Myoelectric intestinal disturbances inEscherichia coli endotoxic shock in rats. Involvement of platelet-activating factor

Administration of BN 52021 (50 mg/kgi.v.), a specific antagonist of platelet-activating factor (PAF), significantly reduced the intestinal myoelectric disturbances induced byE. coli endotoxin injection (50 μg/kgi.v.) by 62%. Thus, PAF may be involved in the intestinal motor alterations observed in endotoxic shock. When given in combination with indomethacin (10 mg/kgi.p.), BN 52021 inhibited endotoxic shock intestinal disturbances. Indomethacin alone also reduced PAF induced (25 μg/kgi.p.) disruption of migrating myoelectric complexes. Endotoxins may act on intestinal motilityvia release of endogenous PAF and prostaglandins, the effects of PAF being mediated through the release of prostaglandins. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor (PAF) receptor antagonists inhibit arachidonic acid induced platelet aggregation in rabbit whole blood

The potency of several platelet-activating factor (PAF) receptor antagonists was measured by observing their inhibitory effects against PAF induced platelet aggregation. Their selectivity was assessed by monitoring their effect on platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP). The antagonists inhibited platelet aggregation induced at the PAF EC₅₀ (0.023 μM) with the following rank order of potency: WEB 2086> WEB 2170> SRI 64–412> SRI 63–675> BN 52021>kadsurenone> SRI 63–441> alprazolam. While the antagonists had no inhibitory effect at the EC₅₀ for ADP (10 μM), they did inhibit platelet aggregation induced at the EC₅₀ for AA (55 μM). However, there was considerable variability in the slope of the inhibitory response and the relative potency of each antagonist against PAF induced platelet aggregation as compared to AA induced platelet aggregation. The antagonist IC₅₀ (μM) against PAF and AA were as follows, with those that showed significantly different (p<0.01) slopes indicated by an asterisk: SRI 63-441^* (3.8, 15.1); SRI 63-675 (1.4, 36.2); SRI 64-412 (0.5, 10.5); BN 52021^* (2.4, 58.9); kadsurenone^* (2.8, 28.3); alprazolam^* (10, 25); WEB 2086 (0.055, 0.220), and WEB 2170 (0.107, 0.534). Therefore, in rabbit whole blood the antagonists were potent, although not completely selective, inhibitors of PAF induced platelet aggregation. These results suggest that the mode of action of PAF and AA induced platelet aggregation may share some common features. However, since the slope of the inhibitory response against PAF and AA for some antagonists differed, mechanistic differences in their action appear to exist. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellulary. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I₂ (PGI₂), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI₂) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. Based on a paper presented at the Third International Conference on Platelet Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of the hetrazepinoic platelet-activating factor antagonist bepafant (WEB 2170) in models of active and passive anaphylaxis in mice and guinea pigs

The selective hetrazepinoic platelet-activating factor (PAF) antagonist WEB 2170 (Bepafant) was used to study the pathophysiological role of PAF in several models of anaphylaxis in mice and guinea pigs. In actively sensitized mice, the PAF antagonist WEB 2170 (1.0–10 mg/kgp.o.) protected mice from anaphylactic death in a dosedependent manner when the anaphylactic response was potentiated by the beta-receptor antagonist propranolol. When active anaphylaxis in guinea pigs was induced intravenously by 100 mg/kg ovalbumin (OA) in the presence of small doses of the antihistamine mepyramine, additional treatment with oral or intravenous WEB 2170 protected the guinea pigs from anaphylactic death. Also, the remaining anaphylactic bronchoconstriction and blood pressure changes (including anaphylactic hypotension) were attenuated. When guinea pigs were passively sensitized with a heterologous antibodyvia the tracheal route and then challenged by ovalbumin (100 mg/kgi.v.) 24 hr after sensitization in the presence of 0.003 mg/kgi.v. mepyramine, additional treatment with tracheal WEB 2170 at 0.1–1 mg/kg protected the guinea pigs dosedependently not only from anaphylactic death but also from a further decrease of respiratory flow and changes of blood pressure. Increased levels of PAF-like activity (20–50 ng PAF/whole lung) were detected in lungs removed from antigen-challenged animals. The results suggest a causative role for PAF in active and passive anaphylaxis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Discovery and preliminary pharmacology of Sch 37370, a dual antagonist of PAF and histamine

From a series of amide analogs of the histamine H₁ antagonist, azatadine, a potent, orally active, dual platelet-activating factor (PAF) and histamine antagonist, Sch 37370, namely 1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo-[5,6]cyclohepta[1,2-b]pyridin-11-ylidine)piperidine, was discovered. Sch 37370 selectively inhibits PAF-induced aggregation of human plateletsin vitro (IC₅₀=0.6 μM), andin vivo inhibits PAF- and histamine-induced bronchospasm in guinea pigs with ED₅₀ values of 6.0 and 2.4 mg/kg p.o., respectively. Sch 37370 is expected to be more efficacious than single mediator antagonists in allergic diseases, such as asthma. Based on papers presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Phorbol ester stimulates PAF synthesisvia the activation of protein kinase C in rat leukocytes

When rat pleural mononuclear leukocytes were stimulated with 1 μM phorbol myristate acetate (PMA), platelet-activating factor (PAF)-like activity was detected in the supernatant and the cellular fractions of the incubation mixture, as measured by rabbit platelet aggregation. C₁₆PAF activity peaked at 30 min in both fractions. Acetyltransferase activity in the microsomal fraction of the stimulated cells also increased rapidly and showed a peak at 10 min. A protein kinase C inhibitor, staurosporine, and an inhibitor of phospholipase A₂,p-bromophenacylbromide, inhibited stimulated PAF formation in both fractions. Staurosporine also inhibited PMA induced acetyltransferase activity. The data suggest that PMA stimulates PAF synthesis by the remodeling pathway in rat pleural cells through activation of both phospholipase A₂ and acetyltransferase, and that the acetyltransferase, in turn, may be activated through activation of protein kinase C. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Reversible or irreversible modification of [3H]PAF binding on rabbit platelet membranes differentiates various PAF receptor antagonists

[³H]Platelet-activating factor (PAF) binding to rabbit platelet membranes was examined before and after 20 min preincubation at 25°C in the presence of PAF, lysoPAF, or of five different PAF receptor antagonists (L 652731, BN 52021, WEB 2086, BN 52111 and BN 52115). When platelet membranes were not washed after preincubation with PAF or PAF antagonists, no significant specific binding of [³H]PAF was observed, which suggests full occupancy of the binding sites. When membranes were extensively washed, full recovery of specific [³H]PAF binding was attained with L 652731 and partial recoveries (60%, 55% and 30%) were reached with BN52021, WEB 2086 and PAF, respectively; no recovery was seen with the dioxolanes BN 52111 and BN 52115. Scatchard analysis of the binding data indicated that no significant change in the dissociation constant (K_d) and maximum number of binding sites (B_max) occurred after preincubation of platelet membrane with L 652731, whereas a reduction of B_max was observed when PAF and BN 52021 were measured. When platelet membranes were preincubated with WEB 2086, B_max and K_d significantly increased. The data suggest differing binding properties for PAF and the PAF antagonists. Some of the PAF antagonists may tightly bind to the PAF receptor site(s) and/or irreversibly modify or downregulate PAF recognition sites. Our results also suggest that the interaction of PAF receptor antagonists with PAF receptor can be divided into at least two components, namely a reversible component and an irreversible one. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Synthesis and antimalarial activities of a diverse set of triazole-containing furamidine analogues

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper‐catalyzed “click chemistry” was used for the synthesis of 1,4‐ and 4,5‐substituted triazoles, aryl magnesium acetylide reagents for the 1,5‐substituted triazoles, with a thermal dipolar addition reaction employed for the 2,4‐substituted triazoles. In vitro antimalarial activity against two different PfCRT‐modified parasite lines (Science 2002 , 298, 210–213) of Plasmodium falciparum and inhibition of hemozoin formation were determined for each compound. Several diamidines with potent nanomolar antimalarial activities were identified, and selected molecules were resynthesized as their diamidoxime triazole prodrugs. One of these prodrugs, OB216, proved to be highly potent in vivo with an ED₅₀ value of 5 mg kg^?1 (po) and an observed 100 % cure rate (CD₁₀₀) of just 10 mg kg^?1 by oral (po) administration in mice infected with P. vinckei.     

Inhibition by cardiolipins of platelet-activating factor-induced rabbit platelet activation

Evidence is presented that cardiolipin, a naturally occurring phospholipid, inhibits the aggregatory effect of platelet-activating factor (paf) on rabbit plateletsin vitro. Bovine heart cardiolipin was shown to inhibit the aggregation of washed rabbit platelets induced by 1×10⁻¹⁰ M and 2×10⁻¹⁰ M paf with IC₅₀ values (doses for half-maximal inhibition) of 8.4±0.8×10⁻⁷ M and 2.6±0.6×10⁻⁶ M, respectively. Phosphonocardiolipin was also able to inhibit platelet aggregation induced by 1× 10⁻¹⁰ M paf with an IC₅₀ value of 3±1×10⁻⁷M. Both compounds, in concentrations up to 1×10⁻⁵ M, were unable to aggregate washed rabbit platelets and failed to inhibit the aggregation induced by 0.9 and 1.8 μM adenosine diphosphate or 0.2–1.0 μM arrchidonic acid. By contrast, the acetylated derivative of cardiolipin exerted an aggregatory effect on aspirin-treated rabbit platelets in the presence of creatine phosphate/creatine phosphokinase. This aggregation was inhibited by the specific paf antagonists BN 52021 and WEB 2086. Also, platelets treated with acetyl-cardiolipin were insensitive to the aggregatory effect of paf. Phosphatidic acid, phosphatidylglycerol,bis(dipalmitoylglycero)phosphate and their phosphono analogues were totally inactive. Similar data were obtained when platelet-rich plasma was used instead of washed rabbit platelets. Our results support the hypothesis that the effect of cardiolipin is mediated through specific paf receptors that act on the rabbit platelet membrane.     

Effect of the selective PAF antagonist SM-10661 on an asthmatic model. 1. Effect on passive anaphylactic bronchoconstriction in guinea pigs

The effect of SM-10661, a selective antagonist of platelet-activating factor (PAF), on passive anaphylactic bronchoconstriction was examined in guinea pigs. A challenge of ovalbumin to passively sensitized guinea pigs induced bronchoconstriction, which peaked at 4 min. When SM-10661 was administered intravenously 2 min before ovalbumin challenge, bronchoconstriction was inhibited dose-dependently with an ID₅₀ of 68 mg/kg. In guinea pigs pretreated with 15 μg/kg mepyramine which is a suboptimal dose, antigen-induced bronchoconstriction peaked at 4–6 min, but was inhibited by SM-10661 with an ID₅₀ of 21 mg/kg. When guinea pigs were pretreated intravenously with 2.5 mg/kg mepyramine, 1 mg/kg indomethacin and 0.01 mg/kg propranolol, the antigen-induced bronchoconstriction peaked at 6 min. SM-10661 inhibited the response with an ID₅₀ of 45 mg/kg. Histamine- and leukotriene D₄-induced bronchoconstrictions were unaffected by up to 100 mg/kg SM-10661. Ovalbumin challenge of minced lungs from passively sensitized guinea pigs triggered the release of leukotrienes and histamine. SM-10661 had no effect on the antigen-induced release of peptide leukotrienes or histamine up to 10⁻⁴ M. These results indicate that SM-10661 may be a useful tool to investigate the role of PAF in antigen-induced anaphylactic bronchoconstriction. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of fortified milk on lyso‐platelet‐activating factor acetyltranferase and lipoprotein‐associated phospholipase A2 in hypercholesterolemic adults

Hypercholesterolemia is associated with subclinical inflammation, characterised by elevated proinflammatory mediators. Lyso‐platelet‐activating factor acetyltransferase (lyso‐PAF AT) and lipoprotein‐associated phospholipase A₂ (Lp‐PLA₂) are two key metabolic enzymes of platelet‐activating factor (PAF), a potent inflammatory lipid mediator. Little information is available concerning the efficacy of a dietary intervention on the metabolism of PAF. The objective of the study was to evaluate the effect of fortified milk on the activity of these enzymes. Forty‐three adults (mean age 49.8 ± 8.1 years) with body mass index <35 kg/m², and total cholesterol >200 but <310 mg/dL were randomised to two groups; (i) intervention group received 500 mL/day (two glasses) of a low‐fat milk fortified with phytosterols, linoleic and alpha linolenic acids, vitamin C, vitamin E, vitamin A, vitamin B6, vitamin B12, folic acid, magnesium and selenium (n = 22), and (ii) placebo group received 500 mL/day of a conventional low‐fat milk (n = 21) for 3 months. Outcome measures were the activities of lyso‐PAF AT from leukocytes and serum Lp‐PLA₂ determined with established methods. None of the activities changed significantly during the study in the intervention group, lyso‐PAF AT (95% confidence interval: ?1.7, 2.3 nmol/min/mg; p = 0.246), and Lp‐PLA₂ (?7.8, 5.8 nmol/min/mL, p = 0.591). No difference was observed between the two groups. In conclusion, daily intake of two glasses of phytosterols, antioxidants, linoleic and linolenic acids via fortified milk for three months had no effect on the activity of either lyso‐PAF AT or Lp‐PLA₂. Practical applications: Platelet‐activating factor (PAF) was the first intact phospholipid known to have messenger functions in which the signaling results from the molecule binding to specific receptors on the plasma membrane or other membranes of the cell. It has a number of pro‐inflammatory properties, and affects several critical points of atherogenesis including thrombosis, inflammation, and oxidation. Fortification of milk with nutrients that possess anti‐inflammatory properties and administration to adults with elevated blood cholesterol could provide a means to controlling inflammatory process through the synthesis and degradation of PAF in a population group at risk for cardiovascular morbidity and mortality.     

Differential inhibition by castanospermine of various insect disaccharidases

The indolizidine alkaloid, castanospermine (1,6,7,8-tetrahydroxy-octahydroindolizidine—a stereochemical mimic of glucose found in the Australian legumeCastanospermum australe), differentially inhibited cellobiose, lactose, maltose, sucrose, and trehalose hydrolyzing enzymes from a broad taxonomic spectrum of insects (19 species from 12 different families). It was a potent inhibitor of cellobiase activity of all insects tested (50% inhibition at <3.2 × 10^–5 M castanospermine). With one exception, it also inhibited lactase activity of all insects examined. Only in the sap-feeding Homoptera did castanospermine inhibit all disaccharidase activities assayed. Trehalase activity of the Lepidoptera and Diptera was generally inhibited by castanospermine, whereas inhibition of trehalase activity of the Coleoptera by castanospermine was exiguous or not detectable. Castanospermine was a significant feeding deterrent towards pea aphids,Acyrthosiphon pisum, with an ED₅₀ of 1 × 10^–4 M in artificial diets. Two compounds stereochemically related to castanospermine, deoxynojirimycin and 6-epicastanospermine, were each slightly active at deterring the feeding of green peach aphids,Myzus persicae, (ED₅₀=2.5 × 10^–3 M) and greenbugs,Schizaphis graminum (ED₅₀=5 × 10^–3 M), respectively. Among the insects studied there was no distinct relationship between enzyme inhibition and adaptation to host plants containing castanospermine or other toxic alkaloids.     

L-659,989: A useful probe in the detection of multiple conformational states of PAF receptors

L-659,989 is a potent, specific and competitive plateletactivating factor (PAF) receptor antagonist. The 2,5-tritium labeled L-659,989, similar to [³H]PAF, specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (K_D) of 1.60 (±0.20) nM in 10 mM MgCl₂. However, guanosine 5′-triphosphate (GTP) and several cations affect the specific binding of [³H]PAF and of [³H]L-659,989 to rabbit platelet membranes in different ways. K⁺, Mg²⁺, Ca²⁺ and Mn²⁺ potentiate the specific binding of both ligands. Na⁺ and Li⁺ inhibit the specific [³H]PAF binding, but enhance the binding of [³H]L-659,989; GTP reduces the [³H]PAF binding but has no effect on the binding of [³H]-L-659,989. Ni²⁺ inhibits the [³H]L-659-989 binding, but has no effect on the binding of [³H]PAF. In the presence of 150 mM NaCl, [³H]L-659,989 exhibits identical K_D and detectable binding sites (B_max) values as those in the presence of 10 mM MgCl₂, while K _d And B_max values of [³H]PAF are dramatically reduced in the presence of 150 mM NaCl compared to those in 10 mM MgCl₂. These results suggest the existence of multiple conformational states of the PAF specific receptor and that PAF and L-659,989 bind differently to those states. In the presence of 150 mM NaCl and 1 mM GTP, receptors appear to exist in a single conformational state with an equilibrium dissociation constant (K_B) of 0.93 μM for PAF as derived from the Schild plot. In isolated rabbit platelets pretreated with 10 μM ETH 227, a Na⁺-specific ionophore, the detectable [³H]PAF binding sites drop from 260 to 100 binding sites per platelet, but the binding sites for [³H]L-659,989 remain roughly the same. The Na⁺ binding sites which modulate the conformation of PAF receptors are therefore protected from extracellular Na⁺ until ionophore is added, and are probably located on the cytoplasmic side of the plasma membrane. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Toxicity of three F‐substituent aromatics in anaerobic systems

BACKGROUND: The commercial use of organofluorine compounds has dramatically increased over the past few years. However, little information has been reported on the potential toxicity of organofluorine compounds to anaerobic digestion processes. In this work, the effects of 4‐fluorophenol (p‐FP), 4‐fluorobenzoic acid (p‐FB) and 4‐fluoroaniline (p‐FA) on methanogenesis and biodegradability were evaluated using sucrose‐fed systems. RESULTS: The anaerobic biodegradation of three test compounds was not observed in the study. Adsorption of p‐FP, p‐FB and p‐FA to the sludge fitted the linear model well (r² > 0.94). The partition coefficient K_d was 25 L kg^?1 for p‐FP, 16 L kg^?1 for p‐FB and 26 L kg^?1 for p‐FA. Both methanogensis and hydrolysis acidification were inhibited in the presence of three test compounds. The half maximal inhibitory concentrations (IC₅₀s) of methanogenic activity were 339, 1390 and 1907 mg L^?1 for p‐FP, p‐FB and p‐FA, respectively. A significant linear correlation (R² = 0.99, P < 0.05) was obtained between the half maximal inhibitory concentrations and the most negative atomic charges of molecules (q^?) of the three F‐substituent aromatics. CONCLUSIONS: Three F‐substituent aromatics had specific effects on methanogensis, hydrolysis acidification and syntrophic cooperation in anaerobic systems. Copyright © 2012 Society of Chemical Industry     

PAF inhibitory activity of diketopiperazines: Structure-activity relationships

FR900452, a natural product isolated from the culture broth ofStreptomyces phaeofaciens No. 7739, was found to inhibit PAF-induced rabbit platelet aggregation with an IC₅₀ of 3.7×10⁻⁷M. FR900452, 1-methyl-3-[1-[5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-ylidene)-2-piperazinyl]ethyl]-2-indoline, has an oxocylopentylidene group incorporated as a vinylogous amide in a diketopiperazine skeleton. This unique structure led us to synthesize diketopiperazine derivatives, 3-arylalkyl-6-substituted-piperazine-2,5-diones. their observed PAF inhibitory activity suggest that the D-D configuration of diketopiperazine is an important factor for anti-PAF activity and that the hydrophobic aromatic portion may play a specific role in the binding of the diketopiperazine to the PAF receptor. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Ether Lipids Tokyo, Japan, May 1989.     

Chemical synthesis and physiological activity of sulfonium analogues of platelet activating factor

Phosphatidylsulfocholine (PSC), the sulfonium analogue of phosphatidylcholine (PC), occurs naturally in some diatoms. The replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ results in an increase in the polar head group size in PSC relative to that of PC, consistent with the observed increase in permeability of PSC bilayers towards urea. It was of interest to see whether replacement of the −N⁺(CH₃)₃ group in platelet activating factor (PAF) by an −S⁺(CH₃)₂ group leads to any change in platelet aggregation or other physiological activity. Synthesis of the sulfonium analogue of PAF was carried out by suitable modifications of known procedures. The PAF-sulfonium analogue was found to have almost the same platelet aggregating activity as PAF itself, in the concentration range 1–20 μM, but a much lower activity in the range 0.01–1 μM. The analogue had little or no effect on the platelet aggregation activity of PAF when added in the concentration range 0.01–1 μM and had about half the hypotensive activity of PAF towards hypertensive CDF male rats. The sulfonium analogue, however, was much more cytotoxic to HL-60 cells than PAF itself, in the concentration range 0–15 μM; replacement of the acetate group by a benzyl group increased the cytotoxicity to the level of that of the methoxy analogue of PAF. Thus, replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ group in the polar head group region of PAF results in a relatively small change in its platelet aggregation activity and a decrease in its hypotensive activity, but greatly increases its antitumor activity. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 8–12, 1989.     

Calcium channel blockade inhibits platelet activating factor production by human umbilical vein endothelial cells

An increase in intracellular calcium level is an important signal in the regulation of cellular responses under normal and pathological conditions. Because two key enzymes in the synthetic pathway of platelet activating factor (PAF), phospholipase A₂ and acetyltransferase, are calcium dependent, we hypothesized that calcium channel blockade may inhibit agonist-induced PAF synthesis. Primary cultures of human umbilical vein endothelial cells (EC), pre-incubated with [³H]acetate, were exposed to thrombin (5 U/mL) and PAF production was quantitated by incorporation of radiolabel into the EC lipid fraction co-migrating with exogenous PAF in thin-layer chromatography. The effect of pre-incubation with calcium channel blockers (verapamil, diltiazem, 10⁻⁴ M) or buffer was determined. Results (triplicate experiments,^*P<0.05 vs buffer, † P<0.05 vs thrombin) demonstates that pre-incubation with calcium channel blocker markedly inhibits thrombin-induced PAF production (verapamil: buffer 273±122, thrombicin 10,735±1524^*, thrombin+verapamil 178±91 † cpm/plate; diltiazem: buffer 1097±581, thrombin 15,283±2661^*, thrombin+diltiazem 280±56 † cpm/plate). The effect of dialtiazem was dosedependent (% inhibition: 10⁻⁷ M, 46%; 10⁻⁵ M, 60%; 10⁻⁴ M, 98%). Diltiazem also inhibited bradykinin (10⁻⁸ M) induced PAF synthesis. In calcium-free medium or in the presence of LaCl₃ (10⁻³ M), the PAF response of EC to thrombin was blunted (buffer 582±360, thrombin 5394±1069, thrombin+calcium free medium 1055 ±571, thrombin+LaCl₃ 1271±58 cpm/plate). We conclude that calcium channel blockers present agonist-induced PAF synthesis, possibly by preventing cellular calcium influx and activation of PAF synthetic enzymes. We speculate that this mechanism may underlie, at least in part, the beneficial effect of calcium channel blockade under various pathological conditions. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Synthesis and Enantioselective Pharmacokinetic/Pharmacodynamic Analysis of New CNS-Active Sulfamoylphenyl Carbamate Derivatives

We recently reported a new class of carbamate derivatives as anticonvulsants. Among these, 3-methylpentyl(4-sulfamoylphenyl)carbamate (MSPC) stood out as the most potent compound with ED₅₀ values of 13 mg/kg (i.p.) and 28 mg/kg (p.o.) in the rat maximal electroshock test (MES). 3-Methylpropyl(4-sulfamoylphenyl)carbamate (MBPC), reported and characterized here, is an MSPC analogous compound with two less aliphatic carbon atoms in its structure. As both MSPC and MBPC are chiral compounds, here, we studied the carbonic anhydrase inhibitory and anticonvulsant action of both MBPC enantiomers in comparison to those of MSPC as well as their pharmacokinetic properties. Racemic-MBPC and its enantiomers showed anticonvulsant activity in the rat maximal electroshock (MES) test with ED₅₀ values in the range of 19–39 mg/kg. (R)-MBPC had a 65% higher clearance than its enantiomer and, consequently, a lower plasma exposure (AUC) than (S)-MSBC and racemic-MSBC. Nevertheless, (S)-MBPC had a slightly better brain permeability than (R)-MBPC with a brain-to-plasma (AUC) ratio of 1.32 (S-enantiomer), 1.49 (racemate), and 1.27 (R-enantiomer). This may contribute to its better anticonvulsant-ED₅₀ value. The clearance of MBPC enantiomers was more enantioselective than the brain permeability and MES-ED₅₀ values, suggesting that their anticonvulsant activity might be due to multiple mechanisms of action.     

The metabolism of 1-acyl-PAF in rabbit platelets and its possible interaction with PAF

The metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF), a naturally occurring analogue of platelet activating factor (PAF), was investigated in rabbit platelets. Our studies showed that 1-acyl-[³H]PAF (1-palmitoyl-2-acetyl-sn-glycero-3-phospho[N-methyl-³H]-choline) was converted by platelets into phosphatidyl-[³H]choline ([³H]PC) in a time-dependent fashion. The formation of [³H]PC occurred at a rate similar to that observed when lyso-[³H]PC (palmitoyl-sn-glycero-3-phospho[N-methyl-³H]choline) was used as substrate. In addition, a time-dependent increase in the level of water-soluble radioactivity was observed during the incubation of platelets with either 1-acyl-[³H]PAF or lyso-[³H]PC. This increase was parallel to the formation of [³H]PC and was not observed in the presence of [¹⁴C]PAF (1-octadecyl-2-acetyl-sn-glycerol-3-phospho[N methyl-¹⁴C]choline). Analysis by thin-layer chromatography showed that the soluble radioactivity was mainly associated with glycerophosphocholine (GPC). On the other hand, the preincubation of platelets with phenylmethylsulfonyl fluoride, an inhibitor of the acetylhydrolase, reduced the hydrolysis of 1-acyl-[³H]PAF to [³H]GPC with a concomitant accumulation of radioactivity in 1-acyl-PAF. These findings suggest that 1-acyl-PAF is converted into PC through deacetylation-reacylation with lysoPC as an obligatory intermediate. The findings also indicate that the lysoPC resulting from 1-acyl-PAF is either reacylated to phosphatidylcholine (PC) or hydrolyzed to GPC by lysophospholipase. Finally, we showed that the stimulation of platelets with PAF led to a time- and concentration-dependent increase in the conversion of 1-acyl-[³H]PAF to [³H]PC. The stimulatory effect of PAF was not observed when platelets were lysed before incubation, suggesting that PAF enhances the metabolism of 1-acyl-PAF, probably by accelerating its translocation through the plasma membrane.