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K B Glaser

Publications and source records attributed to K B Glaser.

At least 19 recordsLinked to original sources
PubMed2004

Differential protein acetylation induced by novel histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.

Amides↗
PubMed1999

Enhancement of the surface expression of tumor necrosis factor alpha (TNFalpha) but not the p55 TNFalpha receptor in the THP-1 monocytic cell line by matrix metalloprotease inhibitors.

The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor alpha (TNFalpha) and both TNFalpha receptors (p55 and p75) upon exposure to bacterial lipopolysaccharide (LPS). The broad-spectrum matrix metalloprotease (MMP) inhibitors [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)succinyl]-L-p henylalanine-N-methylamide (GI-129471) and marimastat [2S-[N4(R*),2R*,3S*]]-N4[2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N 1,2-dihydroxy-3-(2-methylpropyl)butanediamide (BB-2516) were effective inhibitors of LPS-induced TNFalpha (soluble) release with IC50 values of 0.2 and 4.0 microM, respectively. Upon LPS stimulation, the expression of pro-TNFalpha (membrane associated) on the cell surface (FACS analysis) could not be observed. However, in the presence of GI-129471, a concentration-dependent increase in TNFalpha surface expression was observed. Peak expression (percentage of cells expressing pro-TNFalpha and mean fluorescence units) in the presence of GI-129471 was at 2 hr, and steadily declined to return to near control levels by 8 hr. This time course was similar to TNFalpha release, which also peaked at 2-4 hr after LPS exposure and then declined. Stimulation of THP-1 cells with LPS + phorbol myristate acetate increased the percentage of cells expressing pro-TNFalpha by 10-fold. In the presence of GI-129471, these increases were augmented further and peaked between 2 and 4 hr, but also returned to near control levels of expression by 24 hr. This was in contrast to the release of soluble TNFalpha, which continued to accumulate over a 24-hr time course. TNFalpha receptor I (p55, TNFRI) and II (p75, TNFRII) shedding was also inhibited by GI-129471 (IC50 = 1.5 and 3.1 microM, respectively) and BB-2516 (IC50 = 14 and 15 microM, respectively). Unlike pro-TNFalpha surface expression, surface expression of both TNFalpha receptors steadily increased over 72 hr. In contrast to pro-TNFalpha surface expression, TNFRI surface expression was not augmented by these MMP inhibitors in THP-1 cells after LPS stimulation. Surface expression of TNFRII was augmented by these MMP inhibitors. These results suggest that even in the continued presence of LPS stimulation and an inhibitor of TNFalpha processing, the augmented surface expression of TNFalpha is transient. The potential "deleterious" implications of high levels of surface pro-TNFalpha expression in the presence of these inhibitors may be lessened by its transient nature.

Cell Line↗
PubMed1999

Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation: correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-alpha, thromboxane B2, and metalloprotease release.

The effects of lipopolysaccharide (LPS) on the central nervous system, one of the first organs to be affected by sepsis, are still incompletely understood. Rat microglia (BMphi) constitute the main leukocyte-dependent source of reactive oxygen species in the central nervous system. The in vitro effect of LPS on agonist-stimulated superoxide (O2-) generation from BMphi appears controversial. Our purpose was to determine the time- and concentration-dependent effect of Escherichia coil LPS on phorbol-12 myristate 13-acetate-stimulated O2- generation from BMphi. Our results demonstrate that BMphi O2- generation in vitro peaked 17 h after stimulation of with .3 ng/mL LPS. Furthermore, stimulation of BMphi with LPS for 17 h resulted in the following concentration-dependent responses: .1-1 ng/mL LPS induced no prior mediator generation but potently enhanced subsequent phorbol-12 myristate 13-acetate-stimulated O2- generation; 3-10 ng/mL LPS caused nitric oxide, tumor necrosis factor-alpha (TNF-alpha), thromboxane B2 and matrix metalloproteinase-9 release although partially inhibiting ensuing phorbol-12 myristate 13-acetate-stimulated O2- generation; 30-100 ng/mL LPS, maximized nitric oxide, TNF-alpha, thromboxane B2, matrix metalloproteinase-9 generation with concomitant lactic dehydrogenase release although strongly deactivating successive phorbol-12 myristate 13-acetate-stimulated O2 production. Our in vitro studies suggest that enhanced release of these four mediators (nitric oxide, TNF-alpha, thromboxane B2, and matrix metalloproteinase-9) during stimulation of BMphi with LPS might play a critical role in the subsequent ability of BMphi to generate O2- in vivo. Potential clinical implications of our findings are suggested by the fact that LPS levels similar to the ones used in this study have been observed in cerebrospinal fluid both in Gram-negative meningitis and sepsis.

Animals↗
PubMed1998

Sirolimus (rapamycin, Rapamune) and combination therapy with cyclosporin A in the rat developing adjuvant arthritis model: correlation with blood levels and the effects of different oral formulations.

OBJECTIVE AND DESIGN: To determine whole blood levels of sirolimus, a macrolide antibiotic in the rat developing adjuvant arthritis (AA) model after dosing orally with two different vehicles, and whether combinational doses of sirolimus and cyclosporin A (CsA) produced additive or synergistic inhibitory effects in this model. MATERIAL: Male Lewis rats (150-180g). TREATMENT: Arthritis was induced by the injection (0.5 mg/ rat) of heat-killed Mycobacterium butyricum suspended in light mineral oil. Drugs were administered orally either in fine suspension (0.5% Tween 80) or in emulsion (phosal 50 PG in 1% Tween 80) at doses of 0.1 to 5 mg/kg in a 7 day, MWF or daily regimen. METHOD: Paw volumes (ml) were measured by automated mercury plethysmograph and sirolimus concentrations in whole blood were quantitated by liquid chromatography/ mass spectroscopy. RESULTS: At 72h (7 days after adjuvant) after receiving the third oral dose (4.5 mg/kg p.o.), the phosal vehicle resulted in higher sirolimus blood levels (2.5 ng/ml) than in Tween 80 (1.6 ng/ml). After the rats received the last oral dose on day 14, (7 total doses of sirolimus at 4.5 mg/kg) the sirolimus blood levels (2h after the last dose) were about 2 times higher for the phosal dosed rats (9.8 ng/ml) compared to Tween 80 dosed rats (4.6ng/ml). Even 24h after the last dose, sirolimus blood levels were still elevated in the phosal dosed rats (0.8 ng/ml) relative to 0.5% Tween 80 dosed rats (0.5 ng/ml). At day 16 in the rat developing model, sirolimus, when given in phosal vehicle, produced an ED50 of 0.28 mg/ kg (i.e. inhibition of uninjected paw edema) that was about 5.5 times lower than using 0.5% Tween 80 as the suspending agent (ED50 = 1.6mg/kg). When combining sirolimus and CsA using precalculated doses for producing an additive effect in this adjuvant model, an additive inhibitory effect on uninjected paw edema was observed at equal combinational doses of 0.5 and 2 mg/kg, respectively. CONCLUSIONS: The phosal vehicle used in administering sirolimus increases the absorption and whole blood levels in the rat and the elevated blood levels correlated positively with the therapeutic effect in the rat developing AA model. In addition, combination therapy using sirolimus and CsA produced an additive effect in rat developing AA.

Administration, Oral↗
PubMed1998

Pharmacological characterization of the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae.

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.

Animals↗
PubMed1997

Induction of prostaglandin H synthase-2 and tumor necrosis factor-alpha in human amnionic WISH cells by various stimuli occurs through distinct intracellular mechanisms.

These studies examined the signal transduction mechanisms by which prostaglandin (PG) E2 production can occur in human amnionic WISH cells in response to the stimuli okadaic acid, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, phorbol-12-myristate-13-acetate (PMA) or combinations of PMA with IL-1beta or TNF-alpha. We also investigated whether WISH cells are capable of producing TNF-alpha or IL-1beta in response to stimulation, because these cytokines can be produced in an autocrine fashion to perpetuate an inflammatory response. Our data indicate that the magnitude of PGE2 production induced by a given stimulus correlated temporally with the level of PGH synthase-2 (PGHS-2) protein. PMA or IL-1beta induced PGE2 production 2 to 4 hr after treatment, whereas the combination of these agents produced the most rapid induction 2 hr after treatment. Only okadaic acid induced the production of both PGE2 and TNF-alpha, after a lag of 12 to 18 hr. PGE2 production by all stimuli was inhibited by dexamethasone, the IL-1 receptor antagonist (IL-1ra), the specific PGHS-2 inhibitor NS-398 and the protein kinase inhibitor staurosporin. In contrast, TNF-alpha production in response to okadaic acid was inhibited by the TNF-converting enzyme inhibitor GI 129471 and staurosporin but was unaffected by either IL-1ra, dexamethasone or NS-398. We conclude that WISH cells are capable of producing bioactive proinflammatory mediators such as TNF-alpha and PGE2 through separable intracellular signal transduction mechanisms. The ability of IL-1ra to reduce PGE2 production caused by all stimuli used suggests an autocrine role for IL-1 in PGHS-2 induction in these cells.

Amnion↗
PubMed1996

Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2.

The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.

Arachidonic Acid↗
PubMed1995

Regulation of prostaglandin H synthase 2 expression in human monocytes by the marine natural products manoalide and scalaradial. Novel effects independent of inhibition of lipid mediator production.

The marine natural products manoalide and scalaradial are potent anti-inflammatory agents that inactivate the enzyme phospholipase A2 (PLA2) in vitro. To study the mechanism of inhibition of prostaglandin E2 (PGE2) production in human monocytes by manoalide and scalaradial, lipopolysaccharide (LPS)-induced prostaglandin biosynthesis and induction of prostaglandin H synthase (PGHS) were evaluated. LPS (10 ng/mL) and interleukin-1 beta (IL-1 beta, 50-1000 ng/mL) but not tumor necrosis factor alpha (TNF alpha, 300 ng/mL) induced the expression of the PGHS-2 isoform as determined by immunoblot analysis with a specific polyclonal antibody for PGHS-2. Manoalide and scalaradial (1-10 microM) inhibited LPS-induced endogeneous PGE2 production, reduced the LPS-induced PGHS activity, and reduced the expression of PGHS-2. Indomethacin [a PGHS inhibitor (0.01 to 0.1 microM)], zileuton [a 5-lipoxygenase inhibitor (3-10 microM)], and WEB-2806 [a platelet-activating factor (PAF) antagonist (30 microM)] did not affect the LPS-induced expression of PGHS-2 in human monocytes. These results suggest that modulation of lipid mediator production by manoalide or scalaradial may not be involved in the observed effects on the expression of PGHS-2. Manoalide and scalaradial also inhibited the release of IL-1 beta and TNF alpha from LPS-stimulated monocytes. Expression of PGHS-2 induced by either LPS or IL-1 beta was blocked by the IL-1 receptor antagonist (IL-1ra, 2 micrograms/mL) but not by rolipram, a phosphodiesterase IV inhibitor that inhibits TNF alpha but not IL-1 beta release. Similar to LPS, IL-1 beta-induced PGHS-2 expression was apparently not regulated by lipid mediators such as prostaglandins, leukotrienes or PAF as determined with specific inhibitors and antagonists. Scalaradial and to some extent manoalide were capable of blocking the IL-1 beta-induced expression of PHGS-2. These results indicate that IL-1 beta is the predominant cytokine responsible for the induction of PGHS-2 in the human monocyte. Furthermore, marine natural products such as scalaradial have novel effects on the IL-1 beta-mediated induction of PGHS-2 in human monocytes, which appears to be independent of effects on lipid mediator production.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells.

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4 degrees C) of the large molecular weight PDE4 denoted "Peak 2 PDE4" with mixed phosphatidic acids (PAs) produced a 2-fold increase in its Vmax without changing its Km (approximately 2 microM) for cyclic AMP. This "activation" was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Regulation of phospholipase A2 enzymes: selective inhibitors and their pharmacological potential.

The area of PLA2 research has grown immensely over the past 20 years. There is a better understanding of the kinetics, or factors that affect the kinetics, of the different forms of PLA2. New forms of PLA2 are being discovered, such as the cPLA2, which fit the role of an intracellularly regulated enzyme. Multiple forms of PLA2 tend to complicate the elucidation of the cellular mechanisms that regulate AA release and the subsequent eicosanoid production. Because of the factors that affect PLA2 kinetics and the unknown nature of the PLA2 that regulates AA release (there may be more than one), it has been difficult to design or isolate specific inhibitors. This review discussed selected classes of inhibitors because these have generated the most intense research in the field. There is a multitude of structurally diverse compounds reported in the literature that have been reported to be inhibitors of PLA2 in vitro and some have been reported to have anti-inflammatory activity (Wilkerson, 1990; Connolly and Robinson, 1993a). It is clear from a brief survey of the literature that the bulk of PLA2 inhibitors have topical anti-inflammatory activity. This may be due to the nature of these inhibitors: because they are hydrophobic they may be more readily absorbed in the skin whereas when given orally they may not be absorbed. To data, manoalide has been clinically evaluated in man and a new Bristol-Myers Squibb retenoid derivative may enter clinical trials for psoriasis (BMS-181162 (XVI)); however, there are no PLA2 inhibitors on the market or significantly advanced in clinical development (Table III). This indicates the lack of understanding of this enzyme for the development of relevant inhibitors, which is related to the lack of understanding of the relevant PLA2 that regulates AA release and eicosanoid biosynthesis. The concept of regulation of eicosanoid biosynthesis by PLA2 inhibition and decreased AA availability still remains a viable therapeutic approach for the treatment of inflammatory diseases. The proof of this concept has not been obtained because of the complex nature of PLA2 and the multiple forms of PLA2 in the cell. Clinical results with cyclooxygenase inhibitors and recent clinical results with inhibitor of 5-lipoxygenase demonstrate that if inhibition of PLA2 results in reduction in both lipid mediators, a good anti-inflammatory compound should result. The added advantage of PLA2 inhibitors would be the reduction of PAF levels; however, the clinical results with potent and specific PAF antagonists has been less encouraging about the potential benefits of reduction in PAF levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Cellular and topical in vivo inflammatory murine models in the evaluation of inhibitors of phospholipase A2.

Several novel inhibitors of human synovial fluid phospholipase A2 (HSF-PLA2) were evaluated in cellular models of inflammatory mediator release (murine macrophage and human neutrophil) and topical in vivo inflammatory skin models in mice to ascertain the scope of effects which might be observed for PLA2 inhibitors. Potent inhibition of HSF-PLA2 in vitro can be observed with compounds such as scalaradial and ellagic acid, which both have IC50 values of 0.02 microM (using autoclaved [3H]-arachidonic-acid (AA)-labelled Escherichia coli membranes as substrate). Luffariellolide, a manoalide analog, and aristolochic acid are less potent (IC50 = 5 and 46 microM, respectively) in this assay. An interesting observation is that ellagic acid in cellular assays does not inhibit macrophage eicosanoid production and only 30% inhibition of PAF biosynthesis can be obtained at 50 microM in the human neutrophil. Possibly due to its irreversible mechanism of action, scalaradial retained its potent activity in both the macrophage (IC50 for PGE2 production = 0.05 microM) and neutrophil assays (IC50 for PAF biosynthesis = 1 microM). Aristolochic acid is active in these cellular assays (macrophage IC50 = 2.5 microM and neutrophil IC50 = 100 microM), but is consistently less active than either scalaradial or luffariellolide. The relative potencies of these compounds were determined in several murine in vivo inflammatory models such as oxazolone contact hypersensitivity, AA-induced ear edema and phorbol ester (PMA)-induced ear edema. In the mouse model of oxazolone contact hypersensitivity, these PLA2 inhibitors have little effect (< or = 30% inhibition at 400 micrograms/ear) with scalaradial and luffariellolide being less effective than either aristolochic or ellagic acid. PMA-induced ear edema was effectively inhibited by scalaradial, luffariellolide and aristolochic acid (ED50 = 70, 50 and 50 micrograms/ear, respectively) whereas ellagic acid was less effective (ED50 = 230 micrograms/ear). In AA-induced ear edema, these PLA2 inhibitors had minimal effects, as would be expected for compounds which inhibit PLA2. These results, especially those of ellagic acid, suggest that caution should be taken in the extrapolation of potency against a purified human extracellular type PLA2 to the scope of activities these compounds might have in the cellular and in vivo models. The consistency of scalaradial and luffariellolide may be inherent to their irreversible mechanism of action, which is a factor to be accounted for in the extrapolation of enzyme data to cellular and in vivo models.

Animals↗
PubMed1995

Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s).

Ninety minutes after i.v. injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of in vivo LPS-treated alveolar macrophages (AM phi) when compared with saline (SAL)-treated AM phi. The purpose of this investigation was to dissect the in vitro mechanism of PMA-stimulated O2- generation in both LPS and SAL-treated rat AM phi, with a panel of inhibitors of protein kinase C (PKC), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), phospholipase A2 (PLA2), cyclooxygenase (CO) and 5-lipoxygenase (5-LO). The following agents blocked PMA-stimulated O2- generation in both LPS- and SAL-treated AM phi (expressed as percentage of control): 1) PKC inhibitors: staurosporine: 100 nM, 7.0% (LPS) and 5.6% (SAL); sphingosine: 10 microM, 21% (LPS) and 10.5% (SAL); 2) PTK inhibitor: genistein: 100 microM, 44% (LPS) and 31% (SAL); 3) PTP inhibitors: phenylarsine oxide, 10 microM, 12.1% (LPS) and 18% (SAL); diamide, 1000 microM, 10.1% (LPS) and 10.5% (SAL); and 4) PLA2 inhibitors: manoalide: 1 microM, 29.3% (LPS) and 5.2% (SAL); scalaradial: 1 microM, 7.7% (LPS) and 7.1% (SAL); and WAY 125,984: 10 microM, 17.1% (LPS) and 14.5% (SAL). In addition, it was observed that exogenously added arachidonic acid (AA)-stimulated O2- generation in a time- and dose-dependent manner in both LPS and SAL-treated AM phi. The following inhibitors enhanced or did not affect PMA-stimulated O2- generation in LPS- and SAL-treated AM phi (expressed as percentage of of control): 1) PSP inhibitors: okadaic acid: 0.5 microM, 117% (LPS) and 153% (SAL); calyculin A: 1 microM, 112% (LPS) and 101% (SAL); 2) CO and 5-LO inhibitors: indomethacin: 10 microM, 107% (LPS) and 90% (SAL); WY 50, 295: 1 microM, 99% (LPS) and 103% (SAL); and 3) the PTP inhibitor orthovanadate upon prolonged preincubation. In both in vivo LPS- or SAL-primed AM phi, PMA-stimulated O2- generation appears to be modulated by PKC, PLA2, AA, PTK, PTP and PSP. No modulatory role was evident for either CO or 5-LO metabolites. These findings might bear on the design of therapeutic approaches for the modulation of O2- release by AM phi in the early stages of sepsis and adult respiratory distress syndrome.

Animals↗
PubMed1993

Regulation of eicosanoid biosynthesis in the macrophage. Involvement of protein tyrosine phosphorylation and modulation by selective protein tyrosine kinase inhibitors.

The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-activating factor-stimulated prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-primed P388D1 macrophage-like cells (Glaser et al., J Biol Chem 265: 8658-8664, 1990). Therefore, the role of PTK in eicosanoid biosynthesis was investigated in murine resident peritoneal macrophages using genistein and tyrphostin-25, selective PTK inhibitors. Genistein, a competitive inhibitor of ATP binding on PTK, inhibited PGE2 production (IC50 = 20 microM) in response to zymosan, calcium ionophore A23187, and phorbol myristate acetate stimulation. Genistein also inhibited leukotriene C4 (LTC4) production in response to zymosan and calcium ionophore A23187 (IC50 = 10 and 15 microM, respectively) stimulation. Tyrphostin-25, a competitive inhibitor of substrate binding on PTK, inhibited zymosan-stimulated PGE2 and LTC4 production, IC50 = 20 and 7 microM, respectively. Neither genistein nor tyrophostin-25 had any effect on human synovial fluid phospholipase A2 (PLA2) activity in vitro or on cyclooxygenase activity in the intact macrophage; however, tyrphostin-25 did affect 5-lipoxygenase activity (determined from the metabolism of exogenously applied arachidonic acid). These results suggest PTK-mediated phosphorylation as a common event in the signal transduction mechanisms of different stimuli which activate PLA2 for arachidonic acid release and subsequent eicosanoid biosynthesis. Immunoblot analyses of zymosan-stimulated peritoneal exudate cells with the phosphotyrosine monoclonal antibody clone 4G10 demonstrated an increase in protein phosphotyrosine levels in eight major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: p59, 71, 76, 90, 100, 112, 125 and 150. Maximal phosphorylation of these protein substrates occurred after 1-2 min stimulation. Zymosan and LPS stimulation of peritoneal exudate cells produced similar patterns of protein tyrosine phosphorylation. Zymosan-stimulated tyrosine phosphorylation was inhibited by tyrphostin-25 in a concentration-dependent manner between 10 and 60 microM, demonstrating a similar concentration response between effects on tyrosine phosphorylation and eicosanoid biosynthesis in the murine peritoneal macrophage. The use of selective PTK inhibitors suggests a common role for PTK and tyrosine phosphorylation in eicosanoid biosynthesis in the murine peritoneal macrophage.

Animals↗
PubMed1993

Pharmacological characterization of WAY-121,520: a potent anti-inflammatory indomethacin-based inhibitor of 5-lipoxygenase (5-LO)/phospholipase A2 (PLA2).

WAY-121,520 inhibited human synovial fluid PLA2 (HSF-PLA2) (IC50 = 4 microM) using arachidonic acid-labeled E. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC4, IC50 = 4 nM) and rat PMN (LTB4, IC50 = 10 nM) and an ability to antagonize LTD4 binding to isolated guinea-pig trachea (pKB = 6.0). In vivo anti-inflammatory activity was noted in murine TPA-induced (ED50 = 91 micrograms/ear) and arachidonic acid-induced (66% inhibition at 400 micrograms/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA2 with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA2 and LTD4 antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.

Animals↗