The role played by platelet-activating factor (PAF) and tumor necrosis factor (TNF-alpha) in myocardial ischaemia-reperfusion injury was investigated. Pentobarbital anaesthetized rats were subjected to left main coronary artery ligation (1 h) followed by reperfusion (1 h; MI/R). Sham-operated rats were used as controls (Sham MI/R). Myocardial ischaemia-reperfusion injury produced a marked myocardial injury (necrotic area/area-at-risk = 60 +/- 5%; necrotic area/total area = 50 +/- 6%), high serum creatine phosphokinase activity (Sham MI/R = 25 +/- 10 U/ml; MI/R = 190 +/- 12 U/ml), a severe leukopenia (Sham MI/R = 10367 +/- 630 WBC x mm3; MI/R = 4123 +/- 120 WBC x mm3) and elevated myocardial myeloperoxidase activity (investigated as an index of leukocytes adhesion and accumulation) in the area-at-risk (6.2 +/- 0.5 U x 10(-3)/g tissue) and in necrotic area (6.6 +/- 0.7 U x 10(-3)/g tissue. Plasma PAF and serum TNF-alpha were significantly increased only during reperfusion. The peak of PAF plasma levels (6.5 +/- 1.2 pmol/ml) occurred earlier (15 min of reperfusion) than the peak of serum TNF-alpha (150 U/ml at 30 min of reperfusion). At the end of reperfusion, macrophage TNF-alpha was also enhanced (Sham MI/R = undetectable; MI/R = 148 +/- 12 U/ml). The administration of CV 6209, a specific PAF receptor antagonist (5 mg/kg, 5 min after occlusion), significantly reduced myocardial injury (necrotic area/area-at-risk = 27 +/- 3%, P < 0.001; necrotic area/total area = 10 +/- 2%, P < 0.001), blunted the increase in serum creatine phosphokinase (70 +/- 12 U/ml), partially restored leukopenia (8234 +/- 143 WBC x mm3) and lowered myeloperoxidase activity in area-at-risk (2.3 +/- 0.3 U x 10(-3)/g tissue; P < 0.001) and in necrotic area (2.8 +/- 0.5 U x 10(-3)/g tissue). In addition, administration of CV 6209 reduced the serum and macrophage levels of TNF-alpha. The results of this study, therefore, suggest that PAF and TNF-alpha are key mediators of myocardial ischaemia-reperfusion injury and that PAF plays a permissive role in inducing the release of other factor(s) relevant to reperfusion injury.
Human non-pancreatic PLA2 has been the object of intense scrutiny for a relatively short period of time. Its role in physiology remains enigmatic. While PLA2 may serve to remodel or remove peroxidised or senescent phospholipids, the enormous magnitude of its upregulation during infectious or inflammatory episodes is consistent with a role in host defense. However, the nature of this role remains elusive. Attempts to relegate this enzyme to the genre of acute phase reactants have not been helpful in unravelling its role. Difficulty in obtaining adequate amounts of native snp-PLA2 prior to the availability of recombinant snp-PLA2 led to the widespread use of snake venom homologs, particularly in studies of the biology of PLA2. This review has underscored the pitfalls inherent in that approach given the major differences between some venom PLA2s as compared to snp-PLA2. In addition, it bears reiterating that the complex composition of venom allows for potentiation of PLA2 activity by other constituents present in venom. Whether human host defense networks employ this interactive strategy is largely unknown. Nonetheless, in spite of these reservations, some very compelling data have emerged in recent years implicating snp-PLA2 in the initiation or potentiation of local and systemic inflammatory processes. These include sepsis and associated acute lung injury as well as inflammatory arthritides, with rheumatoid arthritis as the prototype. The mechanisms of snp-PLA2 homeostasis are considerably better understood, and it has become apparent that snp-PLA2 is an integral part of a larger network of proinflammatory cytokines, growth factors and lipid mediators. The interrelationship between the functions of secretory and cytosolic PLA2s remains to be defined. A number of selective PLA2 inhibitors have been identified which will allow for discrimination between the actions of these classes of PLA2. The availability of synthetic inhibitors in conjunction with endogenous modulators of PLA2s will shift the biology of PLA2 from the realm of the inferential to that of the mechanistic.
We investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the 5-lipoxygenase (5-LO) component of the leukotriene (LT) biosynthetic pathway of human neutrophils, in order to better understand the mechanism whereby the cytokine primes for LT synthesis. We found that GM-CSF increased 5-LO activation elicited by platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, LTB4, IL-8 and calcium ionophore A23187, as determined by using an exogenous substrate. A close correlation was observed between the priming kinetics of GM-CSF on 5-LO activation and on LT synthesis; moreover, the effects of the cytokine on both 5-LO activation and LT synthesis were inhibited when the cells had been exposed to either the protein synthesis inhibitor, cycloheximide (CX), or the transcription inhibitor, actinomycin D (AD), prior to incubation with GM-CSF. These results raise the possibility that the priming by GM-CSF of LT synthesis may involve an effect of the cytokine on 5-LO protein synthesis and gene expression.
In the present study, we have compared the responses to platelet-activating factor (PAF) of A/J and BALB/c inbred mouse strains. Two PAF-induced events were analyzed: increased vasopermeability, measured by extravasation of Evans blue dye (EB), and mortality. PAF injected into the peritoneal cavity induced a bell-shaped dose-response curve of EB extravasation in both strains of mouse. In A/J mice, maximal EB extravasation was reached with 0.1 microgram of PAF/mouse, whereas in BALB/c mice maximal extravasation was attained at a 10-fold greater PAF concentration. PAF-induced mortality also differed among these mouse strains; the LD50 was 12.1 micrograms/kg in A/J and 21.2 micrograms/kg in BALB/c mice. Thus, these strains differ significantly regarding both events mediated by PAF. Surprisingly, the F1 hybrid (A/J x BALB/c) mice were as sensitive as the A/J strain to PAF-induced extravasation but were as resistant as the BALB/c mice to PAF-induced mortality. The effects of the PAF antagonists BN 52021 and WEB 2086 were compared in the F1 hybrids. It was found that 1.0 mg/kg of WEB 2086 affected PAF-induced extravasation at almost all PAF doses tested (0.03-3.0 micrograms) while 15 mg/kg of BN 52021 was only effective at doses of PAF below 0.3 microgram. Both antagonists prevented PAF-induced mortality. Our results indicate that the two events induced by PAF may be controlled by different genes.
Cardiovascular and respiratory responses to a 2 h intravenous constant infusion of PAF (5 and 10 ng/kg per min) were studied in strain 13 guinea pigs. PAF decreased arterial blood pressure, left systolic ventricular pressure, and cardiac output (CO). These cardiovascular changes were dose-dependent. The PAF-induced hypotension returned to a pre-infusion level spontaneously with increased total peripheral resistance despite continuous infusion of PAF. The decreased CO was most striking, and did not recover to pre-infusion levels due to depressed cardiac contractility and impaired ventricular relaxation. Respiratory responses to PAF infusion at these doses were mild and only occurred after serious cardiovascular dysfunctions developed. A higher dose of PAF (20 ng/kg per min) produced drastically decreased CO and dynamic lung compliance (C(dyn)), increased pulmonary airway resistance, hypoventilation and apnea within 10-40 min. BN 52021. a PAF receptor antagonist, administered as a single i.v. dose (6 mg/kg) 15 min after PAF infusion, reversed most of cardiopulmonary dysfunctions and prevented death by increasing cardiac contractility, CO, and minute volume from extremely low values. The data suggest that marked cardiopulmonary disturbances induced by intravenous PAF infusion reflects certain pathophysiological mechanisms of diseases that may involve the cellular release of PAF. The administration of BN 52021 or other potent PAF antagonists may be beneficial under these circumstances.
The local release of sensory neuropeptides from capsaicin-sensitive primary afferents elicits prominent motor and inflammatory actions in mammalian airways. This neurogenic inflammation can contribute to the pathophysiology of asthma and airway hyperreactivity. In this review evidence will be presented regarding the involvement of this peptidergic neural pathway in the mediation of some pulmonary actions of lipid mediators such as eicosanoids and PAF.
The ability of a furofuran lignan, epiyangambin, to inhibit PAF-induced rabbit platelet aggregation in vitro and thrombocytopenia in rats was investigated. Epiyangambin dose-dependently inhibited PAF-induced platelet aggregation without modifying the amplitude of the maximal response, indicating a competitive antagonism. The IC50 value of epiyangambin for 10(-9) M PAF-induced aggregation was 6.1 x 10(-7) M and the Schild analysis provided a pA2 of 6.91 +/- 0.2 with a slope of 0.98 +/- 0.25 (n = 4) and a pKb of 6.94 +/- 0.19. Epiyangambin had no effect upon the platelet aggregation induced by collagen, thrombin or ADP. The in vivo administration of the lignan at 20 mg/kg significantly inhibited PAF-induced thrombocytopenia in rats. These data indicate that epiyangambin is a potent and selective antagonist of PAF both in vitro and in vivo.
The cDNA encoding the platelet-activating factor (PAF) receptor was cloned from a guinea pig lung cDNA library by using a Xenopus laevis oocyte expression system. The human PAF receptor cDNA was isolated from a human leukocyte cDNA library using a 0.8-kbp fragment of the guinea pig PAF receptor cDNA as a probe. Both receptors have the same number of amino acids (342 residues) with seven putative transmembrane spanning domains, and belong to the G-protein-linked receptor superfamily. Overall amino acid identity between the two receptors was 83%:91% in the transmembrane domains. Seven threonine and three serine residues conserved in the cytoplasmic loops of both receptors may function as phosphate acceptors, as related to the homologous desensitization of the receptor. Activation of the PAF receptor yielded inositol 1,4,5-trisphosphate production in the PAF receptor expressed COS-7 cells and oocytes, and guanosine 5'-O-(2-thio)bisphosphate injected into the oocytes inhibited PAF-induced Cl- current, providing evidence that PAF stimulates phosphoinositide turnover via G-protein(s).
Biosynthesis of thromboxane A2 (TxA2) during cell-cell interactions between platelets and alveolar macrophages, eosinophils and natural killer cells from guinea pigs has been investigated. The stimulation of platelets incubated with varying numbers of macrophages or eosinophils or NK cells with 5 microM ionophore A23187 has induced an increase of TxA2 synthesis in comparison to the sum of TxA2 production by each cell population stimulated alone. These results suggest that transcellular biosynthesis of TxA2 or cell cooperation occurred between platelets and the other cell population studied, which could correspond to cell-cell interactions of type IA and/or type III, according to the classification proposed by Marcus ((1986) Prog. Hemost. Thromb. 28, 127-42). When varying concentrations of platelets were incubated with the supernatant of cells previously stimulated with the ionophore A23187, an enhancement of TxA2 release was also observed. However, the supernatant of platelets stimulated with the ionophore A23187 did not induce a significant increase of TxA2 synthesis by the other cell populations. It is suggested that the biosynthesis of TxA2 during cell-cell interactions could account for the large amount recovered in biological fluid in various pathological conditions such as inflammatory and hypersensitivity reactions.
Plasmid vectors were constructed which allowed expression of the mouse prostaglandin endoperoxide (PGH) synthase-1 and PGH synthase-2 isozymes in cos-1 cells. Efficient expression of the PGHS-2 isozyme required the truncation of the entire 3'-untranslated region of the PGHS-2 cDNA, possibly due to the presence of multiple AUUUA sequences which may destabilize the PGHS-2 mRNA. The length of the 3'-untranslated regions of the murine and ovine PGHS-1 isozymes, which do not contain AUUUA sequences, did not affect the efficiency of expression of these proteins. The murine PGHS-2 isozyme catalyzes the same cyclooxygenase and hydroperoxidase activities as the ovine and murine PGHS-1 isozymes. The maximal activities of the mouse enzymes expressed in cos-1 cells was about equal, but both were only about a third that seen with the sheep enzyme. Whether this reflects differences in the turnover rate of the mouse and sheep enzymes, or differences in the efficiency of expression in cos-1 cells is not known.
The receptors involved in the ocular hypotensive activity of prostaglandins (PG) E2 and F2 alpha in dogs and monkeys was investigated by examining the effects of putative receptor selective agonists on intraocular pressure. A diverse variety of receptor selective agonists lowered intraocular pressure in these species. Thus, FP-receptor agonists (17-phenyl PGF2 alpha, fluprostenol), agonists with potent activity at the EP3 receptor (MB 28767, sulprostone) and a prostanoid with activity at the EP2 receptor (11-deoxy PGE1) were all potent ocular hypotensives when administered as a single dose to dogs and monkeys or b.i.d. for 5 days in monkeys. These findings were regarded as surprising and prompted us to re-examine some aspects of the current classification for prostanoid receptors. At present certain receptor subtypes, notably EP2, EP3 and FP receptors, are defined only according to the potency rank order for agonists. In these studies, we employed radioligand binding studies to determine the degree of competition between prostanoid agonists claimed to be selective on the basis of functional assays. Competition studies with a diverse variety of prostanoids at the binding site for PGE2 and sulprostone on the myometrial plasma membrane prepared from the rat uterus were consistent with the presence of an EP3 receptor. Thus, EP3-receptor agonists (MB 28767 and sulprostone) potently inhibited PGE2 and sulprostone binding, whereas FP agonists (17-phenyl PGF2 alpha, fluprostenol), a DP agonist (BW 245C), an EP1 antagonist (AH 6809), an EP2 agonist (AH 13205) and TP-receptor ligands (BM 13505, I-BOP) afforded little or no inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
A high affinity binding site for 14(R),15(S)-EET, one of the major cytochrome P-450 metabolites of arachidonic acid (AA) in blood vessels, liver, kidney and urine of patients with pregnancy-induced hypertension, has been identified in a membrane preparation from guinea pig mononuclear (GPM) cells. Using a radioligand assay, binding of 14(R),15(S)-[3H]EET to its receptor site was saturable, specific and reversible. Scatchard analysis of saturation binding studies yielded a dissociation constant (Kd) of 5.7 x 10(-9) M, and maximum number of binding sites (Bmax) of 2.4 pmol/mg membrane protein. The specificity of the binding site was determined by competition studies. 14(S),15(R)-EET and 8,9-EET had a Ki of 6.3 and 8.8 nM, respectively, followed by 12(R)-HETE and LTD4. 12(S)-HETE and 5,6-EET were even less effective as a competitive inhibitor of radioligand and binding with Ki values from 2 to 20 microM. Receptor antagonists for TxA2, LTB4, LTD4 and PAF failed to displace 14(R),15(S)-[3H]EET from its binding site on GPM cell membranes. The results correlate well with the reported biological functions of 14,15-EET. In view of its potent biological activities, 14,15-EET may exert its cellular function through the binding and activation of its stereo-specific cell surface binding sites or receptor.
Low levels of 5-lipoxygenase (5-LO), the first committed enzyme in the synthesis of leukotrienes (LTs), have been reported in the porcine pancreas. We have quantitated 5-LO activity in subcellular fractions of pancreas samples from three human donors. 5-LO activity was detectable in all samples, although enzyme activity was lower than in human leukocytes. 5-LO in human pancreas samples displayed highest specific activity in membrane fractions, and did not require arachidonic acid (AA) addition for activity. These unusual characteristics of pancreatic 5-LO appear to be due, at least in part, to the presence of unesterified AA in the pancreas samples. Western blot analysis demonstrated that the human pancreas contains low levels of 5-lipoxygenase-activating protein (FLAP) in addition to 5-LO.
The administration of platelet-activating factor (PAF) to human subjects triggers asthma-like responses. We investigated whether a bronchoconstrictive reaction was accompanied by the release of PAF in the circulation of allergic asthmatics. The appearance of PAF was assessed by measuring the number of freely accessible PAF-receptors on platelets in vitro, assuming that the contact between platelets and PAF in vivo would reduce the receptor binding of [3H]PAF in vitro. 16 asthmatics were challenged twice, first with buffer and the next day with allergen. A comparison between receptor binding after provocation with the data of the same patients after allergen challenge revealed a significant difference in PAF binding (P = 0.034), with an average decrease of 14% immediately after allergen challenge followed by a return to control values after about 4 h and a transient increase of 9% at 7 h after provocation. The decrease in accessible PAF receptors was accompanied by a slight decrease in platelet count in peripheral blood between 30 min and 4 h after allergen challenge. The platelet counts recovered to the original values afterwards. These data support the concept that in patients with allergic asthma PAF is secreted in the circulation. The contact between PAF and the platelets may trigger the transient sequestration of platelets, possibly in the lung. Thus, PAF and platelets may contribute to the pathogenesis of allergic asthma.
D-3-Hydroxybutyrate dehydrogenase (BDH), an inner mitochondrial protein, is a well-known phospholipid dependent enzyme. It is a primary dehydrogenase of the oxidative phosphorylation system and is involved in the redox balance of the NAD+/NADH pool. The preparation of fluorescent phospholipids and newly synthesized bifunctional phospholipid analogues (fluorescent and photoactivatable) allowed us to study the structural requirement for lipid activation of the purified enzyme. This paper reports the chemical synthesis protocols to prepare these new phospholipids and their characterization. Illumination experiments of complexes between bifunctional phospholipids and BDH which lead to a cross-linked polypeptide indicate that both the polar head and the hydrophobic moiety of phospholipids interact with BDH. The bifunctional phospholipids were also tested on other lipid-binding proteins, i.e., horse cytochrome c and bovine serum albumin, and demonstrated the promising potential of this new type of photoactivatable molecules which can be followed merely by fluorescence without radioactive labeling.
We measured circulating platelet factor 4 (PF4) and heparin-releasable platelet factor 4 (HR-PF4) in 13 symptomatic patients suffering from bronchial asthma and in 10 matched normal controls. Neither a difference between patients and controls, nor a correlation between platelet count and HR-PF4 was found (PF4: controls 2.1 +/- 2.9; patients 3.6 +/- 4.4 ng/ml (n.s.); HR-PF4: controls 127 +/- 49, patients 101 +/- 34 ng/ml (n.s.). Therefore, the pathogenetic role of PF4 in the mechanism of bronchial asthma, elsewhere hypothesized, cannot definitely be established.
