Species difference in the specific receptors of platelet activating factor

Relative potencies of platelet activating factor (PAF) and PAF analogs and several PAF receptor antagonists when inhibiting the [3H]PAF specific binding to human and rabbit platelet membranes and membrane fragments of human lung tissues were compared. In rabbit platelets, L-652,731 was found to be most potent in the list of PAF receptor antagonists with an equilibrium inhibition constant (Ki) of 9.83 (+/- 2.92) X 10(-9) M followed by L-653,150 greater than kadsurenone congruent to Ono-6240 greater than ginkgolide B greater than CV-3988 greater than L-651,142, whereas in human platelets the relative potencies of these PAF receptor antagonists were as follows: Ono-6240 greater than L-653,150 congruent to L-652,731 congruent to kadsurenone greater than ginkgolide B greater than CV-3988 greater than L-651,142. Ono-6240 was the most potent one with a Ki of 4.86 (+/- 1.44) X 10(-8) M which was roughly two times more potent than that in rabbit platelets, whereas the affinity of L-652,731 was about ten times less in human platelets (Ki = 1.03 (+/- 0.15) X 10(-7) M) compared to that in rabbit platelets (Ki = 9.83 (+/- 2.92) X 10(-9) M). These variations between species among PAF antagonists strongly suggest that there exists a species difference at or near the binding site of the receptor of platelet activating factor. The relative potency of these PAF receptor antagonists in human lung membranes differed very little from that in human platelets and was found to be Ono-6240 greater than L-653,150 congruent to kadsurenone congruent to L-652,731 greater than ginkgolide B greater than CV-3988 greater than L-651,142. Even though C16-PAF showed slightly higher potency in human lung, and CV-3988 and Ono-6240 showed slightly lower, the difference was too small to suggest that there is a difference in the PAF receptors between human platelets and human lung tissues.     

High affinity specific binding sites for tritiated platelet-activating factor in canine platelet membranes: counterparts of platelet-activating factor receptors mediating platelet aggregation

In canine platelet membranes, tritiated platelet activating factor (PAF) labels in a saturable and reversible manner a single population (nH = 0.97) of binding sites. The affinity of this binding was high (Kd = 0.23 +/- 0.02 nM, n = 4, and 0.21 +/- 0.05, n = 8, determined by kinetics or saturation experiments, respectively), and the density of binding sites (Bmax) was 911 +/- 31 fmol/mg of protein (n = 8). [3H]PAF binding was entirely reversed by unlabeled PAF (10 microM). [3H]PAF exhibited stereoselective discrimination inasmuch as it was poorly displaced by enantio-PAF, the PAF enantiomer that does not occur naturally. Furthermore, the displacing potency of the (+)-enantiomer of the PAF antagonist 52770 RP against [3H]PAF was 45 times higher than that of the (-)-enantiomer. [3H]PAF binding displayed a remarkable specificity in that it was not affected by a variety of classical pharmacological agents. However, this binding was displaced by several PAF receptor antagonists such as 59227 RP, CV-6209, Ro 19-3704, 52770 RP, brotizolam, WEB 2086, SRI 63-441, L-652,731, alprazolam, triazolam, and BN 52021. The Ki of the 16 studied antagonists ranged from 7.9 nM (59227 RP, most potent) to 16.8 microM (BN 52021, least potent). The possible biological significance of our binding procedure was assessed by correlating the potencies of 16 PAF antagonists as [3H]PAF displacers in dog platelet membranes and as inhibitors of PAF-induced platelet aggregation in washed canine platelets. This analysis revealed the existence of a highly significant correlation (r = 0.82, p less than 0.001) between biochemical and functional tests. However, two compounds (Ro 19-3704 and BN 52021) were found to be located outside the confidence limits when the probability level of belonging to the regression line was set at 0.01. In conclusion, this study provides evidence that [3H]PAF binding in canine platelet membranes exhibits the required properties for a valid binding procedure. Furthermore, the labeled sites are likely to be the counterparts of platelet receptors that, when activated by PAF, induce aggregation.     

Desensitization of platelet-activating factor-stimulated protein phosphorylation in platelets

Treatment of 32P-labeled rabbit platelets with platelet-activating factor (PAF) caused a time- and dose-dependent phosphorylation of several proteins including five major phosphorylated proteins with apparent molecular weights of 20,000, 35,000, 40,000, 65,000, and 150,000. Both PAF and thrombin caused a rapid increase followed by a decrease in phosphorylation of proteins, indicating the occurrence of a phosphorylation-dephosphorylation process. Four separate PAF receptor antagonists, CV-3988, CV-6209, SRI-63-441, and SRI-63-675 drastically reduced the PAF-stimulated protein phosphorylation. The order of potency was SRI-63675 greater than SRI-63441 greater than or equal to CV-6209 greater than CV-3988. These antagonists had no effect on thrombin-stimulated protein phosphorylation. Pretreatment of platelets with PAF (0.1 nM) completely abolished any further protein phosphorylation by the same concentration of PAF. PAF pretreatment shifted the dose response of protein phosphorylation by about 2 log units, to the right. When platelets were treated with PAF (10 nM) for 10 min, this abolished phosphorylation of proteins by any concentration of PAF. These studies indicated a homologous desensitization of protein phosphorylation. Interestingly, PAF-pretreated platelets still exhibited phosphorylation of proteins by thrombin. On the other hand, a lack of protein phosphorylation by PAF or thrombin was observed in platelets preexposed to thrombin and this demonstrated a heterologous desensitization. It is concluded that phosphorylation of proteins by PAF is a PAF receptor-coupled event and that this process is desensitized in platelets preexposed to PAF. The fact that both the activation of phosphoinositide-specific phospholipase C and the phosphorylation of proteins are desensitized in PAF-pretreated platelets suggests that a close "regulatory" intercommunication between these processes exists.     

Inhibition by CV-3988 of the binding of [3H]-platelet activating factor (PAF) to the platelet

The inhibitory effects of CV-3988, a specific antagonist of PAF, on the binding of [3H]-PAF to washed platelets of various species including human were examined. The dissociation constant (Kd), binding capacity (Bmax), and the number of receptor/platelet for the specific binding site of rabbit platelets were 2.2 +/- 0.2 nM, 93.7 +/- 8.3 fmoles/10(8) platelets, and 568 +/- 50, respectively. CV-3988 selectively inhibited the specific binding of [3H]-PAF to rabbit platelets with an IC50 of 7.9 X 10(-8) M, and it slightly increased the Kd value (2.5 +/- 0.8 nM) and decreased the binding capacity for PAF (Bmax: 54.3 +/- 16.3 fmoles/10(8) platelets). The Ki value of CV-3988 for the specific binding of [3H]-PAF to rabbit platelets was 1.2 X 10(-7) M. CV-3988 had no effects on the binding of [3H]-5-hydroxytryptamine (5-HT) to rabbit platelets and on the shape change of the platelet induced by 5-HT. CV-3988 also inhibited the specific binding of [3H]-PAF to human and guinea-pig platelets with IC50 values of 1.6 X 10(-7) and 1.8 X 10(-7) M, respectively. CV-3988 inhibited the PAF-induced aggregation in rabbit, guinea-pig, and human platelets. These findings show that CV-3988 is a specific antagonist of PAF at the receptor site(s) of platelets and, in these species, inhibits PAF-induced platelet aggregation by inhibiting the binding of PAF to the "PAF receptor". No specific binding of [3H]-PAF to the platelet of rats and mice was observed, indicating that these species lack a PAF receptor.     

Differential actions of platelet-activating factor (PAF) receptor antagonists on the vasodilator and vasoconstrictor effects of PAF in the rat perfused heart.

Selectivity for blocking the coronary vasodilator and vasoconstrictor effects of platelet-activating factor (PAF) in the rat perfused heart was observed with different PAF antagonists. CV-6209 showed selectivity for blocking the vasodilator effect of PAF and a higher concentration (10 fold) was required to block the vasoconstrictor effect. The remaining PAF antagonists (FR-900452, WEB 2086 and BN-50739) showed selectivity for blocking the vasoconstrictor effect of PAF (10, 200 and 1000 fold respectively). A combination of low concentrations of CV-6209 (10 nM) with FR-900452 (5 microM) or WEB 2086 (0.5 microM) was effective in blocking both the vasodilator and vasoconstrictor effects of PAF. CV-6209 and WEB 2086 did not affect the vasodilator action of leukotriene B4 (LTB4) and the vasoconstrictor action of LTC4 and LTD4. Our results support the hypothesis that the functionally opposite effects of PAF in the rat perfused heart may be mediated by different PAF receptor subtypes.     

Prehispanolone, a novel platelet activating factor receptor antagonist from Leonurus heterophyllus.

1. Using an in vitro radioligand binding assay for the platelet activating factor (PAF) receptor, we have identified a novel, specific PAF antagonist, prehispanolone, from a Chinese medicinal herb Leonurus heterophyllus. 2. The presence of sodium ions inhibited specific [3H]-PAF binding to rabbit platelet membrane with an IC50 of 5.2 mM, decreased the inhibitory potency of PAF but increased the inhibitory potency of prehispanolone. 3. Prehispanolone and several of its derivatives inhibited the binding of [3H]-PAF to rabbit platelets with potencies closely resembling that of inhibition of PAF-induced aggregation. 4. The integrity of the tetrahydrofuran ring of prehispanolone is critical for its interaction with the PAF receptor. 5. By hydrogenating the dihydrofuran ring and replacing the keto group of prehispanolone with a hydroxyl group, we obtained a compound, LC5507, that is more stable and more active than prehispanolone as a PAF receptor antagonist.     

Mechanism of the contractile response to Platelet-Activating Factor (PAF) of the rat stomach fundus. I. PAF-induced contractile response and calcium mobilization

• 1.1. Platelet-activating factor (PAF) caused contraction of the rat stomach fundus in a concentration-dependent manner in the presence of atropine, guanethidine, chlorpheniramine, methysergide, indomethacin, nordihydroguaiaretic acid and tetrodotoxin.
• 2.2. PAF produced phasic contraction followed by tonic contraction. The PAF-induced tonic contraction was significantly reduced by treatment with CV-6209, an antagonist of PAF, but phasic contraction induced by PAF was rather resistant to CV-6209.
• 3.3. The contraction induced by PAF was markedly reduced when tissues were previously exposed to PAF (desensitization).
• 4.4. Nicardipine reduced the PAF-induced phasic contraction but not of the tonic contraction.
• 5.5. PAF-induced contractions were almost abolished in Ca²⁺-free medium.
• 6.6. The Ca²⁺-contraction in Ca²⁺-free solution was significantly augmented by PAF, whereas the Ca²⁺-contraction in Ca²⁺-free, isotonic high K⁺ (60 mM) medium was unaffected by PAF.
• 7.7. These results suggest that PAF-induced contractile response in the rat stomach fundus is due to an influx of Ca²⁺ through voltage-dependent Ca²⁺-channels (VDC) and receptor-operated Ca²⁺-channels (ROC). It is further suggested that PAF may depolarize the stomach fundus and this depolarization may open the VDC, whereas PAF may not act directly on the VDC.

     

Salutary consequences of blockade of platelet activating factor in hemorrhagic shock

We studied the effects of a potent, specific platelet activating factor (PAF) antagonist, CV-6209, in a murine model of hemorrhagic shock. Hemorrhaged rats treated with CV-6209 (1 mg/kg) maintained post-reinfusion mean arterial blood pressure (MABP) at significantly higher values than rats receiving either 0.9% NaCl or a lower dose (0.2 mg/kg) of CV-6209 (final MABP 88 +/- 4 vs. 57 +/- 4, vs. 61 +/- 7 mm Hg, respectively). CV-6209 (1 mg/kg) also significantly attenuated the increase in plasma cathepsin D activity following hemorrhage compared with hemorrhaged rats receiving only its vehicle (i.e. 0.9% NaCl). CV-6209 (1 mg/kg) also significantly decreased the plasma accumulation of free amino-nitrogen compounds and the plasma activity of a myocardial depressant factor (MDF) compared to hemorrhaged rats receiving 0.9% NaCl. Rats receiving CV-6209 (1 mg/kg) exhibited a significantly increased survival rate and survival time post-reinfusion compared to rats receiving only the vehicle. These data indicate that PAF is an important mediator of hemorrhagic shock in the rat and that PAF receptor antagonists may be useful in hemorrhagic shock states.     

Involvement of nitric oxide pathway in the PAF-induced relaxation of rat thoracic aorta.

1. The mechanism of the vasorelaxant effect of platelet activating factor (PAF) on rat thoracic aorta and the effect of aging on the PAF-induced relaxation were investigated. 2. PAF at concentrations causing relaxation induced marked increases in guanosine 3':5'-cyclic monophosphate (cyclic GMP) production, but did not induce an increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP). 3. Removal of the endothelium by mechanical rubbing, and treatment with the PAF antagonists CV-3988, CV-6209 and FR-900452, the nitric oxide biosynthesis inhibitor, NG-nitro L-arginine, the radical scavenger, haemoglobin, and the soluble guanylate cyclase inhibitor, methylene blue, inhibited PAF-induced relaxation and abolished or attenuated PAF-stimulated cyclic GMP production. 4. The relaxation was greatest in arteries from rats aged 4 weeks. With an increase in age, the response of the arteries to PAF was attenuated. 5. Endothelium-dependent cyclic GMP production also decreased with increase in age of the rats. 6. These results suggest that PAF stimulates production of nitric oxide from L-arginine by acting on the PAF receptors in the endothelium, which in turn stimulates soluble guanylate cyclase in the smooth muscle cells, and so increases production of cyclic GMP, thus relaxing the arteries. Age-associated decrease in PAF-induced relaxation may result from a reduction of cyclic GMP formation.     

Influence of plasma protein content and platelet number on the potency of PAF and its antagonist RP 59227 in rabbit platelet preparations.

1. The potency of platelet activating factor (PAF) as a pro-aggregatory agent and of RP 59227, a selective antagonist of PAF-induced platelet aggregation, was determined in several types of rabbit platelet preparations. 2. PAF produced concentration-dependent responses irrespective of whether the suspension medium for the platelets (200,000 per microliter) was undiluted plasma (PRP), saline-diluted plasma (dil. PRP) or a salt solution (WP: washed platelets). The potency of PAF, expressed as pD2, was 3 fold higher in WP than PRP or diluted PRP (dil. PRP) for which the ratio (v/v) of total plasma to saline was 1:1.5. In PRP and WP preparations, an increase in the number of platelets in the reaction medium from 200,000 to 600,000 enhanced the potency of PAF slightly (2.3 fold). Furthermore, PAF was 3 times more potent in WP than PRP when studied in preparations containing either 200,000, 400,000 or 600,000 platelets per microliter. 3. RP 59227, like the reference compounds WEB 2086 and CV-6209, behaved as a competitive antagonist against PAF-evoked platelet aggregation in PRP, WP and dil. PRP (200,000 platelets per microliter). Their potency was slightly greater (1.6 to 2.6 fold more) in dil. PRP than in WP, but in PRP it was 3.5 to 4.3 times lower than in WP. RP 59227 was 2.3 and 5.0 times less potent when the platelet number of the PRP suspension was increased from 200,000 to 400,000 and 600,000 per microliter, respectively, whereas the potency of RP 59227 in WP was not modified by these changes in platelet number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of specific platelet-activating factor binding sites in the rat retina

The specificity of the effects of platelet-activating factor (PAF) on the electrophysiology of the retina suggests the existence of specific sites for PAF in this tissue. In this study, we report the presence of tritiated PAF ([3H]PAF) specific binding sites in membrane preparations of the retina of albino rats. The binding of [3H]PAF was saturable, specific, time-dependent and reversible. Scatchard analysis of the data revealed that the high-affinity retinal binding site possessed a Kd of 2.9 +/- 0.4 nM and Bmax of 0.85 +/- 0.16 pmol/mg protein. These values are comparable with those found for the membranous PAF receptor sites in platelets, neutrophils, lung tissue and brain. We have recently reported that PAF dose dependently modulates the b-wave of the electroretinogram (ERG) obtained from the isolated rat retina. The results of the present study suggest that such PAF-induced disturbances of the ERG may be mediated via specific receptors located in the retina.     

Comparative study of the effect of CV-6209, a specific PAF-antagonist, on rat paw edema caused by different phlogogen agents.

Comparative effects of CV-6209, a potent and specific platelet activating factor (PAF) antagonist, on PAF-acether-, carrageenin-, histamine-, serotonin-, compound 48/80-, dextran-, zymosan A- and arachidonic acid-induced rat paw edema were investigated. CV-6209 has proven to be effective in PAF-induced edema, but it also showed a significant activity in other models of paw edema, except those induced by zymosan A and arachidonic acid. Other drugs tested, namely indometacin, mepyramine, methysergide and nordihydroguaiaretic acid showed a more selective inhibitory profile. These results suggest an important role of PAF in the inflammatory process caused by intraplantar injection of different phlogogenes in the rat paw.     

High affinity receptor binding of platelet-activating factor in rat peritoneal polymorphonuclear leukocytes.

Rat peritoneal polymorphonuclear leukocytes (PMNs) showed a single class of high affinity binding sites for platelet-activating factor (PAF) with an equilibrium dissociation constant (KD) of 4.74 (+/- 2.59) nM. Each cell contained 2.79 (+/- 1.40) x 10(4) receptors. In the isolated membranes at pH 7.0 in 10 mM MgCl2, 10 mM Tris and 0.25% bovine serum albumin, the KD value was 0.61 (+/- 0.1) nM, which is roughly identical to the KD values reported previously for various membrane systems under identical ionic conditions. The receptors were highly specific for PAF. Several receptor antagonists that are reported to inhibit the binding of [3H]PAF and the PAF-induced function in platelets could fully displace the binding. The biologically inactive enantiomer (enantio-C16-PAF), a PAF analog, azido-PAF, and an indene derivative of the PAF receptor antagonist, L-651,142, had different potencies to inhibit [3H]PAF binding to rat and human PMN membranes. L-652,731, a tetrahydrofuran analog of the PAF receptor antagonist was about 10 times more potent to inhibit the binding in rat liver tissues than in rat PMNs. These results suggest that PAF receptors on human and rat PMNs are not identical and that PAF receptor subtypes may exist in rat liver and PMNs.     

PAF binding sites. Characterization by [3H]52770 RP, a pyrrolo[1,2-c]thiazole derivative, in rabbit platelets

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H,3H-pyrrolo[1,2-c]thiazole -7-carboxamide, displaces in a potent, specific and competitive manner [3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for Kd, 0.2 pmol/5 X 10(7) platelets and 3.66 pmol/mg protein for Bmax and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (-)-isomer at displacing [3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [3H]52770 RP binding. Studies carried out using [3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency (Ki) for several analogues of 52770 RP, to displace [3H]PAF from its binding sites, was highly correlated (r = 0.96) to their ability to antagonize [3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.     

Platelet activating factor antagonists: synthesis and structure-activity studies of novel PAF analogues modified in the phosphorylcholine moiety

New analogues of platelet activating factor (PAF), in which the phosphate and trimethylammonium moieties were replaced with an acylcarbamoyl moiety and a quaternary cyclic ammonium group, were synthesized. Their biological activities as PAF antagonists were evaluated by the inhibition of PAF-induced rabbit platelet aggregation in vitro and protective effects on PAF-induced hypotension in rats and PAF-induced death in mice. Investigation of structure-activity relationships revealed that PAF antagonist activity is strongly influenced by the acyl substituent of the nitrogen atom on the carbamoyl group and the nature of the polar head group at the 3-position of the glycerin backbone. Among the compounds tested, 2-[[N-acetyl-N-[[2-methoxy-3-[ (octadecylcarbamoyl)oxy]propoxy]-carbonyl]amino] methyl]-1-ethylpyridinium chloride (21, CV-6209) was one of the most potent compounds in the in vitro assay (IC50 = 7.5 X 10(-8) M) and the most potent and long-lasting in the in vivo assays. (R)-(-)-21 and (S)-(+)-21 were also synthesized, and no significant differences were observed in PAF antagonist activity in vitro and an inhibitory effect on PAF induced hypotension in vivo between (RS)-21 and its enantiomers.     

PAF, rather than histamine, participates in mouse anaphylactic hypotension

We determined the roles of platelet-activating factor (PAF) and histamine in anaphylactic hypotension in ovalbumin-sensitized anesthetized BALB/c mice. The effects of PAF and histamine on hemodynamic variables were studied by measuring the systemic arterial (Psa), portal venous (Ppv) and central venous (Pcv) pressures. Intravenous PAF evoked a biphasic Psa response, an initial rapid and transient drop followed by marked hypotension, accompanied by a decrease in Pcv. Histamine caused only mild systemic hypotension. Both agents similarly increased Ppv by approximately 4 cm H(2)O at high doses. After an injection of antigen, Psa initially increased slightly and then decreased from the baseline of 94 +/- 1 mm Hg to 46 +/- 1 mm Hg at 10 min after antigen administration, with Pcv decreasing by 2.5 cm H(2)O. Ppv increased by 3.5 cm H(2)O at 5 min after antigen injection. Pretreatment with either CV-6209 (PAF receptor antagonist, 1 mg/kg) or diphenhydramine (histamine H(1) receptor antagonist, 20 mg/kg) significantly attenuated an antigen-induced decrease in Psa. The inhibitory action of CV-6209 was greater than that of diphenhydramine, and the combination of these 2 antagonists almost completely inhibited the anaphylactic hypotension. In contrast, the antigen-induced increase in Ppv was attenuated by CV-6209 alone but augmented by diphenhydramine. It is concluded that anaphylactic hypotension is mainly mediated by PAF and, to a lesser extent, by histamine in anesthetized BALB/c mice.     

Disubstituted tetrahydrofurans and dioxolanes and PAF antagonists

A new series of disubstituted tetrahydrofuran and dioxolane derivatives were prepared and evaluated for their PAF antagonist activity in the PAF-induced in vitro platelet-aggregation and in vivo hypotension tests. Several of these compounds exhibited more potent activity than the structurally related 2-[N-acetyl-N-[[[[2-methoxy-3-[(octadecylcarbamoyl) oxy]propoxy]carbonyl]amino]methyl]-1-ethylpyridinium chloride (CV-6209, 3) in the in vitro assay, whereas all showed less potency in the in vivo test. The role of both the substituent nature and the placement and number of oxygen atoms in the ring are discussed. A quantitative SAR study carried out on these nuclei.     

Possible participation of intracellular platelet-activating factor in tumor necrosis factor-alpha production by rat peritoneal macrophages.

Stimulation of rat peritoneal macrophages by thapsigargin (46.1 nM) increased levels of tumor necrosis factor-alpha and prostaglandin E2 in the conditioned medium. Platelet-activating factor (PAF) was not detected in the conditioned medium, but the level of cell-associated PAF was increased transiently by thapsigargin. The PAF receptor antagonists such as E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopro-panecarbonyl-8,11-dim ethyl-2,3,4,5-tetrahydro-8 H-pyrido[4',3':4,5]thieno [3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine), L-652,73 1 (2,5-bis(3,4,5-trimethoxyphenyl) tetrahydrofuran) and CV-6209 (2-[N-acetyl-N-(2-methoxy-3-octadecyl-carbamoyloxy propoxycarbonyl)aminomethyl]-1-ethylpyridinium chloride) inhibited thapsigargin-induced production of tumor necrosis factor-alpha. The cyclooxygenase inhibitor indomethacin inhibited prostaglandin E2 production, and further enhanced thapsigargin-induced tumor necrosis factor-alpha production in parallel with further increase in cell-associated PAF production. The enhancement of tumor necrosis factor-alpha production induced by thapsigargin plus indomethacin was also inhibited by E6123, L-652,731 and CV-6209. However, exogenously added PAF up to 100 nM did not stimulate production of tumor necrosis factor-alpha. The level of tumor necrosis factor-alpha mRNA was increased by thapsigargin, but was lowered by the PAF receptor antagonist E6123, suggesting that the inhibition of tumor necrosis factor-alpha production by the PAF receptor antagonist is induced at the level of mRNA for tumor necrosis factor-alpha. These findings suggested that concurrently produced cell-associated PAF in thapsigargin-stimulated macrophages up-regulates production of tumor necrosis factor-alpha by acting as an intracellular signaling molecule and the PAF receptor antagonists might penetrate into the cells and antagonize the action of intracellular PAF.     

PAF-acether-induced mortality in mice: protection by benzodiazepines

Intraperitoneal injection to mice of platelet-activating factor (PAF) induces severe shock symptoms, leading to death. The effects of various drugs which are potential specific or non-specific PAF inhibitors were tested. Salbutamol, a beta 2-agonist, showed a partial protection, whereas the specific PAF antagonists CV 3988 or BN 52021 induced an important beneficial action against PAF-induced lethal effects. The effects of drugs related to benzodiazepines were also studied: alprazolam administered p.o. prior to PAF challenge provides almost complete protection against PAF toxicity. Thus PAF-induced death in mice represents a useful model of systemic anaphylaxis; moreover, studies of benzodiazepine-related compounds may be interesting for investigating the mechanisms of the biological actions of PAF.     

Bakkenolide G,a Natural PAF-receptor Antagonist

Because platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) participates in many physiopathological responses, including inflammatory reaction, endotoxic shock, allergic diseases and platelet aggregation, PAF-receptor antagonists are important in the treatment of these diseases. A biologically active compound, bakkenolide G, extracted from the plant Petasites formosanus selectively and concentration-dependently inhibited PAF-induced platelet aggregation and ATP release. The IC50 of bakkenolide G for PAF (2 ng mL^?1)-induced platelet aggregation was 5.6 ± 0.9 μm . Bakkenolide G also concentration-dependently inhibited PAF-induced intracellular signal transductions, including thromboxane B₂ formation, and increased intra-cellular calcium concentration and phosphoinositide breakdown without affecting those caused by thrombin (01 units mL^?1), collagen (10 μg mL^?1), arachidonic acid (100 μm ) and U46619 (1 μm ). Bakkenolide G shifted the concentration-response curves of PAF-induced platelet aggregation parallel to the right; the Schild plot slope and the pA₂ value were 1.31 ± 0.31 and 6.21 ± 0.75, respectively. Moreover, bakkenolide G concentration-dependently competed with [³H]PAF binding to platelets, with an IC50 value of 2.5 ± 0.4 μm . These data strongly indicate that bakkenolide G is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.