A comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis

The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B4 (LTB4) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-14C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thin-layer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxis of PMNs towards LTB4 was measured in a modified Boyden chamber. Timegadine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10(-5) M), and of chemotaxis (IC50 3 x 10(-4) M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.     

Modulatory effect of bucillamine (SA96) on interleukin-1-and/or -2-induced proliferation of T lymphocytes

Bucillamine [SA96:N-(2-mercapto-2-methylpropanoyl)-L-cysteine], a synthetic SH compound, has recently been developed as remission-inducing agent for rheumatoid arthritis (RA), and its clinical usefulness for RA has been proved in Japan. Bucillamine suppressed the mitogen-induced proliferation of murine lymphocytes in vitro. The present study was undertaken to clarify the effect of bucillamine primarily on the release of interleukin (IL)-1 from monocytes and on the proliferation of T cells. Bucillamine significantly inhibited IL-1-induced thymocyte proliferation in a dose-dependent manner. And, bucillamine also inhibited IL-2-induced proliferation at the concentration of 1 x 10(-4) M, but augmented proliferation at the concentration of 1 x 10(-5) M. In contrast, D-penicillamine (an analogous SH compound to bucillamine) did not show any significant effect at similar concentrations.     

Arachidonic acid release and prostaglandin E2 synthesis as irritant index of surfactants in 3T6 fibroblast cultures

Surface-active agents are components of many drugs and cosmetics. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. As a result of membrane disintegration or enzymatic catalysis, it is released to the medium and [3H]AA metabolites can be measured by the scintillation technique. In order to validate the use of this test to predict the irritant and toxic effects of surfactants in vitro, various surfactants were examined using this methodology and the neutral red test. Benzalkonium chloride and sodium dodecylsulfate were more irritant and cytotoxic than amphoteric and non-ionic surfactants such as cocoamidopropylbetaine and Tween 80, respectively. The rank order of the toxicity/irritancy of these agents was similar to that given by other tests in vivo and in vitro. Moreover, our results confirm that the measurement of [3H]AA release in 3T6 fibroblast cultures is a sensitive assay of membrane damage.     

The generation and metabolism of leukotrienes in the ionophore-stimulated blood of normal and asthmatic subjects

The generation and metabolism of leukotrienes (LTs) 1341 C₄, D4, and E₄ were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB₄ and LTC₄ in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB₄, which are significantly higher in the AA blood than in N blood. Incubations of ³H-LTB₄ in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB₄ and 20-COON-LTB₄.LTC₄ is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p < 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC₄ elicits significantly greater amounts of LTD₄, and consistently higher levels of LTE₄, in the AA blood. Parallel incubations of 3H-LTC₄ in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB₄ and LTC₄ in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB₄ and LTC₄ generation and was equipotent in N and AA blood.     

Avena Rhealba inhibits A23187-stimulated arachidonic acid mobilization, eicosanoid release, and cPLA2 expression in human keratinocytes: potential in cutaneous inflammatory disorders

The aim of the present study was to examine the effects of Avena Rhealba (AR) oatmeal extract on the metabolism of arachidonic acid (AA) and eicosanoids as well as on the expression of cytosolic phospholipase A(2 )(cPLA(2)) in the human keratinocyte cell line HaCaT. For this purpose, we examined the effects of AR on basal and A23187-triggered release of [(3)H]-AA from phospholipids and on the production of [(3)H]-labeled metabolites of the cyclooxygenase (CO) and 5-lipoxygenase (LO) pathways. AR was found to inhibit A23187-triggered [(3)H]-AA mobilization from phospholipids (p<0.05) and production of [(3)H]-labeled metabolites of CO (p<0.05) and LO (p<0.05) pathways. These results suggest AR decreases PLA(2)-dependent mobilization of AA from phospholipids. A closer examination of the effects of AR on prostaglandin 6KF1alpha (6KPGF1alpha), the stable metabolite of prostacyclin, revealed dose-dependent inhibition of this AA metabolite. AR also decreased A23187- and tumor necrosis factor alpha-induced cPLA(2) overexpression, as shown by cPLA(2) immunodetection and mRNA expression. These results demonstrate the high potential of AR in the treatment of inflammatory diseases of the skin.     

Novel inhibitor of p38 MAP kinase as an anti-TNF-alpha drug: discovery of N-[4-[2-ethyl-4-(3-methylphenyl)-1,3-thiazol-5-yl]-2-pyridyl]benzamide (TAK-715) as a potent and orally active anti-rheumatoid arthritis agent

The p38 mitogen-activated protein (MAP) kinase has been implicated in the proinflammatory cytokine signal pathway, and its inhibitors are potentially useful for the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA) and inflammatory bowel disease. To develop a new drug for RA, we synthesized a novel series of 4-phenyl-5-pyridyl-1,3-thiazoles and evaluated their inhibition of p38 MAP kinase, lipopolysaccharide (LPS)-stimulated release of tumor necrosis factor-alpha (TNF-alpha) from human monocytic THP-1 cells in vitro, and LPS-induced TNF-alpha production in vivo in mice. During the course of the study, we found that these compounds risk the inhibition of cytochrome P450 (CYP) isoforms by coordination of the 4-pyridyl nitrogen with heme iron. We therefore investigated the effects of substitution at the 2-position of the pyridyl ring on the inhibitory activity of p38 MAP kinase and CYPs in more detail. As a result, N-[4-[2-ethyl-4-(3-methylphenyl)-1,3-thiazol-5-yl]-2-pyridyl]benzamide (8h, TAK-715) exhibited potent inhibitory activity in these assays (inhibition of p38alpha, IC50 = 7.1 nM; LPS-stimulated release of TNF-alpha from THP-1, IC50 = 48 nM; LPS-induced TNF-alpha production in mice, 87.6% inhibition at 10 mg/kg, po) and no inhibitory activity for major CYPs, including CYP3A4. This compound also showed good bioavailability in mice and rats and significant efficacy in a rat adjuvant-induced arthritis model. Compound 8h was selected as a clinical candidate and is now under clinical investigation for the treatment of RA.     

Effects of tenidap on intracellular signal transduction and the induction of proinflammatory cytokines: a review

Tenidap is a novel, once-daily antirheumatic drug which has shown promising results against rheumatoid arthritis in extensive clinical trials. It combines NSAID-like cyclooxygenase inhibition with suppression of the acute phase response. In macrophages, tenidap inhibits the lipopolysaccharide-induced synthesis of interleukins-I and -6, but it tends to potentiate the lipopolysaccharide-induced synthesis of tumor necrosis factor α, due to its cyclooxygenase inhibition. In macrophages, tenidap is a potent inhibitor of zymosan-induced responses, not only the induction of proinflammatory cytokines, but also arachidonate mobilization, protein phosphorylation, and inositol phosphate formation, possibly through interference with the receptor-mediated upregulation of phospholipase C. Tenidap also acts as an intracellular acidifier in many cell types, which may explain at least some of its other effects. Recent studies have indicated that, in addition to modulation of prostanoid and cytokine formation, tenidap has many other effects beneficial in rheumatic disease. It has been shown to inhibit bone resorption, neutrophil adhesion and degranulation, the interleukin-l-induced suppression of glycosaminoglycan synthesis, as well as the production of active metalloproteinases from chondrocytes.     

Increase of phosphatidylinositol arachidonic acid incorporation induced by mepacrine

In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.     

Inhibition of prostacyclin synthesis in cultured bovine aortic endothelial cells by vitamin K1

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation, is the predominant metabolite of arachidonic acid (AA) in endothelial cells derived from large blood vessels. Vitamin K1 (1-100 microM) inhibited the release of PGI2 and prostaglandin E2 (PGE2) by bovine aortic endothelial cells in culture, as measured by radioimmunoassay of 6-keto-prostaglandin F1 alpha (6-keto-PGE1 alpha) and PGE2. The conversion of exogenous AA to PGI2 was not affected by vitamin K1 as measured by radioimmunoassay and high performance liquid chromatography of radiolabeled AA metabolites. Similarly, vitamin K1 did not affect the conversion of prostaglandin H2 (PGH2) by in vitro enzyme systems. However, vitamin K1 inhibited the calcium ionophore A23187-induced release of [3H]AA from membrane phospholipids of bovine aortic endothelial cells. Inhibition of [3H]AA release from other cells of vascular origin was also observed after exposure to vitamin K1, but this effect was not observed in cells of non-vascular origin, including platelets. Therefore, vitamin K1 modulates the release of AA in vascular cells and thus inhibits the capacity of blood vessels to synthesize PGI2.     

Suppression of adjuvant arthritis by hesperidin in rats and its mechanisms

The citrus flavonoid hesperidin has been reported to possess a wide range of pharmacological properties. We have investigated the preventive and therapeutic effects of hesperidin on the development of adjuvant arthritis (AA), a rat model of rheumatoid arthritis (RA). Freund's complete adjuvant was used to induce AA in rats. Secondary paw swelling, polyarthritis index and histopathological assessment of ankle joints were used to evaluate the effects of hesperidin on AA rats. Concanavalin-A-induced T-lymphocyte proliferation and interleukin (IL)-2 production by splenocytes were measured using the MTT assay. Levels of IL-1, IL-6 and tumour necrosis factor (TNF)-alpha secreted by peritoneal macrophages (PM) were measured by RIA. Intragastric administration of hesperidin significantly attenuated secondary paw swelling and reduced the polyarthritis index of AA rats in a dose-dependent manner. In addition, hesperidin clearly ameliorated the pathological changes in AA rats. Hesperidin also restored the suppression of T-lymphocyte proliferation and IL-2 production, and downregulated production of IL-1, IL-6 and TNF-alpha by PM in AA rats. Our results suggest that hesperidin improves AA by downregulating the function of over-active macrophages and by up-regulating the activities of dysfunctional T lymphocytes. Hesperidin may therefore have therapeutic value for the clinical treatment of RA. Further research is required to clarify the detailed mechanisms of the protective effects of hesperidin on AA.     

A comparison of effects of sulfasalazine and its metabolites on the metabolism of endogenous vs. exogenous arachidonic acid

Sulfasalazine and, to a lesser extent, 5-aminosalicylic acid and N-acetyl-aminosalicylic acid, were found to block production of 5-hydroxy-6,8,11,14-eicosatetraenoic acid, leukotriene B4 (LTB4), and LTB4 stereoisomers from both exogenous and endogenous [14C]arachidonic acid (14C-AA) in ionophore A23187 (1 microgram/ml)-stimulated human neutrophils. Lipids were assessed by thin-layer chromatography and reverse-phase high-pressure lipid chromatography. Sulfasalazine blocked the synthesis of these metabolites from both exogenous and endogenous AA, but was more effective in blocking the metabolism of exogenous than endogenous AA. The IC50 for sulfasalazine in blocking the synthesis of LTB4 was 0.8 mM when exogenous AA was the substrate and 2.8 mM when endogenous AA was the substrate. N-Acetyl-aminosalicylic acid showed a similar pattern, but was less effective than sulfasalazine (IC50 for exogenous AA was 5.4 mM, and for endogenous AA was 8.0 mM). 5-Aminosalicylic acid had similar effects with an IC50 of 6.0 and 6.4 mM respectively. Sulfasalazine but not 5-aminosalicylic acid inhibited the incorporation of arachidonic acid into phospholipids and triglycerides. Sulfasalazine, but not its metabolites, inhibited the release of 14C-AA from membrane phospholipids in a dose-dependent manner (46.0% inhibition with 4 mM sulfasalazine). Sulfasalazine also blocked the metabolism of exogenously added LTB4 to 20-OH LTB4 and 20-COOH LTB4 with an IC50 of 2 mM. Our findings suggest that under physiologic conditions, with endogenous AA as a substrate, sulfasalazine acts as an inhibitor of lipoxygenase, of phospholipase A2 and of LTB4 metabolism, whereas 5-aminosalicylic acid and N-acetyl-aminosalicylic acid inhibit only lipoxygenase.     

Arachidonic acid metabolism of cultured peritoneal rat macrophages and its manipulation by nonsteroidal antiinflammatory agents

[3H]Arachidonic acid (AA) metabolism by cultured rat peritoneal macrophages (M phi) was examined. Ninety percent of incorporated [3H]AA localized in phospholipids (phosphatidylcholine, 30%; phosphatidylinositol, 23.9%; phosphatidylethanolamine, 23.7%) whereas 8.3% and 1.5% was found in the free fatty acid and neutral lipid fractions. 12-O-tetradecanoate phorbol-13-acetate (TPA) induced the reduction of label from phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine (42%, 46% and 47%, respectively) and an accumulation of label into free fatty acids (34%) or neutral lipid (61%) fractions. Simultaneously, 12% of the incorporated label was released into the medium as [3H]6-keto prostaglandin (PG)F1 alpha, [3H]thromboxane B2 (TxB2), [3H]PGE2, [3H]hydroxyheptadecanoic acid (HHT), [3H]15-hydroxyeicosatetraenoic acid (HETE), [3H]12-HETE and [3H]AA. Exposure to 0.3-3 microM indomethacin reduced TPA-induced label release into the medium which was distinguished by dose-dependent reductions in all [3H]prostanoids as well as [3H]15- and [3H]12-HETE and a reciprocal increase in [3H]AA. Nordihydroguaiaretic acid (NDGA) altered AA metabolism at concentrations which approached its toxic dose (greater than 20 microM). Cells exposed to 10 microM NDGA reduced TPA-induced label release into the medium which was characterized by reductions in [3H]-TxB2, [3H]PGE2 and [3H]HHT, no change in [3H]15-HETE, [3H]12-HETE or [3H]AA and the appearance of [3H]PGF2 alpha. Cellular label redistributions of lipid fractions in cells exposed to NDGA or indomethacin were significantly less than that of control cultures indicating inhibition of acylhydrolase activity. Indomethacin or NDGA, therefore modify AA metabolism of cultured rat M phi by influencing more than one target enzyme.     

Evidence for different mechanisms involved in the formation of lyso platelet-activating factor and the calcium-dependent release of arachidonic acid from human neutrophils.

Recent studies suggest that the first step in platelet-activating factor (PAF) biosynthesis, 1-alkyl-2-lyso-GPC (lyso PAF) formation, may be initiated by the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC to an acceptor lyso phospholipid by a CoA-independent transacylase activity (CoA-IT). The present study was designed to determine whether the formation of 1-alkyl-2-lyso-GPC and the release of arachidonic acid can occur by different mechanisms. These experiments examined both the formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC and the release of arachidonic acid from membrane phospholipids as determined by GC/MS in neutrophil homogenates under various conditions. The addition of unlabelled lyso phospholipids to neutrophil homogenates stimulated the time-dependent formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC. Without exogenous lyso phospholipids, little 1-[3H]alkyl-2-lyso-GPC was formed in this reaction. The activity which catalyzed the formation of 1-[3H]alkyl-2-lyso-GPC had characteristics identical to CoA-IT as indicated by the fact that both reactions were: independent of Ca2+, Mg2+, CoA and CoA fatty acids, located in microsomal fractions, and stable in 10 mM dithiothreitol. In sharp contrast to the aforementioned reaction, addition of lyso phospholipids did not affect the quantity of arachidonic acid released from membrane phospholipids. Furthermore, there was a Ca(2+)-independent release of arachidonic acid from membrane phospholipid that was increased 4 to 5-fold after the addition of 5 mM Ca2+. Finally, Ca(2+)-dependent arachidonic acid release was inhibited by putative phospholipase A2 inhibitors, aristolochic acid and scalaradial, at concentrations where neither the production of 1-[3H]alkyl-2-lyso-GPC nor Ca(2+)-independent arachidonic acid release was altered. Together these data imply that there may be different mechanisms involved in the formation of 1-alkyl-2-lyso-GPC and arachidonic acid from membrane phospholipids.     

Apparent involvement of phospholipase A2, but not protein kinase C, in the pro-oxidative interactions of clofazimine with human phagocytes

The anti-leprosy agent, clofazimine, at concentrations of 0.1-5 micrograms/ml caused a dose-related, stimulus-non-specific (N-formyl-methionyl-leucyl-phenylalanine, calcium ionophore, opsonised zymosan, arachidonic acid and phorbol myristate acetate) potentiation of superoxide generation by human neutrophils in vitro without affecting basal oxidative responses. The pro-oxidative interactions of clofazimine with neutrophils were eliminated by the phospholipase A2 inhibitor 4-p-bromophenacyl bromide but not by the protein kinase C (PKC) inhibitor H-7. In support of these observations clofazimine promoted the release of radiolabeled arachidonic acid from neutrophil membrane phospholipids but did not influence the activity of PKC in cytosolic extracts of neutrophils or of purified PKC from rat brain. Pro-oxidative interactions of clofazimine with human phagocytes may contribute to the intraphagocytic antimycobacterial activity of this agent.     

Inhibition of prostaglandin E2 and leukotriene C4 in mouse peritoneal macrophages and thromboxane B2 production in human platelets by flavonoids from Stachys chrysantha and Stachys candida

Seven flavonoids of Stachys chrysantha and Stachys candida have been isolated. The structures of the compounds were elucidated by spectroscopic methods, particularly highfield NMR spectroscopy. The effects of the methanol extracts of these two endemic Greek Stachys sp. and their main flavonoids were examined on arachidonic acid (AA) metabolism in the cellular system (mouse peritoneal macrophages and human platelets). Their cytotoxicity on cells was also investigated. Most samples assayed did not exhibit any significant effect on prostaglandin E2 (PGE2)-release from calcium ionophore-stimulated mouse peritoneal macrophages. Only chrysoeriol-7-O-beta-(3'-E-p-coumaroyl)-glucopyranoside, at the highest non-cytotoxic dose (50 microM), inhibited the release of PGE2, but this effect is not statistically significant. The release of leukotriene C4 (LTC4) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by a crude extract of S. chrysantha, with an IC50 value of 34.3 microg/ml. Xanthomicrol (IC50 = 29.2 microM) and chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside (IC50 = 11.1 microM) also inhibited the release of LTC4, although it showed less potency than the reference compound nordihydroguaiaretic acid (NDGA) (IC50 = 2 microM). However, most samples assayed showed a significant effect on thromboxane B2 (TXB2)-release from calcium ionophore-stimulated human platelets, with inhibition percentages slightly lower than the reference drug ibuprofen (IC50 = 7 microM). The IC50 values are: crude extract of S. candida 23.3 microg/ml; crude extract of S. chrysantha 23.1 microg/ml; xanthomicrol 28.8 microM; calcycopterin 2.66 microM and chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside 8.8 microM. Our results indicate that the selective inhibition of TX-synthase enzyme may be the primary target of action of most of these samples, and one of the mechanisms through which thus exert their antiinflammatory effects.     

山莨菪碱抑制酵母多糖刺激小鼠巨噬细胞释放前列腺素及白三烯

酵母多糖刺激[~3H]AA预标的小鼠腹腔巨噬细胞可释放[~3H]AA代谢物LTC_4,LTB_4,TXB_2,6-keto-PGF_(1α)及PGE_2.山莨菪碱可显著抑制上述刺激释放作用,使总~3H释放量及各[~3H]AA代谢物释放量均显著减少.这种抑制作用随山莨菪碱剂量增加而增强.当其浓度为0.5 mmol/L时.总~3H释放量下降46％(2 h),45％(5 h)及34％(15 h).本实验结果进一步证实山莨菪碱是通过抑制AA释放而减少PG及LT的形成.     

Pharmacokinetic-pharmacodynamic modeling of apratastat: a population-based approach

Apratastat is an orally active, potent, and reversible dual inhibitor of tumor necrosis factor-α converting enzyme (TACE) and matrix metalloproteinases (MMPs). This study characterizes the pharmacodynamic (PD) effect of apratastat following oral administration on tumor necrosis factor-alpha (TNF-α) release. Data were obtained from 3 clinical studies carried out in healthy subjects. Apratastat was administered orally in these studies as single doses or multiple doses (twice daily). The inhibition of TNF-α release by apratastat was investigated in studies of in vitro, ex vivo, and in vivo. Inhibitory E(max) models were used to characterize the inhibition of TNF-α release in both in vitro and ex vivo studies. Apratastat inhibited TNF-α release with a population mean IC(50) of 144 ng/mL in vitro and of 81.7 ng/mL ex vivo, respectively. The relationship between TNF-α and apratastat plasma concentration in the endotoxin-challenged study in healthy subjects was well characterized by a mechanism-based PD population model with IC(50) of 126 ng/mL. Apratastat can potently inhibit the release of TNF-α in vitro, ex vivo, and in vivo. Even though the dosage provided adequate exposure to inhibit TNF-α release, apratastat was not efficacious in rheumatoid arthritis (RA). This inconsistency between TNF-α inhibition and the clinical response requires further investigation.     

Inhibition of platelet-activating factor synthesis in human neutrophils and platelets by propionyl-L-carnitine.

Propionyl-L-carnitine (PrC) has been shown to exert beneficial effects in the treatment of myocardial and peripheral ischemia in man. These conditions are associated with the activation of circulating neutrophils and platelets. To determine whether PrC could affect the synthesis of lipid mediators known to influence neutrophil and platelet functions, we explored the effects of PrC on the synthesis of platelet-activating factor (PAF) and arachidonic acid (AA) metabolites. Preincubation (90 min) of human neutrophils with PrC (0.1-100 microM) inhibited the synthesis of PAF and of a PAF analog (1-alkyl-1'enyl-2-acetyl-sn-glycero-3-phosphoethanolamine: AEGPE) induced in vitro by the calcium ionophore A23187. In contrast, concentrations of PrC up to 100 microM did not influence the uptake of exogenous AA or the A23187-induced release of AA and eicosanoids from neutrophils in vitro. PrC (1 microM) also inhibited PAF synthesis from human platelets stimulated in vitro with thrombin, but had no effect on thrombin-induced aggregation. Oral administration of PrC (2 g/day for two weeks) to five normal volunteers resulted in a significant inhibition of PAF and AEGPE synthesis by neutrophils stimulated with A23187 ex vivo, with no effect on AA or eicosanoid release. These data indicate that PrC selectively inhibits in vitro and ex vivo PAF synthesis from human neutrophils and platelets without influencing AA metabolism or eicosanoid release. This effect of PrC might represent an additional mechanism by which this molecule can exert protective effects in tissue ischemia and in other inflammatory diseases associated with neutrophil and platelet activation.     

Differential effects of inorganic lead and delta-aminolevulinic acid in vitro on synaptosomal gamma-aminobutyric acid release

Several studies have shown that inorganic lead added in vitro does not alter gamma-aminobutyric acid (GABA) release from rat brain synaptosomes. The decrease in GABA release observed following chronic neonatal in vivo lead exposure has been proposed to be an indirect effect mediated by the increase in delta-aminolevulinic acid (ALA) accompanying chronic lead exposure. In the present study the effect of both lead and ALA in vitro on several aspects of [3H]GABA release from superfused rat cortical synaptosomes are examined. The present study demonstrates that lead (1-30 microM) added in vitro induces [3H]GABA release from preloaded cortical synaptosomes in a dose-dependent manner. This lead-induced increase in spontaneous [3H]GABA release does not appear to be mediated by inhibition of the membrane Na-K AT-Pase. ALA also induces a dose-dependent [3H]GABA release, but only at concentrations equal to or greater than 30 microM. Exposure to a combination of 3 microM lead and 100 microM ALA results in an increase in spontaneous [3H]GABA release that is greater than either treatment separately. The depolarization-evoked release of [3H]GABA resulting from a 1-sec exposure to 61 mM potassium chloride is reduced by lead (3 and 10 microM), whereas ALA (30-300 microM) does not alter depolarization-evoked release. These findings indicate that an indirect action of lead (elevated ALA concentrations) need not be proposed to explain the alterations in GABA release observed following chronic lead exposure.     

Increased concentration of arachidonic acid in erythrocyte membranes in chronically lead-exposed men

Animals intoxicated by lead present alterations in the fatty acid composition of red blood cells (RBC). Since this altered fatty acid composition of membranes may be a general reflection of lead toxicosis, we have examined 12 clinically healthy lead-exposed male subjects for fatty acid composition of RBC membranes along with blood lead, serum calcium, and serum iron concentrations. Twelve unexposed age-matched male subjects were used as controls. Significantly increased levels of arachidonic acid (AA) were found as compared to matching healthy controls in the RBC of the lead-exposed subjects. The increase of AA correlated in a dose-dependent manner with elevation in lead, and with serum iron, while a negative correlation was found between AA and serum calcium. The known ability of lead to substitute for calcium, which is essential in activating phospholipase A2 for AA release from membrane phospholipids, may be the main reason for increased AA in RBC membranes.