Pharmacologic profile of BN 50739, a new PAF antagonist in vitro and in vivo

The effect of a new PAF antagonist BN 50739 was studied on PAF-induced [3H]-serotonin release from washed rabbit platelets in vitro and on PAF-induced hypotension in vivo. BN 50739 competitively inhibited PAF-induced [3H]-serotonin release from the platelets in a dose-dependent manner. In the presence of 4, 10 and 50 nM of BN 50739, the concentration of PAF inducing 50% maximal [3H]-serotonin release from the platelets (EC50) increased from 2.15 nM to 5.10, 45.10 and 900 nM, respectively. The IC50 of BN 50739 for PAF (10 nM) induced [3H]-serotonin release was 3.67 nM. Under the same experimental condition, the IC50s of BN 50726, BN 50730, BN 50741, WEB 2086, SRI 63-441 and BN 52021 were 5.40, 4.61, 6.88, 5.98, 40.90 nM and 14.90 microM, respectively. PAF-induced hypotension in conscious rats was also inhibited dose-dependently by i.p. pretreatment of BN 50739 (3 and 10 mg/kg). PAF-induced hypotension was diminished both in magnitude and duration in rats pretreated with BN 50739. These data taken together indicate that BN 50739 is a most potent PAF antagonist in vitro and in vivo.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Alpha-bulnesene, a novel PAF receptor antagonist isolated from Pogostemon cablin

Alpha-bulnesene is a sesquiterpenoid isolated from the water extract of Pogostemon cablin. It showed a potent and concentration-dependent inhibitory effect on platelet-activating factor (PAF) and arachidonic acid (AA) induced rabbit platelet aggregation. In a radioligand binding assay for the PAF receptor, alpha-bulnesene competitively inhibited [(3)H]PAF binding to the PAF receptor with an IC(50) value of 17.62+/-5.68microM. alpha-Bulnesene also dose-dependently inhibited PAF-induced intracellular Ca(2+) increase in fluo-3/AM-loaded platelets (IC(50) values of 19.62+/-1.32microM). Furthermore, alpha-bulnesene inhibited AA-induced thromboxane B(2) (TXB(2)) formation and prostaglandin E(2) (PGE(2)) formation. These results indicate that the inhibitory effect of alpha-bulnesene on platelet aggregation was due to a dual activity; specifically the chemical blocked PAF-induced intracellular signal transduction and interfered with cyclooxygenase activity, which resulted in a decrease in thromboxane formation. This study is the first to demonstrate that alpha-bulnesene is a PAF receptor antagonist as well as an anti-platelet aggregation agent.     

Modulating effects of adenosine on the process of human platelet activation]

The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.     

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

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.     

Phorbol diester enhances calcium ionophore A23187-induced [3H]acetate incorporation into platelet-activating factor in murine macrophages: predominant incorporation into 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine

Pretreatment of macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to enhance the release of arachidonic acid from cell phospholipids in response to agonist stimulation. This study describes the ability of TPA to also alter calcium ionophore A23187-induced incorporation of [3H]acetate into platelet activating factor (PAF). Cultured murine peritoneal macrophages were preincubated with [3H]acetate (25 muCi) and TPA (10 ng/ml) for 10 min, and subsequently incubated with 0.1 microM A23187 for 0.5-10 min. Buffer and cells were then extracted and PAF resolved by normal-phase HPLC. Sequential exposure to TPA and A23187 resulted in a greatly enhanced incorporation (11,861 dpm/10(6) cells) of [3H]acetate into PAF compared to TPA alone, which did not significantly influence [3H]acetate incorporation into PAF, and 0.1 microM A23187, which induced minimal incorporation (688 dpm/10(6) cells). Macrophage-produced [3H]PAF was resolved by HPLC, extracted, treated with phospholipase-C, and acetylated to facilitate quantitation of 1-O-alkyl-2-acetyl-GPC (PAF) from 1-O-acyl-2-acetyl-GPC (acylPAF). A23187 alone (1 microM) produced 72% 1-O-acyl-2-[3H]acetyl-GPC, and A23187 (0.1 microM) following TPA pretreatment produced 81% 1-O-acyl-2-[3H]acetyl-GPC. Less than 2% of the radioactivity of acylPAF was in the acyl moiety. These data support a role for protein kinase C in modulating agonist-induced PAF synthesis. The results also suggest that acetyltransferase of murine macrophages does not possess specificity for 1-O-alkyl-2-lyso-GPC, and that availability of specific species of lyso-phospholipid may determine the type of PAF produced.     

1-O-alkylglycerols improve boar sperm motility and fertility.

1-O-alkylglycerols are naturally occurring ether lipids with potent biological activities. They may interfere with lipidic signaling, and they amplify platelet-activating factor (PAF) biosynthesis in a monocyte cell line. The PAF is produced by mammalian sperm and is an important activator of sperm motility. The aim of this study was to evaluate the effect of in vitro treatment of boar spermatozoa with natural 1-O-alkylglycerols (10 microM) on 1) boar sperm motility; 2) production of PAF and its metabolite, lyso-PAF, by spermatozoa; and 3) fertility in artificial inseminations of breeding sows. Using a computer-assisted spermatozoa analyzer, we found that 1-O-alkylglycerols increased percentage motility as well as velocity parameters after 24 h. These effects were partially or totally reversed by the PAF receptor-antagonist SR 27417. After [3H]-1-O-alkylglycerol incubation with boar spermatozoa, we identified [3H]lyso-PAF by high-performance liquid chromatography. Production of PAF and lyso-PAF was measured with a biological assay using [3H]serotonin release from rabbit platelets. 1-O-alkylglycerols significantly increased lyso-PAF production but had no effect on PAF production. The effect of 1-O-alkylglycerols on fertilization was also evaluated in industrial breedings: 1-O-alkylglycerol-treated or untreated semen dilutions were alternately used for artificial inseminations of sows on 12 farms. 1-O-alkylglycerol treatment increased the number of farrows but had no effect on the mean size of the litters. This study demonstrates that 1-O-alkylglycerol treatment of boar spermatozoa in vitro improves their motility and fertility, and it suggests that this effect is related to PAF metabolism and function in boar spermatozoa.     

Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.

When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.     

Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.     

Decrease in ovarian platelet-activating factor during ovulation in the gonadotropin-primed immature rat

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.     

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium.

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.     

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium.

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.     

(+/-)-trans-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989), a novel, potent PAF receptor antagonist

The title compound, L-659,989, is a highly potent, competitive, and selective antagonist of the binding of [3H]PAF to its receptors in platelet membranes from rabbits and humans. It exhibits equilibrium inhibition constants for PAF binding of 1.1 nM (rabbit) to 9.0 nM (human), values that are at least 1-2 orders of magnitude lower than those of other PAF antagonists tested. L-659,989 potently inhibits PAF-induced aggregation of rabbit platelets and degranulation of rat (ED50 4.5 nM) and human (ED50 10 nM) neutrophils. L-659,989 inhibits PAF-induced extravasation and lysosomal enzyme release in rats, and is active orally in female rats (ED50 0.2 mg/kg) with an extraordinary oral duration of action of 12 to 16 hours at 1.0 mg/kg p.o.     

Verapamil: influence upon basal and stimulated rat growth hormone and prolactin release in vitro

Verapamil is an organic calcium antagonist which is believed to prevent the passage of calcium (Ca2+) across the cell membrane into the cell. In a rat pituitary perifusion-immunoprecipitation system, verapamil (50 microM) prevents the inhibitory effect of increased extracellular Ca2+ (5.4 mM) on basal and stimulated release of stored, prelabeled [3H]GH and [3H]PRL. [3H]GH release from pituitary explants perifused in standard medium (GIBCO Minimum Essential Medium: 1.8 mM Ca2+) is transiently increased by 50 microM verapamil while [3H]PRL release is suppressed. With continued exposure to 50 microM verapamil, [3H]GH release rates fall below (89.8 +/- 2.1% of base) preverapamil levels while [3H]PRL release rates simply remain suppressed (48.2 +/- 7.3% of base). With 250 microM verapamil, poststimulatory inhibition of [3H]GH release occurs more quickly, and after its withdrawal rebound release of both GH and PRL occur. Inhibition of [3H]GH release by 25 nM somatostatin (SRIF) and post-SRIF rebound [3H]GH release is not prevented by 50 microM verapamil. The early, rapid [3H]GH release phase of 1 mM dibutyryl cyclic AMP (dbcAMP) stimulation is potentiated by verapamil pretreatment, but only if the verapamil is continued during dbcAMP stimulation. Potassium (21 mM K+)-stimulated release of both 3H-labeled hormones is inhibited after similar pretreatment 50 microM verapamil. Conclusions: (a) verapamil antagonizes the inhibitory effects of increased extracellular Ca2+ on basal or dbcAMP-stimulated [3H]GH and [3H]PRL release; (b) in standard medium (1.8 mM Ca2+), 50 microM verapamil increases basal [3H]GH release suggesting either a direct effect or an antagonism of 1.8 mM extracellular Ca2+; (c) although verapamil-sensitive Ca2+ movement is not necessary for dbcAMP stimulation of [3H]GH release, verapamil potentiates dbcAMP-stimulated release; (d) because verapamil also inhibits K+-stimulated [3H]GH and [3H]PRL release, these observations support previous suggestions that K+- and dbcAMP-stimulated rapid hormone release occurs from different intracellular sites; and (e) because verapamil does not prevent any phase of SRIF action and since these two agents differentially alter K+- and cAMP-stimulated release, their mechanisms of action must partially differ.