Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1–10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 μg/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 μM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Inhibition of transmembrane movement and metabolism of platelet activating factor (PAF-acether) by a specific antagonist, BN 52021

Incorporation of 1-[3H]-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF-acether) into rabbit platelet phosphatidylcholine (PC) was inhibited by a specific antagonist, BN 52021 (IC50 5.6 X 10(-6) M, maximal effect, i.e 70% inhibition, at 10(-4) M). Under the same conditions, [3H] lyso-PAF-acether incorporation remained 9 fold lower, compared to PAF-acether, without any effect of BN 52021. Upon cell lysis, both phospholipids attained the same rate of metabolic conversion, corresponding to a 1.15-fold and a 12-fold increase for PAF-acether and lyso-PAF-acether, respectively. In none of these cases was BN 52021 effective. It is concluded that transmembrane movement of the two phospholipids represents the limiting step of their metabolism. The higher rate of PAF-acether conversion by intact platelets could involve its binding to a membrane receptor, as suggested by the inhibitory effect of BN 52021, the significance of which is discussed.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1-10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 micrograms/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 microM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a 3 fold greater Ca²⁺-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

Inhibition by BN 52021 (ginkgolide B) of the binding of [3H]-platelet-activating factor to human neutrophil granulocytes

The inhibitory effect of BN 52021, a specific antagonist of platelet-activating factor (PAF) on PAF-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]-PAF to neutrophils were examined. BN 52021 over the range of 10(-9)-10(-4) M inhibited PAF-induced degranulation and superoxide production of PMNLs in a dose-dependent manner with Kd values of 0.6 +/- 0.1 x 10(-6) M and 0.4 +/- 0.1 x 10(-6) M, respectively. BN 52021 (up to 1 mM) did not show any agonistic activity and it did not affect neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. The Ki value of BN 52021 for the specific binding of [3H]-PAF to neutrophils was 1.3 +/- 0.5 x 10(-6) M versus a Ki of 1.1 +/- 0.3 x 10(-7) M for PAF itself. BN 52021 did not affect metabolism of PAF by PMNL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor.     

A prostacyclin analogue, iloprost, augments the ovulatory response of the LH-stimulated in vitro perfused rat ovary

Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinzing hormone (LH; 0.1 μg/ml) in vitro at 0 h of perfusion resulted in 2.4 ± 0.75 (mean ± SEM) ovulatioons per treated ovary, whereas no ovulations occured in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 μM or 0.1 μM, the ovulation rate increase significantly (p<0.05) to 6.6 ± 1.3 and 10.2 ± 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 μM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3′,5′-monophosphate or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positve modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.     

Interference of the PAF-acether antagonist BN 52021 with endotoxin-induced hypotension in the guinea-pig

Because of the potential role of PAF-acether in the pathogenesis of endotoxin shock, we examined the preventive and curative effects of BN 52021, a new PAF-acether antagonist in guinea-pig challenged with S. Typhimurium endotoxin. A biphasic reduction of mean arterial pressure was elicited by i.v. endotoxin (300 micrograms/kg) in control animals, with a rapid drop of blood pressure (maximal decrease within 10 min), partial recovery at 20 min and a second gradual decrease after 30 min. Treatment with BN 52021 injected 15 min prior to endotoxin reduced the initial rapid drop of blood pressure from 38.5 +/- 5 mmHg in vehicle-treated controls (n = 15) to 17 +/- 3 mmHg (p less than 0.01) in animals treated with 1 mg/kg BN 52021(n = 10) and to 9.5 +/- 8 mmHg (p less than 0.01) in guinea-pigs treated with 6 mg/kg BN 52021 (n = 5). The early hypotensive phase was associated with severe thrombocytopenia-leukopenia; only the thrombocytopenia was reduced by BN 52021. The prolonged secondary phase of hypotension was reduced by BN 52021 pretreatment whereas a small increase of hematocrit persisted. The two phases of the arterial pressure profile during endotoxic shock were not observed in animals previously made thrombopenic by rabbit and anti-platelet serum and only the late hypotensive phase persisted. This late hypotension induced by endotoxin in thrombopenic animals was suppressed by BN 52021 pretreatment suggesting that BN 52021 may act via a platelet-independent mechanism. The intravenous injection of BN 52021 during the prolonged secondary phase of shock was followed by an immediate increase of the depressed blood pressure. This increase of blood pressure was dose-dependent, maximum at 6 mg/kg BN 52021, and observed in normal and thrombopenic animals. The interference of BN 52021 with endotoxin shock may be related to its PAF-acether antagonist properties and suggests that PAF-acether is an important participant in endotoxic shock.     

Desensitization and antagonism of rat polymorphonuclear leukocytes stimulated with PAF acether

The activation of rat polymorphonuclear leukocytes (PMN) with PAF-acether (platelet-activating factor) was blocked by two antagonists: 48740 RP and BN 52021. The release of beta glucuronidase was usually better inhibited than that of lysozyme suggesting that the tested antagonists interfere rather with PAF-acether exocytosis of azurophil granules. Inhibition was relatively specific, even though a moderate effect (below 34%) upon PMN activation by the two unrelated agonist n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and leukotriene B4 sometimes reached significance. The partial persistence of inhibition to stimulation with PAF-acether after elimination of both inhibitors suggests that they do not act at the PAF-acether receptor only. Furthermore, the observation of cross desensitization between PAF-acether and fMLP may be related to common pathways and/or metabolites, including PAF-acether itself.     

Inhibitory action of lanthanum(La) on PGF2α- induced contraction of guinea-pig stomach muscle

The effects of lanthanum(La) on contractions induced by prostaglandin F_2α(PGF_2α) or isotonic K⁺ were investigated in the isolated stomach muscle of guinea-pig.Low concentrations of La(0.1–1 μM) inhibited the contraction to PGF_2α 1 μM in a dose-dependent manner, without affecting the tonic contraction to isotonic K⁺.0.1 and 1 μM La shifted the dose-response curve for PGF_2α(0.001 – 1 μM) to the right and reduced the maximum response.The IC₅₀ of La against PGF_2α and K⁺ were 0.6 μM and 30 μM, respectively.These results support the suggestion that PGF_2α -induced contraction in the stomach muscle depends mainly on the intracellular release of sequestered Ca, which would be depleted or immobilized by La.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID₅₀'s for prostaglandin (PG) E₂ synthesis in these cells were 0.1 μM for 2,6-xylenol, 5 μM for tricresol, 6 μM for -cresol, 7 μM for -cresol, 15 μM for 3,5-xylenol, 30 μM for -cresol and 100 μM for phenol. The corresponding values for aspirin and indomethacin were 4 μM and 0.02 μM, respectively.The substituted phenols also inhibited serotonin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG₂.     

Species dependent relaxation of intrapulmonary arteries (IPA) of rabbits, dogs and humans by prostacyclin

Helically cut strips of successive IPA segments of rabbits, dogs and human patients were set up for isometric recording . High tone was produced by norepinephrine (NE, 3 μM). This tone was markedly reduced by prostacyclin (PGI2) in the secondary, tertiary and quaternary branches of human and canine pulmonary trunk. The IC50 values for PGI2 ranged from 22 to 503 nM, the human vessels being more sensitive to prostacyclin than canine IPA. Under these conditions, the primary and secondary branches of the rabbit pulmonary trunk were not relaxed by PGI2. The contractile potency of NE was determined in each pulmonary vessel studied. The secondary segments of rabbit IPA were about ten times as sensitive to NE (EC50 for NE: 38±7 nM) as compared to the secondary IPA from dogs and humans (EC50 values: 370±84 and 440±50, respectively). When high tone was induced by equieffective contractile concentrations of NE (3 μM for canine and human IPA and 0.3 μM for rabbit vessels), PGI2 was still less effective (P<0.01) in relaxing secondary IPA of rabbits (IC25: 220±55) than the corresponding segments of dogs and humans (IC25: 51±12 and 17±4, respectively). The difference between canine and human vessels was also significant (P<0.02). These results indicate that there is an interspecies difference in the sensitivity of IPA to NE and PGI2.     

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential −70 mV and in the presence of 200 μM glycine IC₅₀ value for the blocking action of BN52021 was 270±10 nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC₅₀ value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl⁻ channels by interacting with the pore region and does not compete for the strychnine-binding centre.     

Effects of platelet activating factor (PAF) and its receptor antagonist BN 52021 on isolated perfused guinea-pig heart

The cardiac effects of PAF and its antagonist BN 52021 have been investigated on the isolated perfused guinea-pig heart maintained at a constant hydrostatic perfusion pressure of 80 cm water. In this model, PAF (1 x 10(-11) to 1 x 10(-7) moles) induced a dose-dependent coronary vasoconstriction, a decrease in heart rate and a fall in contractile force. BN 52021 (1 x 10(-6) to 2 x 10(-4) M) dose-dependently inhibited the vasospasm induced by PAF (1 x 10(-10) moles). BN 52021 also antagonized the decrease in coronary flow and heart rate, but not that of contractile force induced by a high dose of PAF (1 x 10(-7) moles). This dose of PAF also significantly (p less than 0.001) provoked a marked release of TxB2 but did not alter the generation of 6 Keto PGF1 alpha, PGE2 or LTC4. The PAF-induced increase in TxB2 release was completely abolished by BN 52021.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 ± 6.2% (p < 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 ± 10.6% (p < 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 μM) were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 μM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF . We conclude that platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils is not a sensitive indicator.     

Rapid and specific high-performance liquid chromatographic method for the determination of iodide in urine

A rapid and specific method for the determination of iodide in urine by high-performance liquid chromatography on an anion-exchange column with electrochemical detection is described. The assay is reproducible as judged by the coefficient of variation of less than 4% at all concentrations used. The limit of detection was 0.1 μ mol, and the calibration graph was linear for concentrations between 0.1 and 200 μmol. Using this method, healthy volunteers were found to excrete 69±39 μmol of iodide per mole of creatinine.     

Measurement of changes in functional muscarinic acetylcholine receptor density in single neuroblastoma cells using calcium release kinetics

Calcium release in response to the activation of muscarinic M1 and histamine H₁ receptors was studied in single N1E-115 cells using Fura-2 imaging. The objective was to relate changes in the kinetics of Ca release with reductions in functional receptor density resulting from receptor desensitization. Calcium release increased and its time course accelerated with increasing carbachol concentration with an EC₅₀ = 96 ± 8 μM. This value is similar to the binding K_D (100 μM) and the similarity shows that the activation of calcium release is limited by the number of muscarinic receptors. In contrast, the EC₅₀ for Ca release in response to histamine is 4.0 ± 0.7 μM while the binding K_D is 8.3 μM and, therefore, H₁ receptors appear to be in approximately 2-fold excess over the minimum number necessary to fully engage the Ca release mechanism.Functional surface receptor number was assayed in the population of cells by counting the total number of cells responding to agonist. A 5 min exposure to 1 mM carbachol caused 12% of cells to lose their ability to respond to carbachol, with no change in their response to histamine. Interpolating from the dose-response curve taken before desensitization, this is equivalent to an average 23% reduction in the number of muscarinic receptors. In individual cells the latency to Ca release is dose-dependent in the absence of excess receptors. The loss of functional receptors was therefore estimated from the increase in latency after desensitization, and varied from 5–48% of receptors (22 ± 18%). Muscarinic desensitization did not depend on IP₃-evoked Ca release, Ca entry, protein kinase C, NO, or cGMP. We conclude that in a population, the number of cells responding and in single cells, the latency to Ca release can serve as measures of functional receptor density.     

Age-, cycle- and topographic dependency of human myometrial prostaglandin (6-keto-PGF1a, PGF2a)-synthesis in vitro

Specimens of human myometrium (isthmus and fundus) freshly obtained at hysterectomy were immediately transferred in ice cold Tyrode solution and placed in superfusion chambers. Spontaneous contractions were recorded, the effluent of the myometrium was analyzed for PGF2a and 6-keto-PGF1a by use of specific radioimmunoassay systems. Dating of the menstrual cycle was achieved by histological evaluation of the endometrium.The PG release rates expressed as ng/min/g wet weight were correlated to the patients age and to the phase of the menstrual cycle. The production rates of 6-keto-PGF1a were negatively correlated to the age of the patients and declined in fundus specimens from 2.89 ± 0.35 ng/min/g wet weight in 39–42 years old patients to 0.52 ± 0.17 ng/min/g wet weight in 48–52 years old women during the secretory phase (p< 0.001). Similar significant correlations were found in specimens obtained from the isthmus uteri.During the proliferative phase fundus specimes produced on average 1.61 ± 0.67 ng/min/g wet weight in 39–42 years old patients and0.49 ± 0.12 n/min/g weight 6-keto-PGF1a in 48–52 years old women respectively (p < 0.001).The PGF2a synthesis in myometrial specimens of fundus or isthmus origin was significantly lower than 6-keto-PGF1a and did not correlate to the age of the patients during the proliferative phase. However, PGF2a release rates during the secretory phase were significantly (p < 0.001) higher in younger women.These results suggest an age-, cycle- and topographic dependency of PGI2 synthesis in human myometrial tissue.