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1.
J Med Chem ; 67(13): 11197-11208, 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-38950284

ABSTRACT

Tropomyosin receptor kinases (Trks) are receptor tyrosine kinases activated by neurotrophic factors, called neurotrophins. Among them, TrkA interacts with the nerve growth factor (NGF), which leads to pain induction. mRNA-display screening was carried out to discover a hit compound 2, which inhibits protein-protein interactions between TrkA and NGF. Subsequent structure optimization improving phosphorylation inhibitory activity and serum stability was pursued using a unique process that took advantage of the peptide being synthesized by translation from mRNA. This gave peptide 19, which showed an analgesic effect in a rat incisional pain model. The peptides described here can serve as a new class of analgesics, and the structure optimization methods reported provide a strategy for discovering new peptide drugs.


Subject(s)
Receptor, trkA , Receptor, trkA/antagonists & inhibitors , Receptor, trkA/metabolism , Animals , Rats , Humans , Structure-Activity Relationship , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Analgesics/pharmacology , Analgesics/chemistry , Analgesics/chemical synthesis , Peptides, Cyclic/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/chemical synthesis , Male , Nerve Growth Factor/metabolism , Phosphorylation , Pain/drug therapy , Rats, Sprague-Dawley
2.
Opt Express ; 32(7): 12160-12171, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38571047

ABSTRACT

Water has significantly high absorption around 3 µm wavelength region, originated by its fundamental OH vibrational modes. Here, we successfully demonstrate an opto-thermal manipulation of particles utilizing a 3 µm mid-infrared Er:ZBLAN fiber laser (adjustable from 2700 to 2826 nm) that can efficiently elevate the temperature at a laser focus with a low laser power. The 3 µm laser indeed accelerates the formation of the particle assembly by simply irradiating the laser into water. By altering the laser wavelengths, the assembling speed and size, instantaneous particle velocity, particle distribution, trapping stiffness and temperature elevation are evaluated systematically. We propose that the dynamics of particle assembly can be understood through thermo-osmotic slip flows, taking into account the effects of volume heating within the focal cone and point heating at the focus.

3.
J Dermatol Sci ; 93(3): 176-185, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30905492

ABSTRACT

BACKGROUND: Retinoic acid receptor-related orphan receptor gamma t (RORγt) has critical roles in the development, maintenance and function of interleukin (IL)-17-producing cells and is a highly attractive target for the treatment of IL-17-mediated autoimmune disease, particularly psoriasis. On the other hand, RORγt is also critical for controlling apoptosis during thymopoiesis, and genetic RORγt ablation or systematic RORγt inhibition cause progressive thymic aberrations leading to T cell lymphomas. OBJECTIVE: We investigated whether topical administration of our novel RORγt inhibitor, S18-000003 has therapeutic potential for psoriasis with low risk of thymic aberrations. METHODS: We evaluated the effect of topical S18-000003 on psoriasis-like skin inflammation and influence on the thymus in a 12-O-tetradecanoylphorbol-13-acetate-induced K14.Stat3C mouse psoriasis model. RESULTS: S18-000003 markedly inhibited the development of psoriatic skin inflammation via suppression of the IL-17 pathway. In the skin, S18-000003 suppressed all subsets of IL-17-producing cells that we previously identified in this psoriasis model: Th17 cells, Tc17 cells, dermal γδ T cells, TCR- cells that probably included innate lymphoid cells, and CD4-CD8- double-negative αß T cells. Notably, neither reduction of CD4+CD8+ double-positive thymocytes nor dysregulation of cell cycling was observed in S18-000003-treated mice, even at a high dose. CONCLUSION: Our topically administered RORγt inhibitor is a potential therapeutic agent for psoriasis with low risk of thymic lymphoma.


Subject(s)
Dermatologic Agents/pharmacology , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Psoriasis/drug therapy , Sulfones/pharmacology , Administration, Cutaneous , Animals , Cells, Cultured , Dermatologic Agents/therapeutic use , Disease Models, Animal , Drug Evaluation, Preclinical , Healthy Volunteers , Humans , Immunity, Innate/drug effects , Interleukin-17/immunology , Interleukin-17/metabolism , Leukocytes, Mononuclear , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nuclear Receptor Subfamily 1, Group F, Member 3/immunology , Primary Cell Culture , Psoriasis/diagnosis , Psoriasis/etiology , Psoriasis/pathology , Severity of Illness Index , Skin/drug effects , Skin/immunology , Skin/pathology , Sulfones/therapeutic use , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/immunology , Tetradecanoylphorbol Acetate/toxicity , Treatment Outcome
4.
Bioorg Med Chem Lett ; 28(22): 3549-3553, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30301676

ABSTRACT

The retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is the master transcription factor responsible for regulating the development and function of T-helper 17 (Th17) cells, which are related to the pathology of several autoimmune disorders. Therefore, RORγt is an attractive drug target for such Th17-mediated autoimmune diseases. A structure-activity relationship (SAR) study of lead compound 1 yielded a novel series of RORγt inhibitors, represented by compound 6. Detailed SAR optimization, informed by X-ray cocrystal structure analysis, led to the discovery of a potent orally bioavailable RORγt inhibitor 25, which inhibited IL-17 production in the skin of IL-23-treated mice by oral administration.


Subject(s)
Amides/chemistry , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Administration, Oral , Amides/pharmacokinetics , Amides/therapeutic use , Animals , Autoimmune Diseases/drug therapy , Binding Sites , Binding, Competitive , Crystallography, X-Ray , Drug Evaluation, Preclinical , Half-Life , Humans , Interleukin-17/metabolism , Interleukin-23/pharmacology , Mice , Molecular Dynamics Simulation , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Protein Binding , Rats , Skin/drug effects , Skin/metabolism , Structure-Activity Relationship , Th17 Cells/cytology , Th17 Cells/drug effects , Th17 Cells/metabolism
5.
Acta Crystallogr C Struct Chem ; 72(Pt 11): 786-790, 2016 Nov 01.
Article in English | MEDLINE | ID: mdl-27811412

ABSTRACT

Tris(pyrazolyl)borate (scorpionate) ligands can be considered as the most prolific ligands in contemporary coordination chemistry due to the availability of various steric and electronic substituents at the pyrazolyl rings that allow fine-tuning of the open-coordination site for metal centres. The thallium(I) complexes of anionic tridentate-chelating scorpionate ligands, namely [tris(3-mesityl-5-methyl-1H-pyrazol-1-yl-κN2)hydroborato]thallium(I) monohydrate, [Tl(C39H46BN6)]·H2O, (I), and [bis(3-mesityl-5-methyl-1H-pyrazol-1-yl-κN2)(5-mesityl-3-methyl-1H-pyrazol-1-yl-κN2)hydroborato]thallium(I), [Tl(C39H46BN6)], (II), show a {TlIN3} coordination, with average TlI-N bond lengths of 2.53 and 2.55 Šin (I) and (II), respectively. The overall TlI coordination geometry is distorted trigonal pyramidal, with the average N-TlI-N angle being approximately 73° for both. The dihedral angle between the planes of the pyrazolyl and benzene rings of the mesityl group is 82° in (I), while the corresponding angles in (II) are in the range 64-104°. The structural differences between the two ligands are expected to contribute to the different reactivities of the transition metal coordination complexes towards activation of small molecules such as dioxygen and ethylene.

6.
Anal Sci ; 32(7): 805-8, 2016.
Article in English | MEDLINE | ID: mdl-27396665

ABSTRACT

Although ice chromatography is a useful probe of ice interfaces, its low separation efficiency has often made difficult to access the ice/water interface. Coupling of this method with shear-driven flow chromatography, which has high separation potential, solves the problems involved in ice chromatography. This paper reports on shear-driven flow ice chromatographic instrumentation, and discusses the separation performance. Electrostatic separation of positively and negatively charged dyes is demonstrated with an OH(-)-doped ice plate as a stationary phase.

7.
J Pharmacol Sci ; 109(3): 431-43, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19276615

ABSTRACT

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].


Subject(s)
Arachidonic Acid/metabolism , Group IV Phospholipases A2/drug effects , Lysophospholipids/pharmacology , Sphingosine/analogs & derivatives , Animals , Cell Death/drug effects , Cell Line, Tumor , Fibrosarcoma/metabolism , Group IV Phospholipases A2/metabolism , Lysophospholipids/chemical synthesis , Lysophospholipids/chemistry , Mice , RNA, Small Interfering/metabolism , Sphingosine/chemical synthesis , Sphingosine/chemistry , Sphingosine/pharmacology , Structure-Activity Relationship , Time Factors
8.
Cell Signal ; 21(3): 440-7, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19101626

ABSTRACT

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.


Subject(s)
Calcium Signaling/physiology , Ceramidases/metabolism , Ceramides/metabolism , Cytosol/enzymology , Group IV Phospholipases A2/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Animals , CHO Cells , Calcium Signaling/drug effects , Ceramidases/antagonists & inhibitors , Ceramides/genetics , Ceramides/pharmacology , Cricetinae , Cricetulus , Cytosol/drug effects , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Group IV Phospholipases A2/drug effects , Humans , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Kinase C/drug effects , Protein Kinase C/metabolism , Protein Transport/drug effects , Protein Transport/physiology , Up-Regulation/genetics
9.
Cell Signal ; 20(5): 815-24, 2008 May.
Article in English | MEDLINE | ID: mdl-18280113

ABSTRACT

Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type alpha of cytosolic phospholipase A(2) (cPLA(2)alpha), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA(2)alpha has not been well established. We investigated the effects induced by transfection with mutant cPLA(2)alpha and inhibitors for CaM and CaM kinase on the Ca(2+)-stimulated release of AA and translocation of cPLA(2)alpha. The mutation of Ser515 to Ala (S515A) did not change cPLA(2)alpha activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H(2)O(2), 1 mM) and A23187 (10 microM) markedly released AA in C12 cells expressing S515A and wild-type cPLA(2)alpha, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA(2)alpha, A23187 caused the translocation of the wild-type, the every mutants, cPLA(2)alpha-C2 domain, and cPLA(2)alpha-Delta397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 microM significantly decreased the release of AA in C12-cPLA(2)alpha cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA(2)alpha-Delta397-749, but not cPLA(2)alpha-C2 domain. Our findings show a possible effect of CaM kinase on cPLA(2)alpha in a catalytic domain A-dependent and Ser515-independent manner.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Group IV Phospholipases A2/chemistry , Group IV Phospholipases A2/metabolism , Amino Acid Substitution , Animals , Arachidonic Acid/metabolism , Base Sequence , Benzylamines/pharmacology , Biological Transport, Active/drug effects , Calcimycin/pharmacology , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Catalytic Domain , Cell Line , Cytosol/enzymology , DNA Primers/genetics , Enzyme Inhibitors/pharmacology , Group IV Phospholipases A2/genetics , Humans , Ionophores/pharmacology , Mice , Mutagenesis, Site-Directed , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/chemistry , Signal Transduction , Sulfonamides/pharmacology
10.
Biochem Pharmacol ; 75(6): 1358-69, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18191813

ABSTRACT

Stimulation of L929 cells with tumor necrosis factor-alpha (TNFalpha) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFalpha-treated L929 cells, its role in the TNFalpha-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A(2) (PLA(2)). To find out the mechanisms underlying the TNFalpha-induced release of AA, we investigated the relationship between TNFalpha stimulation and PLA(2) regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFalpha and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking alpha type of cytosolic PLA(2) (cPLA(2)alpha)). Stimulation with TNFalpha/zVAD also caused the release of AA from L929-cPLA(2)alpha-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA(2)alpha) completely inhibited the TNFalpha-induced release of AA, but only partially inhibited the TNFalpha/zVAD-induced response in L929 cells. The TNFalpha/zVAD-induced release of AA from C12 and L929-cPLA(2)alpha-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA(2) activity, decreased the amount of AA released by TNFalpha/zVAD. TNFalpha/zVAD appears to stimulate release of AA from C12 cells in a cPLA(2)alpha-independent, BHA-sensitive manner. The possible roles of secretory PLA(2) and reactive oxygen species from different pools in the release of AA and cell death were discussed.


Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Arachidonic Acid/metabolism , Caspase Inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antioxidants/pharmacology , Cell Death , Cell Line , Cytosol , Humans , Mice , Oxidants/pharmacology , Phospholipase A2 Inhibitors , Phospholipases A2/genetics , Phospholipases A2/metabolism , Pyrrolidines/pharmacology , RNA Interference
11.
Biochem Pharmacol ; 73(6): 854-62, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17188653

ABSTRACT

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/physiology , Phospholipases A/metabolism , Protein Kinase C/physiology , Vanadates/pharmacology , src-Family Kinases/physiology , Animals , Arachidonic Acid/metabolism , Calcium/physiology , Cells, Cultured , Enzyme Activation/drug effects , ErbB Receptors/physiology , Hydrogen Peroxide/metabolism , Mice , Phospholipases A2 , Protein Transport/drug effects , Signal Transduction
12.
Eur J Pharmacol ; 546(1-3): 1-10, 2006 Sep 28.
Article in English | MEDLINE | ID: mdl-16914136

ABSTRACT

Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.


Subject(s)
Arachidonic Acid/metabolism , Fibroblasts/drug effects , Hydrogen Peroxide/pharmacology , Phospholipases A/metabolism , Protein Kinase C/metabolism , src-Family Kinases/metabolism , Animals , Calcimycin/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cytosol/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Group IV Phospholipases A2 , Humans , Indoles/pharmacology , Ionophores/pharmacology , Maleimides/pharmacology , Mice , Phospholipases A/antagonists & inhibitors , Phospholipases A/deficiency , Phospholipases A/genetics , Protein Kinase C/antagonists & inhibitors , Protein Kinase C-alpha , Protein Transport/drug effects , Pyrimidines/pharmacology , Pyrrolidines/pharmacology , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transfection , src-Family Kinases/antagonists & inhibitors
13.
Biochem Pharmacol ; 71(6): 850-7, 2006 Mar 14.
Article in English | MEDLINE | ID: mdl-16443193

ABSTRACT

Ceramide-1-phosphate (C1P), a novel bioactive sphingolipid, is implicated in the vital cellular processes such as cell proliferation and inflammation. The role of C1P on activity of cytosolic phospholipase A2alpha (cPLA2alpha), a key enzyme for the release of arachidonic acid (AA) and prostanoids, has not been well elucidated. In this study, we investigated the effect of C1P on the release of AA from L929 cells and a variant, which lacks cPLA2alpha expression, C12 cells. C1P at 30 microM alone induced AA release from L929 cells without an increase in intracellular Ca2+ concentration. C1P-induced AA release was marginal in C12 cells, and treatment with an intracellular Ca2+ chelator (BAPTA-AM) or an inhibitor of cPLA2alpha (2 microM pyrrophenone) decreased C1P-induced AA release in L929 cells. C1P increased the enzymatic activity of cPLA2alpha over two-fold in the presence of Ca2+. C1P triggered the translocation of cPLA2alpha and its C2 domain from the cytosol to the perinuclear region in CHO-K1 cells. Interestingly, C1P at 10 microM synergistically enhanced ionomycin-induced AA release from L929 cells. The AA release induced by C1P with and without ionomycin decreased by treatment with protein kinase C (PKC) inhibitor (10 microM GF109203X) and in the PKC-depleted cells. C1P at 10 microM stimulated the translocation of PKC (alpha and delta) from the soluble to the membrane fractions. We propose that C1P stimulates AA release via two mechanisms; direct activation of cPLA2alpha, and the PKC-dependent pathway.


Subject(s)
Ceramides/pharmacology , Cytosol/drug effects , Phospholipases A/biosynthesis , Protein Kinase C/biosynthesis , Animals , Arachidonic Acid/metabolism , Cell Line , Cytosol/enzymology , Dose-Response Relationship, Drug , Drug Combinations , Enzyme Activation , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Group IV Phospholipases A2 , Mice , Protein Biosynthesis/drug effects
14.
J Pharmacol Sci ; 96(3): 324-32, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15539760

ABSTRACT

Tumor necrosis factor-alpha (TNFalpha)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2alpha) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFalpha activates cPLA2alpha remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2alpha, and in C12 cells transfected with cPLA2alpha expression vectors. In transient and stable clones of C12 cells expressing cPLA2alpha, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFalpha was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFalpha-induced AA release, in addition to cPLA2alpha. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2alpha by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2alpha S505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2alpha. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.


Subject(s)
Arachidonic Acid/metabolism , Cytosol/enzymology , Ionophores/pharmacology , Phospholipases A/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha , Animals , Calcimycin/pharmacology , Calcium/metabolism , Cell Line , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Group IV Phospholipases A2 , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , Mice , Phospholipases A/genetics , Phospholipases A2 , Tumor Necrosis Factor-alpha/pharmacology
15.
Eur J Pharmacol ; 484(1): 9-17, 2004 Jan 19.
Article in English | MEDLINE | ID: mdl-14729377

ABSTRACT

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.


Subject(s)
Arachidonic Acid/antagonists & inhibitors , Arachidonic Acid/metabolism , Cytosol/drug effects , Phospholipases A/antagonists & inhibitors , Sphingosine/pharmacology , Animals , Cytosol/enzymology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Group IV Phospholipases A2 , Humans , Mice , PC12 Cells , Phospholipases A/metabolism , Phospholipases A2 , Rats , Sphingosine/analogs & derivatives
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