Peritoneal Polymorphonuclear Leukocytes Research Articles

Splanchnic sympathectomy prevents translocation and spreading of E coli but not S aureus in liver cirrhosis

IntroductionSpontaneous bacterial peritonitis (SBP) is mainly caused by bacterial translocation of enteric Gram-negative bacteria, predominantly Escherichia coli. The sympathetic nervous system (SNS) is activated in advanced cirrhosis, particularly in the...

Effects of the ω‐6:ω‐3 Fatty Acid Ratio of Fat Emulsions on the Fatty Acid Composition in Cell Membranes and the Anti‐Inflammatory Action

This study investigated the effects of parenterally administered fish oil (FO) on the fatty acid composition in rats to determine the optimal omega-6:omega-3 polyunsaturated fatty acid (PUFA) ratio of fat emulsions to achieve an anti-inflammatory effect. Male Sprague-Dawley rats were infused a parenteral nutrition (PN) solution containing fat emulsions with different omega-6:omega-3 PUFA ratios. The fatty acid content of phospholipids in the membranes of splenocytes was analyzed by gas chromatography (experiment 1). In addition, the amounts of leukotriene (LT) B(4) and LTB(5) released from peritoneal polymorphonuclear leukocytes (PMNs) were measured by high-performance liquid chromatography (experiment 2). In experiment 1, after infusion of the fat emulsion containing FO, the omega-3 PUFA content in cell membranes rose to 70% of the peak value on day 1 and nearly reached a plateau on day 3. The highest ratio of eicosapentaenoic acid (EPA) to arachidonic acid (AA) was achieved by administering a PN solution with the smallest omega-6:omega-3 PUFA ratio. In experiment 2, a larger amount of LTB(5) was released from Ca-ionophore-stimulated PMNs taken from rats given a larger quantity of FO. The ratio of LTB(5):LTB(4) released from PMNs correlated positively with the EPA:AA ratio in the membranous phospholipid and in serum. The omega-3 PUFAs were readily incorporated into the cell membrane within 3 days of infusion with the fat emulsion. The EPA:AA ratio in membranous phospholipid in PMNs was positively correlated with the LTB(5):LTB(4) production ratio and was a good indicator of anti-inflammatory effects.

Fungistatic and fungicidal activities of murine polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis

Recently, a novel culture medium for detecting live yeast cells of Paracoccidioides brasiliensis was developed by Kurita et al. Using this culture medium, murine peritoneal polymorphonuclear leucocytes (PMN) were examined for fungistatic and fungicidal activities against P. brasiliensis yeast cells. The magnitude of the antifungal effect of PMN varied depending upon the fungal isolates used. PMN exhibited a killing effect on P. brasiliensis isolate Bt-4 in 2 h of coculture. In contrast, the other three fungal isolates employed were resistant to killing by PMN. However, PMN considerably suppressed the growth of isolates Tatu and Recife in a long-term assay (∼72 h). The growth of isolate Bt-9 was also suppressed by PMN during the first 24 h, but was found to be considerably promoted at 72 h of coculture. Interferon-Γ (IFN-Γ ), but not tumour-necrosis factor-α, significantly augmented the antifungal activity of PMN. IFN-Γ-treated PMN exhibited a killing effect on isolates Tatu, Recife and Bt-9 after 24 h of coculture, and showed an enhanced killing effect on isolate Bt-4. Contact between PMN and fungal cells was required for PMN to exert the antifungal effect. Our results suggest that PMN, whether activated with cytokines or not, might play a critical role in host resistance in early infection with this fungus by buying time for development of more effective immunologic responses.

Isolation of eriocitrin (eriodictyol 7‐O‐rutinoside) as an arachidonate lipoxygenase inhibitor from Lumie fruit (Citrus lumia) and its distribution in Citrus species

AbstractAn inhibitory compound acting against rat platelet 12‐lipoxygenase was isolated from the peel of Lumie fruit (Citrus lumia) by activity‐guided separation. It was identified as eriocitrin (eriodictyol 7‐O‐rutinoside) by spectroscopic analyses. Eriocitrin inhibited 5‐lipoxygenase (IC5029.1 µmol L−1) from rat peritoneal polymorphonuclear leukocytes in addition to 12‐lipoxygenase (IC5022.3 µmol L−1). Its aglycone, eriodictyol (5,7,3′, 4′‐tetrahydroxyflavanone), was a much more potent inhibitor of both 12‐lipoxygenase (IC500.07 µmol L−1) and 5‐lipoxygenase (IC500.20 µmol L−1). It also inhibited the production of leukotriene B4 in intact peritoneal polymorphonuclear leukocytes stimulated with calcium ionophore A23187 (IC5012.7 µmol L−1). The distribution of eriocitrin in 39 citrus fruits was investigated by high‐performance liquid chromatography analysis. Lumie, eureka lemon (Citrus limon), Sambokan (Citrus sulcata), Sudachi (Citrus sudachi) and Koji (Citrus leiocarpa) fruits were found to contain high levels of eriocitrin in both peel and juice vesicles. Copyright © 2006 Society of Chemical Industry

Characterization of Hyaluronan-Binding Proteins on Guinea Pig Polymorphonuclear Leukocytes: Possible Involvement of Complement Receptor Type 3 (CR3, CD11b/CD18) in the Hyaluronan–Leukocyte Interaction

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical ( O 2 − ) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O 2 − production. CD as well as PMA induced the translocation of p47 phox to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCζ antagonist with sequences based on the endogenous PKCζ pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O 2 − production. On the other hand, the production of O 2 − induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.

Enhanced oxidative stress in neutrophils from hyperlipidemic guinea pig

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase are antilipidemic agents (statins) widely used for the prevention of cardiovascular diseases. Recent studies have suggested that the overall benefits of statin therapy cannot be accounted for solely by its antilipidemic effect. To obtain further insight into the mechanism of action of statins, we studied the effect of pitavastatin on the generation of reactive oxygen species (ROS) by peritoneal polymorphonuclear leukocytes (PMN) obtained from control and hyperlipidemic guinea pigs. Flow cytometric analysis revealed that the amount of ROS generated by PMN from the hyperlipidemic animals that had been administered a laurate-containing diet (LD) for 4 weeks was larger than that from the normal diet (ND) group (837% increase, ND; 82.17 arbitrary units, LD; 688.10 arbitrary units, P < 0.01, n = 6). Administration of pitavastatin to the LD group significantly decreased plasma levels of total cholesterol (TC) and low-density lipoprotein (LDL) with a reduction in ROS generation by PMN (19% decrease, LD control; 688.10 arbitrary units, LD+pitavastatin; 556.87 arbitrary units, P < 0.01, n = 6). Western blotting analysis revealed that the expression of protein kinase C α (PKC α) and βI was higher in PMN from the LD group than in PMN from the ND group (PKC α; 74% increase, PKC βI; 339% increase, P < 0.05, n = 4, respectively). Furthermore, expression of NADPH oxidase gp91phox in PMN from the LD group was higher than that in PMN from the ND group (18% increase, P < 0.05, n = 4). By administration of pitavastatin to the LD group, the expression of PKC α, βI and gp91phox was suppressed compared with the control LD group (PKC α; 41% decrease, PKC β; 28% decrease, gp91phox; 56% decrease, P < 0.05, n = 4, respectively). These results indicate that PMN from hyperlipidemic animals is associated with an accelerated respiratory burst of ROS by increasing the expression of PKC α, βI and gp91phox, and pitavastatin inhibits this by suppressing the expression of those proteins.

Intraperitoneal injection of lactoferrin ameliorates severe albumin extravasation and neutrophilia in LPS-induced inflammation in neonatal rats

Lactoferrin (LF) plays various anti-inflammatory roles in inflammation experimentally induced by lipopolysaccharides (LPS). But the effects of LF on albumin extravasation and neutrophilia have not been elucidated. We aimed to study the effects of LF on albumin extravasation, neutrophilia and/or on other symptoms in inflammation caused by LPS in rats. Human lactoferrin (hLF) was injected (10 mg/100 mL in PBS) 18 h, or 15 min prior to, or 60 min after intraperitoneal injection of LPS in 13 days old Sprague Dawley rats. Prophylactic injection of hLF significantly ameliorated albumin extravasation in ascitic fluid at 5 h and neutrophilia in the blood at 24 h after LPS injection, but the after-injection of hLF did not. Interestingly, an injection of rat anti-TNFalpha IgG 15 min prior to LPS injection did not ameliorate albumin extravasation. Prophylactic injection of hLF significantly ameliorated other symptoms like mortality, and the decrease of phagocytotic activity of peritoneal polymorpho-nuclear leukocytes (PMNL), but did not ameliorate the decrease of platelets in the plasma. These findings suggest that hLF may be available as a medical treatment prior to surgery for prophylaxis of side effects like albumin extravasation or neutrophilia.

Effects of different dietary oils on inflammatory mediator generation and fatty acid composition in rat neutrophils

Virgin olive oil (VOO) compared with fish oil (FO) and evening primrose oil (PO) on the ability of stimulated leukocytes to produce inflammatory mediators was investigated in rats. Weaned Wistar rats were fed a basal diet (BD) (2% by weight of corn oil) or diets containing 15% by weight of VOO, PO, or FO. After 8 weeks, glycogen-elicited peritoneal polymorphonuclear leukocytes, mainly neutrophils, were isolated. The calcium-ionophore stimulated neutrophils (2.5 × 10 6 cells/mL) obtained from rats fed the different oils produced a higher release of lysosomal enzymes (β-glucuronidase, lysozyme, and myeloperoxidase [MPO]) compared with those fed BD. The production of reactive oxygen species (ROS) in response to the stimulant, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), by neutrophils from the VOO group (15.44 nmol of O 2 − and 6.56 nmol of H 2O 2) was similar to the BD group (12.01 nmol O 2 − and 8.49 nmol H 2O 2) and significantly lower than the PO (20.90 nmol O 2 − and 10.84 nmol H 2O 2) and FO (20.93 nmol O 2 − and 12.79 nmol H 2O 2) groups. The cyclooxygenase-derived eicosanoid production was reduced by the lipid enrichment of the diets. Whereas the generation of prostaglandin E 2 (PGE 2) was significantly decreased in VOO (5.40 ng/mL), PO (4.95 ng/mL), and FO (1.44 ng/mL) groups compared with BD (8.19 ng/mL), thromboxane B 2 (TXB 2) reduction was especially significant in neutrophils from the FO diet group (14.67 ng/mL compared with 26.69 ng/mL from BD). These experimental data suggest that FO and PO, as well as VOO, could be considered a valuable strategy in preventing the generation of some inflammatory mediators.

Isolation of two triterpenoids and a biflavanone with anti-Inflammatory activity from Schinus molle fruits.

Three compounds with anti-inflammatory activity were isolated from Schinus molle fruits. Two of the compounds were identified as 3- epi-isomasticadienolalic acid ( 1), isomasticadienonalic acid ( 2) and chamaejasmin ( 3). Triterpenes 1 and 2, and biflavanone 3 were tested on two models of mice paw inflammation: one of acute inflammation, induced by subcutaneous injection of either phospholipase A (2) (PLA (2)) or carrageenan in the paws of mice, and one of chronic inflammation in the form of eczema, provoked by repeated administration of TPA to the ears of mice. On the PLA (2)-induced mouse paw oedema, only 2 was active (30 mg/kg, 66 % inhibition at 60 min), whereas all compounds reduced the chronic model of inflammation (48 to 26 % of swelling reduction), but only triterpenes reduced the leukocyte infiltration, measured as tissue peroxidase activity. In the case of the carrageenan-induced mouse paw oedema, only 3 led to a reduction of the swelling 3 h after challenge (50 mg/kg, 46 % oedema inhibition). In addition, 3 inhibited the LTB (4) production in rat peritoneal polymorphonuclear leukocytes with an IC (50) value of 29.8 microM, while triterpenes showed toxicity against cells at 100 microM.

Effect of phosphotyrosine proteins on phorbol myristate acetate-induced NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes.

Phorbol myristate acetate (PMA)-induced superoxide radical (O(2)(-))-production in guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was significantly lower than that in peripheral cells. To determine the role of phosphotyrosine proteins in the lower O(2)(-) production, the effect of ST638 and genistein, tyrosine kinase inhibitors, on PMA-induced O(2)(-) production in peritoneal PMNs was examined. PMA-induced O(2)(-)-production of the cells was increased by the pretreatment with ST638 or genistein, the increment depending on the inhibitor concentration. The p47phox level in the plasma membrane of PMA-stimulated PMNs was increased by the pretreatment with ST638, although the phosphorylated p47phox level in the cells was not altered by ST638. On the other hand, PMA-induced O(2)(-)-production of peripheral PMNs was not affected by the pretreatment with ST638, but that of cytochalasin B (CB)-primed peripheral PMNs significantly increased by further treatment with ST638. The phosphotyrosine protein level of peritoneal PMNs was higher than that of the peripheral cells, especially in cytosolic proteins including 50-60 and 70-85 kDa proteins, and that of the CB-primed peripheral cells was also higher than that of the intact cells in similar cytosolic proteins to those above. Further treatment of CB-primed peripheral cells with ST638 resulted in a lower level of phosphotyrosine proteins. These findings suggest that phosphorylation of some protein(s) at specific tyrosine residues inhibits the translocation of p47phox to the plasma membrane from the cytosol, resulting in lower O(2)(-)-generation in casein-induced peritoneal exudate PMNs.

Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine.

Luminol is a non-radical-specific amplifying molecule which produces light upon interaction with various reactive oxygen intermediates (ROIs). ROI production of rat peritoneal polymorphonuclear leukocytes (PMNLs) elicited by 2.3 microM formyl-methionyl-leucyl-phenylalanine (fMLP) results in a biphasic luminol-dependent chemiluminescence (LDCL) signal. Whereas ROIs are also produced intracellularly, as judged by flow cytometry, addition of non-membrane-permeable catalase reduces the first and second phases of the LDCL signal to around 3% and less than 3%, respectively. This suggests that in the case of fMLP-stimulated rat PMNLs, the LDCL signal is related to the ROIs in the extracellular medium and hydrogen peroxide has a key role in the formation of the LDCL signal. In the presence of the non-specific myeloperoxidase inhibitor Na-azide, the first phase of the LDCL signal decreases slightly (87+/-8%), while the second phase almost disappears (< 3%), indicating the myeloperoxidase dependence of the second phase. The hydroxyl radical scavenger histidine results in an 84+/-4% and a 71+/-4% decrease in the intensity of the first and second phases, respectively. Based on these data, it is concluded that hydrogen peroxide might be the source of hydroxyl radicals directly oxidizing luminol in the first phase of the LDCL signal, while in the second phase it serves as a substrate of myeloperoxidase in the peroxidation reaction of the luminol.

PSK and OK-432-Induced Immunomodulation of Inducible Nitric Oxide (NO) Synthase Gene Expression in Mouse Peritoneal Polymorphonuclear Leukocytes and NO-Mediated Cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/106 PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.

Fungistatic and fungicidal activities of murine polymorphonuclear leucocytes against yeast cells ofParacoccidioides brasiliensis

Recently, a novel culture medium for detecting live yeast cells of Paracoccidioides brasiliensis was developed by Kurita et al. Using this culture medium, murine peritoneal polymorphonuclear leucocytes (PMN) were examined for fungistatic and fungicidal activities against P. brasiliensis yeast cells. The magnitude of the antifungal effect of PMN varied depending upon the fungal isolates used. PMN exhibited a killing effect on P. brasiliensis isolate Bt-4 in 2 h of coculture. In contrast, the other three fungal isolates employed were resistant to killing by PMN. However, PMN considerably suppressed the growth of isolates Tatu and Recife in a long-term assay (approximately 72 h). The growth of isolate Bt-9 was also suppressed by PMN during the first 24 h, but was found to be considerably promoted at 72 h of coculture. Interferon-gamma (IFN-gamma), but not tumour-necrosis factor-alpha, significantly augmented the antifungal activity of PMN. IFN-gamma-treated PMN exhibited a killing effect on isolates Tatu, Recife and Bt-9 after 24 h of coculture, and showed an enhanced killing effect on isolate Bt-4. Contact between PMN and fungal cells was required for PMN to exert the antifungal effect. Our results suggest that PMN, whether activated with cytokines or not, might play a critical role in host resistance in early infection with this fungus by buying time for development of more effective immunologic responses.

Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O 2 −) production after a lag time. The rank order of efficiency of these PAs in triggering O 2 − production was PA 8:0 [1,2-dioctanoyl- sn-glycerol-3-phosphate (GP)]>PA 10:0 (1,2-didecanoyl-GP)>PA 6:0 (1,2-dicaproyl-GP)≫PA 12:0 (1,2-dilauroyl-GP). The O 2 − release from PMN stimulated with PA 10:0 or PA 12:0, but not with PA 6:0 or PA 8:0, was lowered by the addition of 1 mM extracellular Ca 2+. Studies with various inhibitors showed that the mechanism of multiphasic O 2 − production induced by PA 8:0 depended on its concentration: 1 and 3 μM PA 8:0 induced O 2 − production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 μM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 μM TMB-8. Concentrations of 30, 60 and 100 μM PA first elicited transient O 2 − production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O 2 − production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [ 14C]PA 8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O 2 − production via three different mechanisms described above.

Immune responsiveness in a rat model for type II diabetes (Zucker rat, fa/fa): susceptibility to Candida albicans infection and leucocyte function

There is a causal relationship between obesity-associated diabetes and an increased risk of infection. The ability of obese (fa/fa) Zucker rats, a model for non-insulin-dependent diabetes mellitus (NIDDM), to clear Candida albicans from the circulation and tissues was compared to that of lean (Fa/fa, Fa/Fa) Zucker rat controls as a measure of immune function. The ID50 necessary to establish tissue colonisation in lean Zucker rats was 1.18 log10 times greater than that determined for the obese Zucker rats. Nine days after intravenous (i.v.) injection of a yeast suspension, the organs of obese rats had a 10-fold greater yeast/g organ burden than did lean rats. The kidney was determined to be the primary target organ for colonisation. Germ-tube formation by C. albicans occurred at a rate 1.5 times faster in serum from obese rats than in serum from lean rats. Peritoneal polymorphonuclear leucocytes, resident macrophages and thioglycollate-elicited macrophages from lean Zucker rats displayed a significantly higher ability to kill ingested yeast cells than analogous cell populations from obese Zucker rats.

Changes in arachidonic acid metabolism and the aggregation of polymorphonuclear leukocytes in rats with streptozotocin-induced diabetes

Changes in lipid composition were examined in peritoneal polymorphonuclear leukocytes (PMNL) from rats with streptozotocin-induced diabetes. The largest changes associated with the lipids in PMNL prepared from rats with insulin-dependent diabetes mellitus were found in the composition of fatty acids of phospholipids although no consistent changes were noted in the amounts of phospholipids in individual classes. The relative amount of arachidonic acid was significantly reduced and that of linoleic acid was significantly increased in PMNL from diabetic rats. The extent of A23187-stimulated release of arachidonic acid from CGP and IGP in PMNL from diabetic rats was much smaller than that from normal PMNL. Moreover, there were decreases in the production of free arachidonic acid and its metabolites, such as LTB 4 and 5-HETE by A23187-stimulated PMNL from diabetic rats as compared to PMNL from normal rats. Aggregation of PMNL provoked by A23187 was significantly suppressed in PMNL from diabetic rats. However, addition of free arachidonic acid or LTB 4 to normal PMNL and to those from diabetic rats resulted in aggregation to similar extents, a result that supports a role for enhanced availability of endogenous arachidonic acid in the induction of the aggregation of PMNL from diabetic rats. The present results suggest that diabetes-associated changes in arachidonic acid metabolism might play a critical role in the modulation of aggregation of PMNL.

Acetyl-11-keto-beta-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity.

1. 5-Lipoxygenase (5-LOX) products from endogenous arachidonic acid in ionophore-stimulated peritoneal polymorphonuclear leukocytes (PMNL) and from exogenous substrate (20 microM) in 105,000 g supernatants were measured. 2. The effects of natural pentacyclic triterpenes and their derivatives on 5-LOX activity were compared with the inhibitory action of acetyl-11-keto-beta-boswellic acid (AKBA), which has been previously shown to inhibit the 5-LOX by a selective, enzyme-directed, non-redox and non-competitive mechanism. 3. The 5-LOX inhibitory potency of AKBA was only slightly diminished by deacetylation of the acetoxy group or reduction of the carboxyl function to alcohol in intact cells (IC50 = 1.5 vs. 3 and 4.5 microM, respectively) and in the cell-free system (8 vs. 20 and 45 microM). 4. beta-Boswellic acid (beta-BA), lacking the 11-keto function, inhibited 5-LOX only partially and incompletely, whereas the corresponding alcohol from beta-BA, as well as amyrin, acetyl-11-keto-amyrin, 11-keto-beta-boswellic acid methyl ester had no 5-LOX inhibitory activity up to 50 microM in either system. 5. beta-BA only partially prevented the AKBA-induced 5-LOX inhibition, whereas the non-inhibitory compounds, amyrin and acetyl-11-keto-amyrin, almost totally antagonized the AKBA effect and shifted the concentration-inhibition curve for the incomplete inhibitor beta-BA to the right. In contrast, the non-inhibitory 11-keto-beta-BA methyl ester exerted no antagonizing effect. 6. The results demonstrate that the pentacyclic triterpene ring system is crucial for binding to the highly selective effector site, whereas functional groups (especially the 11-keto function in addition to a hydrophilic group on C4 of ring A) are essential for 5-LOX inhibitory activity.

Increased receptor-mediated phospholipase D activation and Ca 2+ mobilization in peritoneal polymorphonuclear leukocytes from streptozotocin-induced diabetic rats

Phospholipase D activation was investigated in peritoneal polymorphonuclear leukocytes from streptozotocin-induced diabetic rats. Stimulation of the cells with formyl-Met-Leu-Phe resulted in a time- and dose-dependent increase in phosphatidylethanol in the presence of ethanol, and this lipid formation in cells prepared from diabetic rats was enhanced as compared to that in the case of nondiabetic rats. Furthermore, the increase in the intracellular Ca 2+ concentration was also enhanced in the stimulated cells from diabetic rats. Under the present conditions, N-acetyl- β-glucosaminidase release and Superoxide generation, which are known to be dependent on phospholipase D activation, were higher in the cells from diabetic rats than those in the cells from control rats. However, there was no difference in the dissociation constant and the number of binding sites for formyl-Met-Leu-Phe between the cells from diabetic and control rats. Phosphatidylethanol formation, N-acetyl- β-glucosaminidase release and Superoxide generation in response to ionomycin or 4β-phorbol 12-myristate 13-acetate were not enhanced in diabetic rat cells, as compared with those in control rat cells. These results suggest that receptor-mediated phospholipase D activation and Ca 2+ mobilization are enhanced in diabetic rat polymorphonuclear leukocytes, which might be due to acceleration of receptor-mediated signaling.

Actions of platelet-activating factor on isolated rabbit basilar artery: modulation by activated polymorphonuclear leukocytes.

In segments of rabbit basilar artery (BA) platelet-activating factor (PAF) initiated a concentration-dependent transitory contraction which was unaffected by indomethacin or nordihydroguaiaretic acid (NDGA). N omega-nitro-L-arginine (NLA) did not change the magnitude of PAF-induced contraction, but in the presence of NLA the contraction tended to be more prolonged. In precontracted BA segments, PAF caused a concentration-dependent relaxation which was unaffected by NDGA. Indomethacin and NLA decreased PAF-induced relaxation by 10-30 and 70-90%, respectively; in combination these agents totally eliminated PAF-induced vessel reactions. Treatment of BA segments by rabbit peritoneal polymorphonuclear leukocytes (PMNLs) activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 20 min led to PMNL adhesion to the vascular endothelium and a significant decrease (60-100%) in acetylcholine-induced endothelium-dependent vessel relaxation. After the treatment of BA segments, PAF induced strong, slow, tonic contraction of either non-precontracted or precontracted vessels which was unaffected by indomethacin or NLA. NDGA decreased this contraction by 30-60%. These results indicate that PAF is an endothelium-dependent vasodilator that can also produce an insignificant constrictor effect. However, when the vessels are affected by activated PMNLs, PAF is transformed to a strong vasoconstrictor, presumably due to the generation of as yet unknown vasoconstrictor stimuli resulting from PMNL-endothelium interactions. Under these conditions the PAF-induced contraction is partly mediated by 5-lipoxygenase metabolites.