Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bürkert, E.
Right arrow Articles by Werz, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bürkert, E.
Right arrow Articles by Werz, O.
(Journal of Leukocyte Biology. 2002;71:477-486.)
© 2002 by Society for Leukocyte Biology

Hypertonicity suppresses ionophore-induced product formation and translocation of 5-lipoxygenase in human leukocytes

Eva Bürkert*, Olof Rådmark2, Bengt Samuelsson2, Dieter Steinhilber* and Oliver Werz*

* Institute of Pharmaceutical Chemistry, University of Frankfurt, Germany; and
{dagger} Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden

Correspondence: Dr. Oliver Werz, Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany. E-mail: o.werz{at}pharmchem.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
5-Lipoxygenase (5-LO) initiates the biosynthesis of proinflammatory leukotrienes from arachidonic acid (AA). Here, we demonstrate that hypertonicity suppresses ionophore-induced 5-LO product formation reversibly in isolated human polymorphonuclear leukocytes (PMNL) and in Mono Mac 6 cells. Hypertonicity blocked the liberation of AA and abrogated translocation of 5-LO to the nuclear membrane. Accordingly, in the presence of exogenous AA, 5-LO product formation was less affected. The effects of hypertonicity were a result of cell shrinkage and not cytosolic hyperosmolarity. Hypertonicity did not inhibit the rapid increase in intracellular Ca2+ induced by ionophores but prevented the ionophore-induced activation of p38 MAPK-regulated MAPKAP kinases, which can phosphorylate and activate 5-LO (and cPLA2). In summary, we show that hypertonicity blocks agonist-induced release of AA, 5-LO product formation, and translocation and in parallel, prevents activation of p38 MAPK and downstream 5-LO kinases in leukocytes.

Key Words: leukotriene • p38 MAP kinase • arachidonic acid • cytosolic phospholipase A2 • cell stress • Mono Mac 6


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukotrienes (LTs) are lipid mediators with pivotal roles in normal immune responses as well as in inflammatory and allergic diseases, such as asthma and arthritis [1 ]. The biosynthesis of LTs is initiated by 5-lipoxygenase (5-LO), which converts free arachidonic acid (AA) first to 5-hydroperoxyeicosatetraenoic acid and subsequently to LTA4, which can be metabolized to LTB4 by LTA4 hydrolase or to LTC4 by LTC4 synthase, depending on the enzymes present [2 ].

Formation of LTs in leukocytes depends on the release of sufficient amounts of AA from endogenous pools by activated phospholipase A2 (PLA2) or via transcellular migration of AA, released from surrounding cells such as platelets or endothelial cells. Depending on the cell type, 5-LO can be present in the cytosol but also in a soluble pool of the nucleus of resting cells (for review on 5-LO, see ref. [3 ]). On cell stimulation by Ca2+-mobilizing agents (e.g., ionophore A23187, thapsigargin), soluble 5-LO translocates to the nuclear membrane where it colocalizes with 5-lipoxygenase-activating protein (FLAP) and cytosolic PLA2 (cPLA2). This migration to the nuclear membrane is believed to be necessary for substantial formation of LTs, and it is assumed that phosphorylation of 5-LO could influence its translocation to the nucleus and/or the association with the nuclear membrane and could stimulate its catalytic activity [4 5 6 ]. Moreover, the capacity of 5-LO to metabolize AA depends on several cofactors, such as Ca2+, adenosine 5'-triphosphate (ATP), phosphatidylcholines (membranes), hydroperoxides, and cellular proteins. In fact, it was demonstrated that 5-LO may interact with Grb-2, a coactosin-like protein, a transforming growth factor-ß (TGF-ß) receptor-associated protein, and a putative RNase III [7 , 8 ].

Hypertonicity (HT) leading to cell shrinkage has been shown to inhibit several neutrophil functions, such as superoxide production, phagocytosis, elastase release, adhesion, and cytokine production. However, the cellular mechanisms underlying these effects remain largely undefined [9 10 11 12 13 14 ]. Osmotic cell shrinkage triggers rapid tyrosine phosphorylation in a variety of cell types and was shown to activate mitogen-activated protein kinase (MAPK) pathways [13 , 15 16 17 18 19 20 ]. It is interesting that HT, which activated MAPK by itself, suppressed the activation of these enzymes by other stimuli [11 , 13 ].

Recently, cellular stress was shown to activate p38 MAPK and downstream MAPK-activated protein kinases (MAPKAPKs), which can phosphorylate 5-LO in vitro, and it was suggested that such phosphorylation increases 5-LO product formation in various leukocytes [6 , 21 , 22 ].

In this study, we present that HT, leading to cell shrinkage, reversibly abrogates ionophore-induced release of AA and 5-LO product formation, as well as translocation of 5-LO to the nuclear membrane in polymorphonuclear leukocytes (PMNL) and Mono Mac 6 (MM6) cells. We show that HT does not prevent rapid Ca2+ influx but prevents the ionophore-induced activation of p38 MAPK-regulated 5-LO kinases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI-1640 medium was from Gibco-BRL (Grand Island, NY), and fetal calf serum was obtained from Boehringer Mannheim (Mannheim, Germany). Insulin was a gift from Hoechst-Marion-Roussel (Frankfurt, Germany). Human TGF-ß1 was purified from outdated platelets as described [23 ]. Calcitriol was kindly provided by Dr. H. Wiesinger (Schering AG). Human recombinant 5-LO was expressed and purified as described [24 ]. [3H]AA was from Biotrend (Colonia, Germany), and [{gamma}-32P]ATP (110 TBq/mmol) was purchased from Amersham (Buckinghamshire, England). Nycoprep was from PAA Laboratories (Linz, Austria); phorbol 12-myristate 13-acetate (PMA), Ca2+ ionophore A23187, and thapsigargin were from Sigma Chemical Co. (Deisenhofen, Germany); and high-pressure liquid chromatography (HPLC) solvents were from Merck (Darmstadt, Germany). Fura-2 AM was from Calbiochem (Bad Soden, Germany).

Cells and cell incubations
MM6 cells were cultured and differentiated with TGF-ß and calcitriol as described [25 ]. Cells were harvested by centrifugation [200 g, 10 min at room temperature (RT)] and washed once in phosphate-buffered saline (PBS), pH 7.4. Human PMNL were isolated immediately from fresh leukocyte concentrates obtained at St. Markus Hospital (Frankfurt, Germany) as described [26 ]. Cells were resuspended in isotonic PBS containing 1 mg/ml glucose and 1 mM CaCl2 (PGC buffer). Hyperosmolar solutions were added prior to stimulation with the indicated agonists at 37°C at a final volume of 1 ml (see below). In some experiments, cells were washed by centrifugation (200 g, 5 min at RT) immediately after treatment with hyperosmolar solutions and were resuspended in 1 ml isotonic PGC buffer.

Determination of 5-lipoxygenase product formation
For assays of intact cells, freshly isolated PMNL (5x106) or differentiated MM6 cells (3x106) were finally resuspended in PGC buffer and preincubated as indicated. The reaction was started by addition of ionophore A23187, with or without exogenous AA at the indicated concentrations. Final volume of the incubations was 1 ml. After 10 min at 37°C, the reaction was stopped with 1 ml methanol and 30 µl 1 N HCl, and 200 ng prostaglandin B1 and 500 µl PBS were added. Formed 5-LO metabolites were extracted and analyzed by HPLC as described [27 ]. 5-LO product formation is expressed as ng 5-LO products per 106 cells, which includes LTB4 and its all-trans isomers, 5(S),12(S)-di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid [5(S),12(S)-DiHETE] and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid [5-H(p)ETE].

Permeabilization of PMNL
Freshly isolated PMNL were permeabilized using nystatin, according to the method of Krump et al. [28 ], with some minor changes. In brief, PMNL were resuspended in KS solution (115 mM KCl, 50 mM sucrose), collected by centrifugation (200 g, 5 min at RT), and finally resuspended in this solution at 5 x 107 cells/ml. After incubation in the absence or presence of 50 µg/ml nystatin for 10 min at 4°C, 0.3 M KCl was added to the cell suspension for 3 min at 4°C. Then, cells were washed by centrifugation and resuspended in KS solution containing 0.3 M KCl to remove nystatin, leading to resealing the plasma membrane. After 5 min at 37°C, cells were centrifuged, transferred into PGC buffer, and challenged by 2.5 µM ionophore in the absence or presence of 0.3 M KCl, and 5-LO translocation was determined as described below.

Determination of release of [3H]-labeled AA from PMNL and MM6 cells
Freshly isolated PMNL or differentiated MM6 cells were resuspended at 2 x 106/ml in RPMI-1640 medium containing 4.8 nM [3H]AA (corresponding to 0.25 µCi/ml, specific activity 200 Ci/mmol) and were incubated for 120 min at 37°C in 5% CO2 atmosphere. Thereafter, cells were collected by centrifugation and washed once with PBS and twice with PBS containing 2 mg/ml fatty acid-free albumin to remove unincorporated [3H]AA. Labeled PMNL (5x106) or MM6 cells (3x106) were resuspended in 1 ml PGC buffer containing 2 mg/ml fatty acid-free albumin and were preincubated as indicated. The reaction was started by addition of ionophore A23187. Final volume of the incubations was 1 ml. After 5 min at 37°C, the samples were put on ice for 2 min, and cells were centrifuged at 200 g for 5 min at RT. Aliquots (200 µl) of the supernatants were measured in a beta-counter (Micro Beta Trilux, Perkin Elmer, Foster City, CA) to detect the amounts of [3H]-labeled AA released into the cell medium.

Subcellular redistribution of 5-LO
Subcellular localization of 5-LO was performed as described previously [29 ]. In brief, MM6 cells (1x107) or human PMNL (3x107) were resuspended in PGC buffer and preincubated as indicated at 37°C. After addition of ionophore at the indicated concentrations, samples were incubated for another 10 min at 37°C, chilled on ice for 3–5 min, and nuclear and nonnuclear fractions were obtained after cell lysis by 0.1% Nonidet P-40 (NP-40). Aliquots of nuclear and nonnuclear fractions were mixed immediately with the same volume of 2 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (SDS-b), heated for 6 min at 95°C, and analyzed for 5-LO protein by SDS-PAGE and immunoblotting.

Measurement of intracellular calcium levels
Freshly isolated PMNL (1x107 in 1 ml PGC buffer) were incubated with 2 µM Fura-2 AM for 30 min at 37°C. Cells were washed, resuspended in PGC buffer (final vol, 1 ml), and transferred into a thermally controlled (37°C) fluorimeter cuvette in a spectrofluorometer (Aminco-Bowman Series 2, Thermo Spectronic, Rochester, NY) with continuous stirring. The fluorescence emission at 510 nm was measured after excitation at 340 and 380 nm, respectively. Intracellular Ca2+ levels were calculated according to the method of Grynkiewicz et al. [30 ], and Fmax (maximal fluorescence) was obtained by lysing the cells with 1% Triton-X 100 and Fmin (minimal fluorescence), by chelating Ca2+ with 10 mM ethylenediaminetetraacetate.

Immunoblot analysis of total cell lysates and subcellular fractions; antibodies
Total cell lysates (20 µl) and aliquots (25 µl) of pair-wise subcellular fractions (cytosol and nucleus, corresponding to equal amounts of cells) were mixed with 4 µl glycerol/0.1% bromphenol blue (1:1, vol/vol) and analyzed by SDS-PAGE using a Mini Protean system (Bio-Rad, Hercules, CA) on a 10% gel. After electroblot to nitrocellulose membrane (Amersham Pharmacia, Little Chalfont, UK), membranes were blocked with 5% nonfat dry milk in 50 mM Tris/HCl, pH 7.4, and 100 mM NaCl Tris-buffered saline (TBS) for 1 h at RT. Membranes were washed and then incubated with primary antibody overnight at 4°C. Anti-5-LO antiserum (AK7, 1551) was affinity-purified on a 5-LO column, and phospho-specific antibodies recognizing p38 MAPK (Thr180/Tyr182) were obtained from New England Biolabs (Beverly, MA) and were used as 1:2000 dilution. Antibody against p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA). The membranes were washed with TBS and incubated with 1:1000 dilution of alkaline phosphatase (AP)-conjugated immunoglobulin G (Sigma Chemical Co.) for 2 h at RT. After washing with TBS and TBS plus 0.1% NP-40, proteins were visualized with the AP substrates, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma Chemical Co.) in detection buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2).

In-gel kinase assay
Incubations were stopped by addition of the same volume of SDS-b and heated for 6 min at 95°C. Total cell lysates of PMNL and MM6 cells corresponding to 0.5 x 106 cells were analyzed for 5-LO kinase activity by in-gel kinase assay using purified 5-LO (0.2 mg/ml) as substrate as described [6 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypertonicity suppresses 5-LO product formation in ionophore-stimulated PMNL and MM6 cells
To investigate the effects of HT on ionophore-induced 5-LO product formation in human leukocytes, hyperosmotic solutions were added to freshly isolated PMNL or PMA-primed MM6 cells (100 nM PMA for 10 min prior to stimulation; compare with ref. [29 ]), which were resuspended in isotonic PGC buffer (approximately 290 mosmol/l). Subsequently, PMNL were stimulated with 2.5 µM and MM6 cells with 5 µM ionophore in the absence or presence of 40 µM exogenous AA, and 5-LO products were determined by HPLC. Figure 1 shows that various hypertonic solutions, such as NaCl, KCl, and Na2SO4, as well as the nonionic impermeable sucrose, strongly suppressed ionophore-induced 5-LO product formation in human PMNL (Fig. 1A) , and PMA-primed MM6 cells (Fig. 1B) . Because different salt solutions and nonionic sucrose suppressed 5-LO product formation, it seems that neither a specific ion nor increase in ionic strength is required for this effect. No significant shift in the ratio of 5-H(p)ETE, LTB4, and its all-trans isomers was observed after treatment with hyperosmotic solutions. Similar effects of HT on 5-LO product formation were observed in PMNL when the Ca2+-mobilizing agents thapsigargin or ionomycin were used as stimuli (unpublished results). It is puzzling that when sorbitol (0.4–0.8 M) was used to increase osmolarity, 5-LO product formation was strongly suppressed in MM6 cells but only marginally reduced in PMNL (unpublished results).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Hypertonicity suppresses ionophore-induced 5-LO product formation in PMNL and MM6 cells. (A) Isolated PMNL. To 5 x 106 cells in PGC buffer, solutions of the indicated compounds were added directly prior to stimulation with 2.5 µM A23187. (B) MM6 cells. Cells (1.5x106) in PGC buffer were preincubated with 100 nM PMA for 10 min at 37°C. Then, solutions of the indicated compounds were added directly prior to stimulation with 5 µM A23187. Final incubation volume was 1 ml, and the samples were incubated at 37°C for 10 min. 5-LO product formation was determined by HPLC. The control values (100%) were 51.8 ± 6.7 ng/106 PMNL and 83.2 ± 6.6 ng/106 MM6 cells. Results are given as mean + SE, n = 3–5.

 
In both cell types, suppression of 5-LO product formation by HT was impaired when exogenous AA was added together with ionophore for cell stimulation. As can be seen from Figure 2 A , the EC50 value for inhibition of ionophore-induced 5-LO product formation in the absence of exogenous AA by NaCl was about 430 mosmol/l and at 690 mosmol/l product formation, was almost abolished completely. Although in the presence of exogenous substrate the onset in inhibition by HT was similar, a further increase in osmolarity gave no enhanced inhibition in the formation of 5-LO products. Similar results were obtained with MM6 cells, where 5-LO product formation was suppressed efficiently by sucrose (at 790 mosmol/l) in the absence of exogenous AA, whereas about 30% inhibition was achieved in the presence of exogenous AA (Fig. 2B) . Also in presence of exogenous AA, HT caused no changes in the 5-LO metabolite pattern. Pretreatment of cells for longer time periods (>10 s–30 min) in hypertonic medium caused the same reduction of 5-LO product formation as simultaneous addition of hypertonic solutions together with ionophore (unpublished results). 5-LO product formation in broken cell preparations was not affected by increased osmolarity.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Effects of exogenous AA on hypertonicity-suppressed 5-LO product formation. (A) Isolated PMNL. NaCl at the indicated concentrations was added to 5 x 106 cells and resuspended in PGC buffer to a final volume of 1 ml, directly prior to stimulation with 2.5 µM A23187 or with 2.5 µM A23187 plus 40 µM AA as indicated. After 10 min at 37°C, 5-LO product formation was determined. The control values (100%) were 51.8 ± 6.7 and 75.4 ± 6.4 ng/106 cells in the absence or presence of exogenous AA, respectively. (B) MM6 cells. When 5-LO product formation was determined in the absence of exogenous AA, cells were preincubated with 100 nM PMA for 10 min at 37°C. Sucrose at the indicated concentrations was added to 1.5 x 106 cells and resuspended in PGC buffer to a final volume of 1 ml. Then, cells were stimulated with 5 µM A23187 or with 5 µM A23187 plus 40 µM AA as indicated at 37°C for 10 min, and 5-LO product formation was determined. The control values (100%) were 83.4 ± 6.6 and 460 ± 25.7 ng/106 cells in the absence or presence of exogenous AA, respectively. Results are given as mean + SE, n = 3–4.

 
Next, we determined whether HT could also terminate ongoing 5-LO product formation after ionophore challenge. PMNL were stimulated with ionophore in isotonic buffer. After various time periods (indicated in Fig. 3 ), aliquots of these incubations were mixed with the same volume of ice-cold methanol to stop product formation immediately or were transferred to tubes containing hypertonic NaCl solution (0.3 M NaCl, final concentration) and incubated for a further 10 min at 37°C. As can be seen in Figure 3 5 -LO product formation was terminated immediately by increasing osmolarity, almost as rapidly as by cell destruction with methanol. Thus, the inhibitory effect of HT appears to be extremely rapid and occurs within seconds upon exposure to HT at all stages of 5-LO product formation. Comparable results were obtained with MM6 cells (unpublished results).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Rapid termination of 5-LO product formation by hypertonicity. Freshly isolated PMNL (2x108), resuspended in 14 ml PGC buffer, were stimulated with 2.5 µM A23187. After the time periods indicated in the figure, aliquots (700 µl) corresponding to 5 x 106 cells were mixed with the same volume of ice-cold methanol or alternatively transferred to tubes containing 300 µl 1 M NaCl solution (0.3 M NaCl, final concentration, 1 ml final volume) and incubated for a further 10 min at 37°C. For all samples, 5-LO product formation was determined by HPLC. Similar results were obtained in two additional independent experiments.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Hyperosmolarity blocks translocation of 5-LO and reduces 5-LO product formation. (A) Isolated PMNL. NaCl at the indicated concentrations was added to 5 x 106 (5-LO product formation) or 3 x 107 (5-LO translocation) cells, resuspended in PGC buffer to a final volume of 1 ml. Then, cells were stimulated with 2.5 µM A23187 or with 2.5 µM A23187 plus 40 µM AA as indicated at 37°C for 10 min. (B) MM6 cells. Cells (1.5x106 for 5-LO product formation or 1x107 for 5-LO translocation) were primed for 10 min at 37°C with 100 nM PMA. Then, NaCl at the indicated concentrations was added, and cells were stimulated with 5 µM A23187 or with A23187 (5 µM) plus AA (40 µM) for 10 min. 5-LO product formation was determined by HPLC. Results are given as mean + SE, n = 3–4. 5-LO translocation was determined by Western blot analysis of 5-LO in subcellular fractions (non-nuclear, nuclear) following cell fractionation by detergent lysis (0.1% NP-40). Pair-wise samples (non-nuclear, nuclear) correspond to the identical cell numbers. Similar results were obtained in two additional, independent experiments.

 
Hypertonicity suppresses the release of [3H]AA from ionophore-stimulated PMNL and MM6 cells
Suppression of 5-LO product formation by HT could be a result of an inadequate supply of endogenous substrate, thus the release of AA from PMNL and MM6 cells prelabeled with [3H]AA was determined. As shown in Figure 4 , NaCl caused a dose-dependent suppression of ionophore-induced release of AA, and after addition of 200 mM NaCl, the ionophore-induced release of AA was abolished. Sucrose gave comparable effects, and similar inhibition of AA release was determined for MM6 cells primed with PMA and subsequently challenged with ionophore (not shown).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Hypertonicity blocks the release of [3H]AA from phospholipids. Freshly isolated human PMNL (2x106/ml in RPMI-1640 medium) were prelabeled with 0.25 µCi/ml [3H]AA for 120 min at 37°C and 5% CO2. After removal of unincorporated [3H]AA, cells (5x106 in 1 ml PGC buffer, containing 2 mg/ml fatty acid-free albumin) were treated with the indicated additives, and 2.5 µM A23187 was added and incubated for 5 min at 37°C. Free (nonesterified) [3H]AA was determined as described in Materials and Methods. Results are given as mean + SE, n = 5.

 
Hypertonicity blocks translocation of 5-LO from the cytosol to the nucleus in PMNL and MM6 cells
Because 5-LO redistribution from the cytosol to the nucleus is a determinant for substantial formation of 5-LO products, we addressed whether HT-mediated 5-LO inhibition was accompanied by reduced 5-LO translocation. Localization of 5-LO was assessed by means of subcellular fractionation using detergent lysis and 5-LO immunoblotting [29 ]. As shown in Figure 5 A , 5-LO in resting PMNL is located mainly in the cytosol, and stimulation with ionophore leads to considerable translocation of the enzyme to the nucleus. HT, induced by NaCl (or sucrose; not shown), blocked ionophore-induced translocation of 5-LO in PMNL, and a significant correlation regarding dose-dependencies for inhibition of 5-LO product synthesis was found. Also, HT blocked 5-LO translocation in PMNL induced by thapsigargin (unpublished results). Similarly, as found for PMNL, ionophore-induced 5-LO translocation in PMA-primed MM6 cells was reduced by NaCl (Fig. 5B) or sucrose (not shown). Although exogenous AA could restore 5-LO product formation partially (Fig. 2) , the presence of exogenous AA did not abolish the HT-induced suppression in 5-LO translocation (Fig. 5) .

Cell shrinkage reversibly blocks translocation and 5-LO product synthesis
We attempted to determine whether cell destruction, intracellular hyperosmolarity, or cell shrinkage reduced translocation and product formation of 5-LO. To determine if HT-mediated effects were reversible, PMNL and MM6 cells were exposed to hypertonic solutions for 5 min and were centrifuged, and hyperosmolar medium was then replaced by isotonic buffer. PMNL were stimulated with ionophore subsequently, and MM6 cells were primed with PMA for 10 min and then stimulated with ionophore. As shown in Figure 6 , reconstitution of isotonic conditions (fresh buffer) restored activity and translocation of 5-LO in both cell types completely. When NaCl-treated PMNL were transferred to fresh buffer and re-exposed to hypertonic NaCl, 5-LO product formation was suppressed again (unpublished results).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Inhibition of 5-LO translocation and 5-LO product formation by hypertonicity is reversible. (A) Isolated PMNL. Sucrose or NaCl (at the indicated final concentrations) was added to 5 x 106 (5-LO product formation) or 3 x 107 (5-LO translocation) cells, resuspended in PGC buffer as indicated in the figure. Cells were stimulated with 2.5 µM A23187 for 10 min at 37°C or alternatively centrifuged, resuspended in fresh PGC buffer, and stimulated with 2.5 µM A23187 for 10 min at 37°C as indicated. (B) MM6 cells. Cells (1.5x106, 5-LO product formation, or 1x107, 5-LO translocation) were primed for 10 min at 37°C with 100 nM PMA. Then, NaCl (0.3 M) was added as indicated, and cells were stimulated with 5 µM A23187 for 10 min at 37°C. Alternatively, non-PMA-primed cells were treated with NaCl (0.3 M, final concentration), centrifuged, resuspended in fresh PGC buffer, and primed with 100 nM PMA for 10 min at 37°C prior to stimulation with 5 µM A23187 for another 10 min at 37°C. 5-LO product formation and 5-LO translocation were determined as described in the legend to Figure 4 . The control values (100%) for 5-LO product formation were 51.8 ± 6.7 and 83.2 ± 6.6 ng/106 PMNL and MM6 cells, respectively.

 
To exclude that an increase in intracellular osmolarity by itself prevented 5-LO translocation and LT synthesis, urea (which in contrast to sucrose and salts passes the plasma membrane without affecting the cell volume) was used to increase cytosolic osmolarity. Addition of 500–800 mM urea to the isotonic incubation buffer (resulting in 790–1090 mosmol/l) did not affect ionophore-induced LT synthesis or 5-LO translocation in both cell types (Fig. 7 ). Also the ionophore-induced release of AA was unaffected by 0.5 M urea (not shown). In control experiments, coaddition of NaCl or sucrose, together with urea, again gave reduced formation of 5-LO products and association with the nucleus (unpublished results).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 7. Cell shrinkage suppresses 5-LO translocation and 5-LO product formation. (A) Isolated PMNL. Sucrose, NaCl, or urea (at the indicated final concentrations) was added to 5 x 106 (5-LO product formation) or 3 x 107 (5-LO translocation) cells, resuspended in PGC buffer as indicated in the figure. Then, cells were stimulated with 2.5 µM A23187 for 10 min at 37°C. (B) MM6 cells. Cells (1.5x106, 5-LO product formation, or 1x107, 5-LO translocation) were primed for 10 min at 37°C with 100 nM PMA. Then, NaCl or urea was added as indicated, and cells were stimulated with 5 µM A23187 for 10 min at 37°C. 5-LO product formation and 5-LO translocation were determined as described in the legend to Figure 4 . The control values (100%) for 5-LO product formation were 51.8 ± 6.7 and 83.2 ± 6.6 ng/106 PMNL and MM6 cells, respectively.

 
Finally, PMNL were permeabilized using nystatin, which forms pores in the plasma membrane, allowing penetration of ions to compensate extracellular hyperosmolarity without affecting the cell volume. Attempts to assess 5-LO product formation using this model failed, because we were unable to detect 5-LO products after this procedure, regardless of whether cells were exposed to HT or treated under isotonic conditions. Also, nystatin (50 µg/ml) inhibited 5-LO activity in cell homogenates incubated with AA, indicating a direct inhibitory effect of nystatin on 5-LO. The result of the investigation of 5-LO translocation is shown in Figure 8 . 5-LO in PMNL, pretreated with (lane 3) or without (lane 1) 50 µg/ml nystatin, translocated to the nucleus upon challenge with ionophore. In contrast to nonpermeabilized cells, where 5-LO translocation was blocked by 0.3 M KCl (lane 2), treatment with nystatin prevented the effects of HT (0.3 M KCl), and 5-LO translocated to the nucleus after ionophore treatment (lane 4). Taken together, the results indicate that the effects of HT are reversible and that cell shrinkage triggers the suppression of 5-LO translocation and accompanied reduction of the formation of 5-LO products in leukocytes.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 8. 5-LO translocation in nystatin-permeabilized PMNL. Freshly isolated PMNL were permeabilized by nystatin as described in Materials and Methods and incubated with 2.5 µM ionophore in the absence or presence of additional 0.3 M KCl for 10 min as indicated in the figure. 5-LO translocation was determined by Western blot analysis of 5-LO in subcellular fractions (non-nuclear, nuclear), following cell fractionation by detergent lysis (0.1% NP-40). In contrast to untreated cells (lanes 1 and 2), hypertonic KCl could not prevent 5-LO translocation in PMNL treated with nystatin (lanes 3 and 4), where cell shrinkage was prevented. Similar results were obtained in two additional, independent experiments.

 
Effects of hypertonicity on Ca2+ mobilization
Because increased intracellular Ca2+ is considered important for the release of AA and the formation of 5-LO products in cells stimulated by ionophores and also by other 5-LO stimuli, such as platelet-activating factor or N-formyl-methionyl-leucyl-phenylalanine (fMLP) [3 ], we determined the effects of HT on the Ca2+ influx in PMNL using the Fura-2 method. As shown in Figure 9 , treatment of PMNL with ionophore (ionomycin, 2.5 µM) caused a rapid and sustained elevation of intracellular Ca2+ (left panel), and addition of 0.3 M NaCl (or 0.5 M sucrose; not shown) prior to stimulation did not prevent this rapid Ca2+ response (middle panel). Also, when cells were first stimulated with ionomycin, and subsequently NaCl (0.3 M) was added after 10–15 s, increased intracellular Ca2+ levels were not abrogated (right panel).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 9. Effects of hypertonicity on ionophore-induced Ca2+ influx. Fura-2-loaded PMNL (1x107 in PGC buffer) were treated as indicated at 37°C, and the fluorescence was measured; final volume was 1 ml. Intracellular free Ca2+ was calculated as described. The monitored curves show one typical experiment out of four to five.

 
Hypertonicity suppresses ionophore-induced activation of p38 MAPK and 5-LO kinases
To determine MAPK activities relevant for 5-LO activation, we performed Western blot experiments using antibodies that detect the activated, dually phosphorylated form of p38 MAPK, as well as in-gel kinase assays using 5-LO as substrate. After cell stimulation for 2.5 min, total cell lysates were analyzed for kinase activation. PMNL shrinkage, induced by 0.3 M NaCl or 0.5 M sucrose, led to phosphorylated (active) p38 MAPK, whereas treatment with cell-permeable urea (0.5 M) gave no p38 MAPK phosphorylation (Fig. 10 A ). Also stimulation of PMNL with ionophore resulted in strong p38 MAPK phosphorylation, whereas addition of NaCl or sucrose prior to ionophore prevented p38 MAPK activation. Urea added together with ionophore did not give this effect. In in-gel kinase assays, ionophore and NaCl (0.3 M) by themselves gave phosphorylated bands at 40, 47, and 55 kDa, resulting from activated p38 MAPK-regulated kinases (MK2 and MK3; see ref. [6 ]), and coaddition of NaCl suppressed these ionophore-induced kinase activities (Fig. 10B) . Sorbitol, which was much less efficient in suppression of 5-LO translocation and 5-LO product formation in PMNL, was also less effective in the prevention of 5-LO kinase activation induced by A23187. Taken together, the results show that HT, particularly when induced by NaCl, counteracted ionophore-induced activation of p38 MAPK and downstream 5-LO kinases in parallel with suppressed 5-LO product formation and translocation.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 10. Hypertonicity prevents activation of p38 MAPK and downstream 5-LO kinases. Freshly isolated PMNL (5x106), resuspended in PGC buffer, were stimulated with the indicated additives at 37°C. Final volume was 100 µl. After 2.5 min, the reaction was stopped by addition of SDS-b, and the samples were boiled at 95°C for 6 min. (A) Determination of p38 MAPK activation. Samples were electrophoresed and analyzed for dually phosphorylated p38 MAPK by immunoblotting (upper panel), and equal amounts of protein were evaluated with anti-p38 MAPK antibodies (lower panel). (B) Activation of p38 MAPK-regulated 5-LO kinases. Samples were electrophoresed on a polyacrylamide gel that contained 0.2 mg/ml 5-LO as substrate. In-gel kinase assay was performed as described in Materials and Methods. Arrows indicate the expected positions for MK2 and MK3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that HT suppresses the synthesis of 5-LO products in PMNL, as well as in differentiated MM6 cells, stimulated with Ca2+-mobilizing ionophore A23187. For both cell types, HT reduced the liberation of endogenous AA and suppressed the translocation of 5-LO from the cytosol to the nucleus. Different salts and sucrose were used to increase tonicity with equal results. The effects of HT were reversible, did not cause cell destruction (Fig. 6) , and were accompanied by reduced activities of p38 MAPK and downstream 5-LO kinases. It appeared that these effects of HT are a result of cell shrinkage, because an increase in the cytosolic osmolarity by the cell-permeable solute urea did not block the release of AA, 5-LO translocation, or 5-LO product generation (Fig. 7) . Also in contrast to NaCl or sucrose, urea did not suppress ionophore-induced p38 MAPK activation (Fig. 10) . Moreover, when cell shrinkage was prevented by permeabilization of PMNL with nystatin, KCl (which can then pass the plasma membrane) no longer suppressed 5-LO translocation.

Particularly in the absence of exogenous substrate, suppression of 5-LO product formation by HT was prominent; thus, limited substrate supply seems to contribute to low 5-LO product synthesis. One reason for limited substrate supply should be impaired activity of cPLA2, which is assumed to release AA at the nuclear membrane for LT formation in PMNL and monocytes [31 ]. In fact, HT strongly suppressed ionophore-induced release of AA (Fig. 4) . Cytosolic PLA2 is regulated by Ca2+ binding to its C2 domain and/or phosphorylation by members of the MAPK family, depending on the cell type and the stimulus used [32 , 33 ]. In this study, HT prevented ionophore-induced activation of p38 MAPK, implying that reduced kinase activities could possibly contribute to low AA release. However, opposite effects were observed for human platelets, where HT increased ionophore-induced liberation of AA and the activity of cPLA2, accompanied by elevated MAPK activities [34 ]. Further studies are necessary to elucidate the mechanisms by which AA release is suppressed in leukocytes by HT.

Another reason for impaired 5-LO product synthesis could be that HT impaired 5-LO translocation. For efficient LTA4 formation in intact cells, 5-LO has to translocate from a soluble compartment to the nuclear membrane, where it colocalizes with cPLA2 and FLAP [35 , 36 ], and an orchestrated interplay between these enzymes is considered important for substantial LT synthesis. HT blocked translocation of 5-LO from the cytosol to the nucleus, with a clear correlation with the dose response for inhibition of 5-LO product formation (Fig. 5) . We found before that in MM6 cells, stimulation with ionophore alone failed to induce 5-LO translocation and product formation, but after priming with PMA, subsequent ionophore stimulation led to 5-LO redistribution to the nucleus, and 5-LO product synthesis from endogenous substrate occurred [29 ]. Nonprimed cells (5-LO remains in the cytosol) gave prominent 5-LO product formation after stimulation with ionophore plus exogenous AA. We concluded that in presence of exogenous AA, 5-LO in the cytosol has ample supply of substrate, and 5-LO product formation can occur without translocation. In this study, also in the presence of exogenous AA, HT still reduced 5-LO product formation to about 60–70% of control, indicating that translocation also may be beneficial for conversion of exogenous substrate. Indeed, it was found that FLAP also stimulated the conversion of exogenous AA [37 ]. Taken together, the strong suppression of 5-LO product formation by HT in cells stimulated with ionophore alone seems to be because of the inhibition of cPLA2, causing low substrate release, but also as a result of the inability of 5-LO to translocate to the nuclear membrane, where AA is presented by FLAP for efficient metabolism.

When Ca2+ ionophore activates 5-LO in PMNL and other types of leukocytes, one important event is Ca2+ binding to the N-terminal C2-like domain of 5-LO [38 ], which in turn promotes membrane association similar to cPLA2 activation by Ca2+ [31 ]. Thus, elevation of intracellular Ca2+ is a determinant for activation of cPLA2 and 5-LO, and it seemed possible that HT could prevent the activation of these enzymes as a result of interference with ionophore-induced Ca2+ influx. However, in agreement with others [13 , 39 , 40 ], HT caused no significant suppression of agonist-induced Ca2+ mobilization compared with isotonic conditions (Fig. 9) . Importantly, elevated Ca2+ levels in ionophore-stimulated cells were not decreased when hypertonic NaCl was added 10–15 s after ionophore stimulation. This is in contrast to 5-LO product formation, which was immediately blocked after NaCl addition (Fig. 3) .

p38 MAPK-regulated MKs can phosphorylate 5-LO in vitro [6 ], and there is increasing evidence that p38 MAPK and MKs lead to activation of 5-LO in PMNL and MM6 cells [6 , 22 ]. Different types of cell stress (sodium arsenite, osmotic stress) could activate p38 MAPK pathways and 5-LO in PMNL, also after chelation of Ca2+. The p38 MAPK inhibitor SB203580 was quite efficient to inhibit cell stress-induced 5-LO product formation (IC50, 5 µM), whereas ionophore-induced 5-LO product synthesis was less sensitive (IC50, 30 µM) [22 ]. Also, in rat fibroblasts transfected with 5-LO-GFP, a high concentration of SB203580 (15 µM, which may not be specific for p38 MAPK) was required to inhibit ionophore-induced 5-LO translocation to the nucleus [41 ]. In comparison, for inhibition of MK activity in PMNL stimulated with ionophore, 3 µM SB203580 was sufficient [6 ]. Taken togeher, Ca2+ mobilizing agents or HT by themselves are sufficient for p38 MAPK activation (Fig. 10) , and each stimulus itself can activate 5-LO for product formation [22 ]. However, it appears that ionophores leading to Ca2+ mobilization activate 5-LO primarily by the Ca2+ effect (compare above), which does not depend on p38 MAPK activity.

In view of the 5-LO stimulatory effects of Ca2+ and HT-induced p38 MAPK activity, it is puzzling that when PMNL were subjected to HT in combination with ionophore, 5-LO product synthesis was downregulated. A change in phosphorylation processes may be one factor involved, because a combination of ionophore and HT gave reduced activity of p38 MAPK and MKs in PMNL (Fig. 10) . However, downregulation of p38 MAPK and MKs cannot be the entire explanation, because SB203580 did not counteract ionophore-induced 5-LO activity in PMNL efficiently [6 , 22 ]. However, the protein-kinase inhibitor calphostin C suppressed ionophore-induced 5-LO product formation from endogenous AA in MM6 cells and PMNL [29 ]. Calphostin C prevented translocation of 5-LO to the nuclear membrane, and when exogenous substrate was added, 5-LO product synthesis was prominent, probably because of cytosolic 5-LO still acting on the added AA. Thus, calphostin C, as HT in combination with ionophore, may also impair other phosphorylation events of importance for cellular 5-LO activity.

In analogy with our observations, others showed that in PMNL, HT alone activates p38 MAPK but at the same time, prevents p38 MAPK activation in response to agonists such as lipopolysaccharide [11 ]. Junger et al. [13 ] showed that in PMNL, HT by itself stimulated p38 MAPK and degranulation, whereas HT blocked activation of p38 MAPK and reduced degranulation in response to fMLP. To date, it is unclear how HT can uncouple ionophore-stimulated p38 MAPK activation in PMNL. It was suggested that HT could render p38 MAPK refractory to other stimuli or could uncouple signaling pathways upstream of p38 MAPK [11 , 13 ]. A further complication is that these effects seem to be cell-type specific. In a human B-cell line (BL41-E95-A), ionophore and HT (or other types of cell stress) were required for substantial p38 MAPK activation, which in turn, was necessary for 5-LO product synthesis. Thus, in B cells, ionophore and HT act in conjunction to stimulate p38 MAPK and 5-LO [21 ]. Remarkably, in the B cells, translocation of 5-LO to the nucleus did not occur under any conditions, and exogenous AA was always required for the cytosolic 5-LO activity.

HT was shown to result in dramatic changes in the cytoskeleton [42 , 43 ], which might cause a sustained interaction of 5-LO with cytoskeletal proteins such as actin, possibly retaining the enzyme within this locus. In fact, 5-LO binds actin in vitro, and it was postulated that in resting leukocytes, 5-LO might be bound to the cytoskeleton [5 , 7 ]. 5-LO interacts with coactosin-like protein (CLP, an actin-binding protein), and in vitro, 5-LO can inhibit actin polymerization and compete with F-actin for CLP binding [8 , 44 ]. It was shown that in neutrophils, HT induced sustained polymerization of actin [43 ], and inhibition of actin polymerization by cytochalasin B increased fMLP-induced LT synthesis [45 , 46 ]. It was speculated before that the translocation process may involve phosphorylation of 5-LO and other proteins and that the cytoskeleton may be involved [5 ]. Possibly, the effects of HT on Ca2+-induced 5-LO activation may involve alterations in the actin cytoskeleton, which prevent nuclear translocation of 5-LO. Moreover, osmotic changes of cytoskeletal structures may also affect other protein interactions and intracellular signal-transduction mechanisms relevant for cellular 5-LO activity. Rearrangement of the actin cytoskeleton was also suggested to be the reason for impaired 5-LO activity in PMNL subjected to sulphatide-induced cell spreading [47 ].

Collectively, we show here that HT, via cell shrinkage, prevents ionophore-induced 5-LO translocation and product formation. Downregulation of p38 MAPK pathways may be one factor involved, but other mechanisms putatively involving the cytoskeleton could also be operative. Inhibition of ionophore-activated LT formation is another example of suppression of Ca2+-induced phagocyte functions by HT.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from the Fonds der Chemischen Industrie, the Swedish Medical Research Council (03X-217), the European Union, and the Verum Foundation. We thank Agneta Nordberg, Astrid Neuss, and Dagmar Szellas for expert technical assistance.

Received October 2, 2001; revised November 22, 2001; accepted November 26, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Samuelsson, B. (1983) Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation Science 220,568-575[Abstract/Free Full Text]
  2. Samuelsson, B., Dahlén, S-E., Lindgren, J-anA., Rouzer, C. A., Serhan, C. N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects Science 237,1171-1176[Abstract/Free Full Text]
  3. Rdmark, O. P. (2000) The molecular biology and regulation of 5-lipoxygenase Am. J. Respir. Crit. Care Med. 161,S11-S15[Free Full Text]
  4. Lepley, R. A., Fitzpatrick, F. A. (1996) Inhibition of mitogen-activated protein kinase kinase blocks activation and redistribution of 5-lipoxygenase in HL-60 cells Arch. Biochem. Biophys. 331,141-144[Medline]
  5. Lepley, R. A., Muskardin, D. T., Fitzpatrick, F. A. (1996) Tyrosine kinase activity modulates catalysis and translocation of cellular 5-lipoxygenase J. Biol. Chem. 271,6179-6184[Abstract/Free Full Text]
  6. Werz, O., Klemm, J., Samuelsson, B., Rdmark, O. (2000) 5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases Proc. Natl. Acad. Sci. USA 97,5261-5266[Abstract/Free Full Text]
  7. Lepley, R. A., Fitzpatrick, F. A. (1994) 5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins J. Biol. Chem. 269,24163-24168[Abstract/Free Full Text]
  8. Provost, P., Samuelsson, B., Rdmark, O. (1999) Interaction of 5-lipoxygenase with cellular proteins Proc. Natl. Acad. Sci. USA 96,1881-1885[Abstract/Free Full Text]
  9. Takahashi, K., Matsumoto, T., Kubo, S., Haraoka, M., Tanaka, M., Kumazawa, J. (1994) Influence of hyperosmotic environment comparable to the renal medulla upon membrane NADPH oxidase of human polymorphonuclear leukocytes J. Urol. 152,1622-1625[Medline]
  10. Sato, N., Kashima, K., Shimizu, H., Uehara, Y., Shimomura, Y., Mori, M. (1993) Hypertonic glucose inhibits the production of oxygen-derived free radicals by rat neutrophils Life Sci 52,1481-1486[Medline]
  11. Rizoli, S. B., Kapus, A., Parodo, J., Rotstein, O. D. (1999) Hypertonicity prevents lipopolysaccharide-stimulated CD11b/CD18 expression in human neutrophils in vitro: role for p38 inhibition J. Trauma 46,794-799[Medline]
  12. Kuchkina, N. V., Orlov, S. N., Pokudin, N. I., Chuchalin, A. G. (1993) Volume-dependent regulation of the respiratory burst of activated human neutrophils Experientia 49,995-997[Medline]
  13. Junger, W. G., Hoyt, D. B., Davis, R. E., Herdon-Remelius, C., Namiki, S., Junger, H., Loomis, W., Altman, A. (1998) Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38 J. Clin. Investig. 101,2768-2779[Medline]
  14. Hampton, M. B., Chambers, S. T., Vissers, M. C., Winterbourn, C. C. (1994) Bacterial killing by neutrophils in hypertonic environments J. Infect. Dis. 169,839-846[Medline]
  15. Han, J., Lee, J. D., Bibbs, L., Ulevitch, R. J. (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells Science 265,808-811[Abstract/Free Full Text]
  16. Galcheva-Gargova, Z., Derijard, B., Wu, I. H., Davis, R. J. (1994) An osmosensing signal transduction pathway in mammalian cells Science 265,806-808[Abstract/Free Full Text]
  17. Itoh, T., Yamauchi, A., Miyai, A., Yokoyama, K., Kamada, T., Ueda, N., Fujiwara, Y. (1994) Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells J. Clin. Investig. 93,2387-2392
  18. Rizoli, S. B., Rotstein, O. D., Kapus, A. (1999) Cell volume-dependent regulation of L-selectin shedding in neutrophils. A role for p38 mitogen-activated protein kinase J. Biol. Chem. 274,22072-22080[Abstract/Free Full Text]
  19. Szaszi, K., Buday, L., Kapus, A. (1997) Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells J. Biol. Chem. 272,16670-16678[Abstract/Free Full Text]
  20. Kapus, A., Szaszi, K., Sun, J., Rizoli, S., Rotstein, O. D. (1999) Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers J. Biol. Chem. 274,8093-8102[Abstract/Free Full Text]
  21. Werz, O., Klemm, J., Rådmark, O., Samuelsson, B. (2001) p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line J. Leukoc. Biol. 70,830-838[Abstract/Free Full Text]
  22. Werz, O., Buerkert, E., Samuelsson, B., R;anadmark, O., Steinhilber, D. (2001) Activation of 5-lipoxygenase by cell stress is calcium-independent in human polymorphonuclear leukocytes Blood in press
  23. Werz, O., Brungs, M., Steinhilber, D. (1996) Purification of transforming growth factor beta1 from human platelets Pharmazie 51,893-896[Medline]
  24. Hammarberg, T., Rdmark, O. (1999) 5-Lipoxygenase binds calcium Biochemistry 38,4441-4447[Medline]
  25. Brungs, M., Rådmark, O., Samuelsson, B., Steinhilber, D. (1995) Sequential induction of 5-lipoxygenase gene expression and activity in Mono Mac 6 cells by transforming growth factor-beta and 1,25-dihydroxyvitamin D3 Proc. Natl. Acad. Sci. USA 92,107-111[Abstract/Free Full Text]
  26. Böyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood Scand. J. Clin. Lab. Investig. 97(Suppl. 21),77-89
  27. Werz, O., Steinhilber, D. (1996) Selenium-dependent peroxidases suppress 5-lipoxygenase activity in B-lymphocytes and immature myeloid cells. The presence of peroxidase-insensitive 5-lipoxygenase activity in differentiated myeloid cells Eur. J. Biochem. 242,90-97[Medline]
  28. Krump, E., Nikitas, K., Grinstein, S. (1997) Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils J. Biol. Chem. 272,17303-17311[Abstract/Free Full Text]
  29. Werz, O., Klemm, J., Samuelsson, B., Rdmark, O. (2001) Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes Blood 97,2487-2495[Abstract/Free Full Text]
  30. Grynkiewicz, G., Poenie, M., Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties J. Biol. Chem. 260,3440-3450[Abstract/Free Full Text]
  31. Leslie, C. C. (1997) Properties and regulation of cytosolic phospholipase A2 J. Biol. Chem. 272,16709-16712[Free Full Text]
  32. Gijon, M. A., Leslie, C. C. (1999) Regulation of arachidonic acid release and cytosolic phospholipase A2 activation J. Leukoc. Biol. 65,330-336[Abstract]
  33. Gijon, M. A., Spencer, D. M., Siddiqi, A. R., Bonventre, J. V., Leslie, C. C. (2000) Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation J. Biol. Chem. 275,20146-20156[Abstract/Free Full Text]
  34. Buschbeck, M., Ghomashchi, F., Gelb, M. H., Watson, S. P., Borsch-Haubold, A. G. (1999) Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2 Biochem. J. 344(Pt. 2),359-366
  35. Peters-Golden, M., McNish, R. W. (1993) Redistribution of 5-lipoxygenase and cytosolic phospholipase-A2 to the nuclear fraction upon macrophage activation Biochem. Biophys. Res. Commun. 196,147-153[Medline]
  36. Woods, J. W., Evans, J. F., Ethier, D., Scott, S., Vickers, P. J., Hearn, L., Heibein, J. A., Charleson, S., Singer, I. I. (1993) 5-Lipoxygenase and 5-lipoxygenase activating protein are localized in the nuclear envelope of activated human leukocytes J. Exp. Med. 178,1935-1946[Abstract/Free Full Text]
  37. Abramovitz, M., Wong, E., Cox, M. E., Richardson, C. D., Li, C., Vickers, P. J. (1993) 5-Lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase Eur. J. Biochem. 215,105-111[Medline]
  38. Hammarberg, T., Provost, P., Persson, B., Rådmark, O. (2000) The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity J. Biol. Chem. 275,38787-38793[Abstract/Free Full Text]
  39. Zhang, F., Warskulat, U., Wettstein, M., Schreiber, R., Henninger, H. P., Decker, K., Haussinger, D. (1995) Hyperosmolarity stimulates prostaglandin synthesis and cyclooxygenase-2 expression in activated rat liver macrophages Biochem. J. 312,135-143
  40. Sha’afi, R. I., Shefcyk, J., Yassin, R., Molski, T. F., Volpi, M., Naccache, P. H., White, J. R., Feinstein, M. B., Becker, E. L. (1986) Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin? J. Cell Biol. 102,1459-1463[Abstract/Free Full Text]
  41. Eom, Y. W., Cho, S. H., Hwang, J. S., Yoon, S. B., Na, D. S., Kang, I. J., Kang, S. S., Song, W. K., Kim, J. H. (2001) Rac and p38 kinase mediate 5-lipoxygenase translocation and cell death Biochem. Biophys. Res. Commun. 284,126-132[Medline]
  42. Chowdhury, S., Smith, K. W., Gustin, M. C. (1992) Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation J. Cell Biol. 118,561-571[Abstract/Free Full Text]
  43. Rizoli, S. B., Rotstein, O. D., Parodo, J., Phillips, M. J., Kapus, A. (2000) Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling Am. J. Physiol. Cell Physiol. 279,C619-C633[Abstract/Free Full Text]
  44. Provost, P., Doucet, J., Hammarberg, T., Gerisch, G., Samuelsson, B., Radmark, O. (2001) 5-Lipoxygenase interacts with coactosin-like protein J. Biol. Chem. 276,16520-16527[Abstract/Free Full Text]
  45. Ham, E. A., Soderman, D. D., Zanetti, M. E., Dougherty, H. W., McCauley, E., Kuehl, F. A. (1983) Inhibition by prostaglandins of leukotriene B4 release from activated neutrophils Proc. Natl. Acad. Sci. USA 80,4349-4353[Abstract/Free Full Text]
  46. Haurand, M., Flohe, L. (1989) Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids Biochem. Pharmacol. 38,2129-2137[Medline]
  47. Sud’ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., Ullrich, V. (2001) Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase Biochem. J. 359,621-629[Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Rakonjac, L. Fischer, P. Provost, O. Werz, D. Steinhilber, B. Samuelsson, and O. Radmark
Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production
PNAS, August 29, 2006; 103(35): 13150 - 13155.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bürkert, E.
Right arrow Articles by Werz, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bürkert, E.
Right arrow Articles by Werz, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS