the latter four species total lysate activity was recovered in 1 M KCl extracts, suggesting that the enzyme occurs either in soluble form or as a peripheral membrane-associated protein. Immune cross-reactivity with monoclonal antibodies against rat liver mitochondrial phospholipase A2 was studied in dot-blot and monoclonal antibody-Sepharose binding experiments. Only sheep and rat platelet extracts contained cross-reactive phospholipase(s) A2. Immuno-affinity chromatography of rat platelet extracts indicated virtually complete binding of total phospholipase A2 activity and yielded pure enzyme in a single purification step. Enzyme visualization by immunogold electron microscopy showed a predominant localization in the matrix of alpha-granules.

PAF-receptor. 1. 'Cache-oreilles' effect of selected high-potency platelet-activating factor (PAF) antagonists.

Abstract: Three-dimensional electrostatic maps were calculated for six potent antagonists of platelet-activating factor (PAF), the antagonists being selected for their apparent structural heterogeneity. The molecules examined were the compact Ginkgolides BN 52020, BN 52021 and BN 52022 (1, 2 and 3), the semi-rigid kadsurenone (4), a flexible synthetic dinor type C furanoid lignan L-652,731 (5a) and the triazolothienobenzodiazepine WEB 2086 (7). Calculation of the electrostatic potential generated around all the above molecules showed the existence of two wells of negative potential or 'cache-oreilles' (ear-muffs), i.e., the isocontours drawn at -10 kcal/mol, located at 180 degrees from each other and separated by a maximum distance of 22-27 A. Except for the synthetic dinor type C furanoid lignan (5a), the molecules also presented a moderate hydrophobic fragment, which constitutes a third point of interaction with the high-affinity binding site in rabbit and human platelets. The findings of the present study allow speculation that this high-affinity acceptor site may be a 'polarized cylinder' with a diameter of 10-12 A.

Elevated plasma levels of platelet-activating factor (PAF) in breast cancer patients with hypercalcemia.

Abstract: Peripheral plasma concentrations of PAF were measured in patients with benign and malignant tumors of the breast, and in healthy female controls with a very sensitive PAF assay. The mean values +/- SEM were significantly higher in the malignant group than in healthy controls (1070 +/- 111 fmol/ml vs. 142 +/- 12 fmol/ml, P less than 0.01) and patients with benign tumors of the breast (1070 +/- 111 fmol/ml vs. 208 +/- 16 fmol/ml). Patients with malignant tumors of the breast and hypercalcemia contributed predominantly to the higher values of PAF (1500 +/- 167 fmol/l). The high levels of PAF were not found to be correlated to the clinical and histopathological data. The surgical removal of the tumor had little effect on the plasma concentration of PAF. In contrast, hypercalcemic patients showed an increase, although not significant, in plasma PAF levels after the surgical operation.

Platelet-activating factor-induced calcium mobilization in human platelets and neutrophils: effects of PAF-acether antagonists.

Abstract: Since one of the first measurable events that occurs as a consequence of receptor-mediated cell activation is an increase in the cytosolic free calcium concentration, the calcium-selective fluorescent indicator fura-2 was employed to monitor increases in cytosolic calcium following PAF-acether stimulation of human neutrophils and platelets. In neutrophils, approximately 70% of the increase in cytosolic calcium was attributed to release from an intracellular source and 30% could be attributed to enhanced influx through the plasma membrane. In platelets, only 30% was released from an intracellular pool and the remainder reflected enhanced influx of calcium. In both cell types, the intracellular source of calcium was a non-mitochondrial, vesicular compartment, probably endoplasmic reticulum. The ability of different compounds to antagonize the PAF-acether-induced increase in cytosolic calcium was investigated using quin2 and fura-2 as calcium indicators. In decreasing order of potency, the following PAF-acether antagonists inhibited the change in platelet cytosolic free calcium elicited by 10 nM PAF-acether: L-652,731, kadsurenone, triazolam, diltiazem and alprazolam. L-652,731, kadsurenone, triazolam and diltiazem were also tested in neutrophils stimulated by 10 nM PAF-acether. While the antagonists were 7-20 times less active in neutrophils as compared to platelets, they all inhibited the rise in free calcium with the same order of potency in both cell types. Each of the antagonists was PAF-acether-specific, inhibited both the rate and magnitude of calcium mobilization and appeared to exhibit competitive antagonism. These data demonstrate that calcium mobilization can provide a rapid, sensitive and quantitative method by which to evaluate agonists such as PAF-acether and PAF-acether antagonists such as kadsurenone in different cell types.

Guanine nucleotides and fluoride enhance carbachol-mediated arachidonic acid release from phosphatidylinositol. Evidence for involvement of GTP-binding protein in phospholipase A2 activation.

Abstract: Release of arachidonic acid (AA) from 1-stearoyl-2-[14C]arachidonyl-glycerophosphoinositol (PI) by plasma membrane-bound enzyme(s) is a calcium-dependent reaction and is markedly activated at 4 x 10(-4) M CaCl2. In the presence of Ca2+, the agonist of the cholinergic receptor (carbachol) enhances, in a dose-related manner, AA release. Moreover, GTP and its non-hydrolysable analogs GTP gamma S and GppNHp and also NaF additionally increase the carbachol-mediated liberation of AA from PI. On the contrary, in the absence of Ca2+ carbachol and GTP gamma S have no stimulatory effect on AA release. Guanosine-5'-O-2-thiodiphosphate GDP gamma S, which inhibits the function of GTP-binding proteins, also suppresses carbachol-mediated activation of AA release from PI. The stimulatory effect of carbachol and guanine nucleotides was observed exclusively in the brain plasma membrane (there was no effect on mitochondria, microsome and cytosolic enzymes). Quinacrine, the inhibitor of phospholipase A2, completely inhibits carbachol- and guanine nucleotide-activated AA release and greatly (by about 60-70%) decreases Ca(2+)-dependent AA liberation from phosphatidylinositol. These results indicate that GTP-binding protein(s) are involved in the regulation of carbachol-mediated AA release. The main pool of this acid is liberated from phosphatidylinositol by phospholipase A2 and only a small pool of AA may be released indirectly as the result of PI hydrolysis by sequential action of phospholipase C and diacylglycerol lipase.

Possible physiological role of myocardial fatty acid binding protein in phospholipid biosynthesis.

Abstract: Accumulation of free fatty acids and their esters resulting from the degradation of membrane phospholipids is one of the major causes for the myocardial dysfunction during ischemia and reperfusion. In this communication, we have studied the possible physiological role played by fatty acid binding protein (FABP) in stimulating key enzymes involved in phospholipid biosynthesis. Purified rat heart FABP bound a maximum of either 2 mol of [1- 14C]palmitoyl coenzyme A (CoA), oleoyl CoA, or oleic acid per mol of FABP as observed by Scatchard analysis. FABP caused a threefold increase in the incorporation of [1- 14C]palmitoyl CoA into phosphatidic acid as compared to only a 1.5-fold increase by bovine serum albumin (BSA). Myocardial FABP also enhanced acyl CoA monoacylglycerophosphorylcholine acyl transferase minimally at a substrate concentration (greater than 200 microM), the activity of this enzyme was enhanced 4.5- and 2-fold by FABP and BSA, respectively. The maximum stimulation of the enzyme activity took place at the fatty acyl CoA concentration where inhibition of the enzyme activity is usually observed due to the surfactive property of acyl CoAs. These results thus indicate that under abnormal pathophysiological conditions such as ischemia, when acyl CoA concentration increases, FABP may protect acyl CoA monoacylglycerophosphorylcholine acyl transferase as well as stimulate glycerophosphate acyl transferase to limit the loss of membrane phospholipids, suggesting a possible role of FABP in phospholipid biosynthesis.

Local effects of PAF in guinea-pig inner ear.

Abstract: The effect of PAF-containing artificial perilymph at 10(-7) and 10(-8) M on the endocochlear potential (EP) was investigated in guinea-pigs. The inner ear was perfused and the EP recorded at the same time. The characteristic decline of the EP which is highly sensitive to all stimuli could be prevented by pretreating the animals with either ginkgolide B (BN 52,021) or BM 13,177 (Sulotraban). Less effective in preventing the EP decline was Daltroban (BM 13,505). The results are discussed with respect to an involvement of PAF in cochlear physiology and in the regulation of ion transport, respectively.

Metabolism of platelet activating factor (PAF) and related ether lipids by neonatal rat myocytes.

Abstract: Suspensions of neonatal rat myocytes were used to investigate the metabolism of [3H]PAF (1-alkyl-2-acetyl-(sn-glycero-3-phosphocholine) (GPC] and [3H]alkyllyso-GPC. [3H]Alkylacyl-GPC consisting of molecular species with four or more double bonds (87%) was the major metabolite formed when either [3H]PAF (4 x 10(-6) - 4 x 10(-9) M) or [3H]alkyllyso-GPC (2 x 10(-7) M) was the precursor. However, substantial amounts of [3H]alkyllyso-GPC (mostly in the media) were also generated from [3H]PAF during the early periods of the incubations. At 2 x 10(-7) M or higher concentrations of either [3H]PAF or [3H]alkyllyso-GPC, [3H]alkylglycerols were also formed (4-12% of the total radioactivity). The [3H]alkylglycerols appear to be produced from [3H]alkyllyso-GPC through the combined actions of lysophospholipase D and a phosphatase. Pretreatment of neonatal rat myocytes with phenylmethylsulfonyl fluoride partially blocked the deacetylation of [3H]PAF and decreased both the formation and subsequent acylation of [3H]alkyllyso-GPC in intact cells. Phenylmethylsulfonyl fluoride also significantly inhibited the activity of cytosolic acetylhydrolase, but it had no direct effect on the microsomal transacylase in vitro. Our data demonstrate that the metabolism of PAF and alkyllyso-GPC by neonatal rat myocytes is qualitatively, but not quantitatively, similar to what occurs in other cell types. In addition, when [3H]alkylacetylglycerol was the precursor, 75% of the label appeared as alkylglycerols in the neonatal rat myocytes, but [3H]PAF was not detected. Therefore, it appears that the de novo pathway via the dithiothreitol-insensitive cholinephosphotransferase step does not contribute to the biosynthesis of PAF in neonatal rat myocytes.

Biologically active ether lipids: incorporation of long-chain precursors into 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N-trimethyl)homoserines and other lipids of Chlorella fusca.

Abstract: The lipids of Chlorella fusca are composed of the ester lipids typical of photosynthetically active cells. In addition, there occurs a class of less common ether lipids, the biologically active 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N- trimethyl)homoserines, at a level of about 1.3% of total lipids. The acyl moieties of the total lipids include saturated as well as mono-, di- and tri-unsaturated species with chain lengths of 16 and 18 carbon atoms, the major constituents being palmitic and oleic acids. In both the diacylglycerophosphocholines, i.e., the major class of ester phospholipids, and the diacylglycero-4'-O-(N,N,N-trimethyl)homoserines palmitic acid is located predominantly at position 1 of the glycerol backbone, whereas oleic acid is almost equally distributed between positions 1 and 2; palmitoleic and polyunsaturated fatty acids are esterified preferentially at position 2. Incubation of C. fusca cultures with 14C-labeled fatty acids leads to their rapid incorporation into various lipid classes. Oleic and palmitic acids are incorporated at a faster rate than stearic acid (18:1 greater than 16:0 much greater than 18:0). 1,2- and 1,3-Diacylglycerols are the most prominent intermediates of early metabolism of the exogenous fatty acids. In the course of time, a steady decrease of radioactive 1,2-diacylglycerols is observed that is accompanied by an increase in labeled triacylglycerols, diacylglycerophosphocholines, and diacylglycero-O-(N,N,N-trimethyl)homoserines. The stereospecific distribution of acyl moieties in the diacylglycero-O-(N,N,N-trimethyl)homoserines in C. fusca indicates that these ether lipids are derived from 1,2-diacylglycerol intermediates. This notion is supported by the finding that during incubation with radioactively labeled fatty acids the formation of diacylglycero-O-(N,N,N-trimethyl)homoserines parallels the biosynthesis of both diacylglycerophosphocholines and diacylglycerophosphoethanolamines, two classes of phospholipids which are known to be derived from 1,2-diacylglycerols. The mechanism of the formation of the ether bond, however, is as yet unknown. Incubation of C. fusca cultures with 14C-labeled fatty acids or alcohols leads to the formation of fair proportions of wax esters that are labeled in both the acyl and the alkyl moieties, indicating that in these algae fatty acids and alcohols are interconverted. 14C-Labeled long-chain alcohols are not incorporated into the alkyl moieties of ether lipids, whereas labeled 1-O-alkylglycerols are used, though to a very small extent, as precursors of ether phospholipids.

Aspirin, platelets and prevention of vascular disease.

Abstract: Aspirin inhibits thromboxane and prostaglandin formation in platelets and in vascular cells. It prevents platelet aggregation by irreversible acetylation of cyclooxygenase, a key enzyme in arachidonic acid metabolism. On the basis of its antiplatelet effect, aspirin has been assessed during the past two decades in patients with a history of myocardial infarction, stroke, transient ischemic attack or unstable angina. A meta-analysis of randomized controlled trials of long-term aspirin treatment for the secondary prevention of vascular disease indicated that aspirin (300-1500 mg daily) significantly reduced fatal and non-fatal vascular events. More recently aspirin (160 mg daily) produced a significant reduction in hospital vascular mortality and in non-fatal events in patients with suspected acute myocardial infarction. The combination of aspirin and streptokinase was significantly better than either drug alone. On the other hand, two primary prevention trials of aspirin in healthy doctors did not show any modification of vascular mortality despite an overall reduction of non-fatal myocardial infarction. Resolution of some problems related to the mechanism of action of aspirin and to selection of trial populations will possibly increase the benefit/risk ratio of aspirin treatment for the prevention of vascular disease.

Involvement of PAF-acether in the Anaphylactic Shock in the Rat

Abstract: In normal Wistar rats, SDZ 63-675 (1.2 mg/kg) inhibited hypotension and hemoconcentration induced by PAF-acether (0.2-0.8 micrograms/kg.min). These effects of PAF-acether were not modified by ketoprofen (3 mg/kg). Only the hemoconcentration was reduced by BW755C (40 mg/kg). Part of the vascular effects of PAF-acether in rats would thus depend on the release of lipoxygenase products. In sensitized rats, SDZ 63-675 (1.2 mg/kg) increased the survival rate, reduced the vascular collapse and suppressed the hemoconcentration induced by intravenous injection of the antigen. BW755C (40 mg/kg) increased the survival rate and slightly reduced the vascular collapse. This anaphylactic shock was neither modified by ketoprofen (3 mg/kg), acetylsalicylic acid (100-200 mg/kg) and indometacin (3-6 mg/kg) nor by the association of methysergide (3 mg/kg), metiamide (35 mg/kg) and mepyramine (3 mg/kg). The inhibitory effect of SDZ 63-675 on the anaphylactic shock was not affected by the association with methysergide, mepyramine and metiamide or with indometacin. It was enhanced by acetylsalicylic acid. Injected simultaneously, SDZ 63-675 and BW755C suppressed the vascular collapse induced by the antigenic challenge. The main vasoactive factors responsible for this anaphylactic shock in the rat would thus be, in decreasing order, PAF-acether, leukotrienes and prostanoids. The association of mepyramine, methysergide and metiamide inhibited hypotension and hemoconcentration induced by the infusion of dextran. Since SDZ 63-675 had no influence on the effects of dextran, it is concluded that dextran did not release significant amounts of PAF-acether from mast cells.

Effect of 1-oleoyl-2-acetyl-sn-glycerol on inositol lipid metabolism of ascites tumor cells in culture.

Abstract: 1-Oleoyl-2-acetyl-sn-glycerol (OAG), the membrane-permeable analogue of 1,2-diacylglycerol (DAG), which stimulates ascites tumor cell proliferation, was used to study its effect on phosphoinositide metabolism. Culturing of ascites cells labeled with [3H]inositol at low serum concentration in the presence of OAG suppressed the radioactivity level of the inositol phosphates, particularly IP3. Membrane-bound, Ca(2+)- and GTP gamma S-sensitive PI- and PIP2-specific phosphodiesterase (phospholipase C) showed much lower activities in OAG-stimulated cells, which could be enhanced by GTP gamma S in these but not in the unstimulated cells. A high susceptibility to Ca2+ of the PI- and PIP2-specific phospholipase C of non-stimulated cells was observed. The PIP-kinase activity was similarly reduced by about 85% in OAG-stimulated cells. These data indicate a negative feedback regulation of the phosphoinositide metabolism mediated by OAG. Reduction in synthesis and degradation of PIP2, which furnishes the two second messengers, DAG and IP3, provides a means of controlling the intracellular level of these molecules, which is important for a balanced proliferation rate.

Measurement of ionized cytoplasmic calcium mobilization with the photoprotein aequorin in human polymorphonuclear leukocytes activated by platelet activating factor (PAF).

Abstract: The use of the sensitive photoprotein aequorin as a Ca2+ indicator in human polymorphonuclear leukocytes (PMN) not pretreated with cytochalasin B and stimulated with platelet activating factor (PAF) may help cast more light on the relative importance of intracellular and extracellular Ca2+ in PMN function. PAF elicits Ca2+ mobilization in PMN (resuspended in the presence of 1 mM extracellular Ca2+), in a concentration-dependent manner. The Ca2+ chelator ethyleneglycoltetraacetic acid (EGTA) abolishes Ca2+ mobilization, suggesting that almost all Ca2+ mobilized by PAF derives from the external medium. Aggregation and enzymatic release parallel the Ca2+ mobilization triggered by PAF. In contrast PAF appears to be only a weak stimulus of superoxide anion production (compared to the phorbol ester phorbol 12-myristate 13-acetate (PMA] and leukotriene B4 (LTB4) synthesis (compared to the Ca2+ ionophore A23187). The fact that PAF elicits Ca2+ mobilization, aggregation, secretion and LTB4 generation in human PMN supports the role of this phospholipid as a powerful mediator of physiopathological events involving PMN activation.

Dietary salt, blood pressure and circulating levels of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine in patients with essential hypertension.

Abstract: The effects of dietary salt on circulating levels of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 PAF) in patients with essential hypertension were studied by gas chromatography/mass spectrometry with negative ion chemical ionization. Circulating levels of C16 PAF in patients with essential hypertension (18.1 +/- 5.3 pg/ml, n = 16) were not changed compared with those in normotensive subjects (17.2 +/- 7.2 pg/ml, n = 14). Although changes in circulating levels of C16 PAF were small with changes in dietary salt, net changes in circulating C16 PAF levels significantly and positively correlated with net changes in mean arterial blood pressure (r = 0.47, P less than 0.05). Changes in C16 PAF levels also correlated with changes in creatinine clearance (r = 0.55, P less than 0.05). However, changes in C16 PAF levels did not correlate with changes in plasma sodium concentration, plasma chloride concentration and plasma volume. These results indicate that C16 PAF plays an antihypertensive role and this may be reflected as small changes in circulating levels of C16 PAF.

Action of platelet-activating factor (PAF) antagonists on the bronchopulmonary effects of PAF in the guinea-pig.

Abstract: Intravenous injection of BN 52021 in anesthetized guinea-pigs, 5 min before challenge, inhibited in a dose-dependent fashion with an IC50 of 0.90 mg/kg the bronchoconstriction induced by PAF (60 ng/kg i.v.). However, BN 52021 did not prevent the leukopenia following PAF injection but significantly inhibited the thrombocytopenia induced by PAF. The dioxolan compound, BN 52111, dose-dependently reduced the bronchoconstriction (IC50 = 0.27 mg/kg) and at doses higher than 1 mg/kg partially antagonized the decrease in the number of circulating platelets and leukocytes induced by PAF. BN 52115 also markedly inhibited the bronchoconstriction (IC50 = 0.36 mg/kg) as well as the thrombocytopenia induced by PAF, but was without significant effect on the leukopenia. These results demonstrate that the two dioxolan compounds, BN 52111 and BN 52115, are more potent than BN 52021 in inhibiting the in vivo bronchopulmonary alterations induced by PAF. Since these alterations are related to the activation of platelets by the autacoid, these blood elements are probably the targets of BN 52111 and BN 52115. Injection of PAF (10 and 100 ng) via the pulmonary artery of ventilated and perfused guinea-pig lungs induced dose-dependent increases in pulmonary inflation pressure (PIP) and pulmonary perfusion pressure (PPP), associated with a dose-dependent release of thromboxane B2 (TxB2). Addition of BN 52021, BN 52111 or BN 52115 (0.1, 1 or 10 microM) to the perfusion medium, 15 min before challenge, dose-dependently inhibited the bronchopulmonary effects of PAF. Although BN 52111 was the more potent in inhibiting the PAF-induced increase in PIP, BN 52021 was the more active with respect to the PAF-evoked generation of TxB2, suggesting that the two phenomena are not directly related.

Prevention of Ca(2+)-induced hepatocyte plasma membrane bleb formation by inhibitors of eicosanoid synthesis.

Abstract: Isolated rat hepatocytes were incubated in the presence of Ca2+ ionophore A23187 to induce the formation of plasma membrane blebs which appear to be a consequence of toxic or ischaemic cell injury. When incubated in the presence of both A23187 and an inhibitor of phospholipase A2, bleb formation was limited to that observed in the absence of A23187. Inhibitors of cyclooxygenase or thromboxane synthetase had similar effects. Incubation of isolated hepatocytes with individual eicosanoids showed that thromboxane B2 caused the formation of many plasma membrane blebs, prostaglandin E2 fewer than thromboxane B2 but more than prostacyclin which did not enhance bleb formation. These observations support the idea that Ca(2+)-induced cell injury and death may be mediated via a Ca(2+)-activated phospholipase A2, arachidonic acid release and eicosanoid formation.

Na+/H+ exchange in PAF-stimulated platelets.

Abstract: In experiments on human platelets, inhibition of Na+/H+ exchange was caused either by equimolar substitution of external Na+ with choline or N-methyl-D-glucamine, by decreasing the pHo to 6.8, or by an inhibitor of the antiport 5-(N-ethyl-N-isopropyl)amiloride (EIPA). In all these cases a considerable inhibition of PAF-induced platelet aggregation and as a rule a more or less marked decrease in the cytoplasmic Ca2+ signal (quin-2-loaded platelets) occurred. Stimulation by 10(-7) M PAF caused biphasic pHi changes in human platelets loaded with the pH-sensitive fluorescent probe BCECF: a small transient decrease, followed by a sustained increase of 0.02 +/- 0.006 pH units, resulted from stimulation of the Na+/H+ exchange. Thrombin (0.1 U/ml) also caused biphasic pHi changes, but the alkalinization step was more pronounced (0.15 +/- 0.03 U). Every means of Na+/H+ exchange inhibition prevented a rise in pHi in stimulated platelets. Activation of the adenylate cyclase system by carbacyclin suppressed the agonist-induced pHi increase. The inhibition of neither cyclooxygenase by 10(-5) M indomethacin nor calmodulin-dependent enzymes by 10(-5) M calmidazolium affected the agonist-induced pHi signals. A decrease in temperature from 37 to 24 degrees C caused a considerable increase in the lag phase of the pHi signal induced by tetradecanoyl phorbol acetate (TPA), but did not affect the kinetics of the pHi signal induced by PAF. An inhibitor of protein kinase C (PKC), compound H-7 (60 microM), completely abolished the TPA-induced increase in pHi but caused only a partial inhibition of the pHi signal in about 50% of the experiments with PAF. On the basis of these results the conclusion is drawn that the activation of PKC is not the only pathway for the PAF-induced stimulation of Na+/H+ exchange. The PAF-induced pHi rise depended both on the presence of extracellular Ca2+ and on the [Ca2+]i increase. On the other hand, inhibition of Na+/H+ exchange decreased the magnitude of the Ca2+i signal in PAF-induced platelets loaded with quin-2, but did not influence the Ca2+ mobilization from intracellular stores as measured by quin-2 or chlortetracycline in experiments with thrombin-stimulated platelets. We conclude that in PAF-activated platelets some initial increase of [Ca2+]i is essential for Na+/H+ exchange activation while activated antiport potentiates a full-scale Ca2+ influx into the cells.

Inhibitory effects of dexamethasone in endotoxic shock and its relation to PAF-acether synthesis in the gastrointestinal tract and lung.

Abstract: Endotoxic shock is accompanied by significant increases in PAF-acether synthesis, particularly by the lung. The onset of tissue damage and increases in vascular permeability in the gastrointestinal tract correlate temporally with the changes in PAF-acether synthesis and have previously been shown to be inhibited by PAF-acether antagonists. In the present study, the effects of pretreatment with dexamethasone on endotoxin-induced hemoconcentration and hypotension were examined in the rat. Furthermore, the effects of dexamethasone on PAF-acether synthesis and gastrointestinal vascular permeability following administration of endotoxin were also studied. Pretreatment with dexamethasone resulted in a significant attenuation of endotoxin-induced hemoconcentration, hypotension and damage in the duodenum and stomach. Dexamethasone also significantly reduced PAF-acether synthesis by the lung. However, dexamethasone pretreatment had no significant effect on endotoxin-induced increases in PAF-acether release and vascular permeability in the gastrointestinal tissues. The mechanism of the protective actions of dexamethasone may be related to inhibition of the release of PAF-acether from the lung. PAF released from gastrointestinal tissues likely contributes little to the systemic disturbances in endotoxic shock.

Purification and characterization of monoclonal antibodies to alpha-linolenic acid.

Abstract: The covalently linked antigenic complex, bovine serum albumin-alpha-linolenic acid, was used to immunize Balb/c mice against the hapten. Hybridization between splenocytes and the myeloma cell line, P 3 X63 Ag 8,651, resulted in stable clones synthesizing monoclonal antibodies (Mab) that were subsequently purified and characterized. Four Mab (A, B, C, D) were retained and their specificities studied by ELISA. Antibody D only recognized 18-carbon fatty acids having a cis,cis,-cis-1,4,7 unsaturated system in the omega-3 position: it was specific for alpha-linolenic acid. B recognized all fatty acids containing the structure cis,cis,cis-1,4,7-octatriene. A and C recognized polyunsaturated fatty acids with a degree of unsaturation superior to two double bonds.

Platelet-activating factor (PAF) antagonistic actions of two new analogs of tetrahydrofurans.

Abstract: Platelet-activating factor (PAF) has been implicated as a mediator involved in the pathogenesis of several types of inflammatory and shock states. The following group of experiments were designed to examine the effects of two new PAF receptor antagonists termed JS-1 and JS-3. The chemical structures of the two compounds were synthesized based on computer modeling of PAF and previously reported PAF receptor antagonists (e.g., tetrahydrofurans). Both JS-1 (1.3 mumols/kg) and JS-3 (9.5 mumols/kg) were found to significantly reverse PAF-induced (0.3 microgram/kg) hypotension in the rat when compared to their respective vehicles. In washed rabbit platelets, pretreatment with JS-1 (10 microM) and JS-3 (60 microM) inhibited aggregation induced by PAF at concentrations from 185 pM to 18.5 nM. These data indicate that both JS-1 and JS-3 are effective receptor antagonists of platelet-activating factor, and that computer modeling of molecular structures can be an important tool in developing analogs of known mediators of circulatory disease.

PAF receptor. 2. Quantitative hydrophobic contribution of the agonist's etheroxid chain.

Abstract: In order to undertake a quantitative assessment of the contribution of the hydrophobic effect of the etheroxid chain to agonistic activity, it was necessary to include in the calculated data a highly lipophilic platelet-activating factor (PAF) analogue. The synthesis of such a compound--racemic 1-O-docosyl-2-O-acetylglycero-3-phosphocholine (C22 PAF)--is described. The regression analysis performed with data of 14 etheroxid analogues (reviewed by Godfroid et al., 1987), combined with data obtained with C22 PAF, is significant with respect to a parabolic evolution between lipophilicity of the chain and the logarithm of relative platelet stimulation. This result is characteristic of a hydrophobic interaction between the agonist and the PAF receptor.

Time-dependent differences in the vasopermeability response to intradermal peptidoleukotrienes.

Abstract: The time course of extravascular albumin accumulation responses elicited by the leukotrienes LTC4, LTD4, LTE4, and histamine in the skin were compared in the conscious guinea pig. During the initial 15-min period, comparison of the dose-response curves revealed that histamine produced a much larger increase in extravascular albumin content than any of the leukotrienes. One hour after intradermal injection and at subsequent time intervals, the response to LTD4 had increased in magnitude so that it equaled the response produced by histamine. This was apparent from comparison of the time courses of extravascular albumin accumulation for intermediate doses of LTD4 and histamine and also from comparison of dose-response relationships at 4 h post intradermal injection. In contrast to LTD4, the magnitude of the microvascular permeability responses to LTC4 and LTE4 remained relatively small even over an extended time scale. Although histamine produced a large initial response, this also remained essentially unchanged over a 4-h period. It appears that LTD4 may produce a unique, time-dependent cutaneous microvascular permeability response and measurements over 15-30-min periods may underestimate its activity as a vasopermeability factor. The time-dependent effects of LTD4 on albumin extravasation cannot be ascribed to leukocyte infiltration into the skin since LTC4, LTD4, and LTE4 were entirely without effect in this regard.