ABSTRACT
The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.
Subject(s)
Apoptosis/immunology , MAP Kinase Signaling System/immunology , Mitogen-Activated Protein Kinases/metabolism , Neutrophils/cytology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Androstadienes/pharmacology , Apoptosis/drug effects , Chemotaxis, Leukocyte/physiology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , Imidazoles/pharmacology , Leukotriene B4/pharmacology , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Morpholines/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/enzymology , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Pyridines/pharmacology , Virulence Factors, Bordetella/pharmacology , Wortmannin , p38 Mitogen-Activated Protein KinasesABSTRACT
Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.
Subject(s)
Calcium-Transporting ATPases/antagonists & inhibitors , Calcium/metabolism , Kidney/metabolism , Receptors, Cell Surface/metabolism , Absorption , Algorithms , Animals , Cattle , Cell Line , Cell Membrane/drug effects , Cell Membrane/enzymology , Electrophysiology , GTP-Binding Proteins/metabolism , Kidney/enzymology , Pertussis Toxin , Receptors, Calcium-Sensing , Type C Phospholipases/metabolism , Virulence Factors, Bordetella/toxicityABSTRACT
Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Neutrophils/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Imidazoles/pharmacology , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase 3 , MAP Kinase Kinase 6 , Mitogen-Activated Protein Kinase Kinases/metabolism , Neutrophils/enzymology , Phosphatidylinositols/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt , Pyridines/pharmacology , Serine/genetics , Threonine/genetics , p38 Mitogen-Activated Protein KinasesABSTRACT
The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b(558), a component of the NADPH oxidase. This study shows that TNFalpha also increases membrane expression of flavocytochrome b(558). Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b(558) are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNFalpha and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b(558), with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNFalpha failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNFalpha and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b(558) through exocytosis of intracellular granules in a process regulated by p38 MAPK.
Subject(s)
Antigens, Neoplasm , Cell Adhesion Molecules , Cytochrome b Group/metabolism , Cytoplasmic Granules/metabolism , Exocytosis , Mitogen-Activated Protein Kinases/metabolism , NADPH Oxidases , Neutrophils/physiology , Respiratory Burst , Antigens, CD , Cell Membrane/drug effects , Cell Membrane/metabolism , Enzyme Activation , GPI-Linked Proteins , Humans , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/biosynthesis , Phagocytosis/drug effects , Receptors, Complement 3b/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein KinasesABSTRACT
The calcium-sensing receptor (CaR) stimulates ERK1 in rat fibroblasts, but its effect on other MAP kinases is not known. We used a model of renal distal tubule, the MDCK cell, to determine the effects of CaR stimulation on Jun kinase (JNK) activity. Stimulation of the CaR with 5 mM Ca(2+) resulted in a time-dependent increase in JNK activity. Activation of JNK occurred preferentially with stimulation on the basal surface relative to the apical surface. Basal administration of the CaR agonist gadolinium (30 microm) also stimulated JNK activity. Pertussis toxin blocked the ability of both CaR agonists to stimulate JNK, indicating that the effect was mediated through G(ialpha) class G proteins. Finally, we used confocal microscopy to determine that the CaR was located predominantly on the basal surface. These studies demonstrate for the first time that the CaR stimulates JNK activity.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Receptors, Cell Surface/metabolism , Animals , Cell Line , Dogs , Enzyme Activation , GTP-Binding Proteins/metabolism , JNK Mitogen-Activated Protein Kinases , Pertussis Toxin , Rats , Receptors, Calcium-Sensing , Virulence Factors, Bordetella/metabolismABSTRACT
Fc gamma Rs mediate immune complex-induced tissue injury. The hypothesis that Fc gamma RIIa and Fc gamma RIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of Fc gamma RIIa and/or Fc gamma RIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. Fc gamma RIIa and Fc gamma RIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both Fc gamma RIIa and Fc gamma RIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic Fc gamma R cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by Fc gamma RIIIb or heterotypic cross-linking, but had no effect on Fc gamma RIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by Fc gamma R cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either Fc gamma RIIa or Fc gamma RIIIb, but cytochalasin D only enhanced H2O2 production stimulated by Fc gamma RIIIb. Our data indicate that Fc gamma RIIa and Fc gamma RIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of Fc gamma RIIIb in neutrophils.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Neutrophil Activation/immunology , Neutrophils/enzymology , Neutrophils/immunology , Receptors, IgG/physiology , Actins/metabolism , Actins/physiology , Enzyme Activation/immunology , Humans , Immunophenotyping , Mitogen-Activated Protein Kinases/physiology , Neutrophils/metabolism , Phosphatidylinositol 3-Kinases/physiology , Respiratory Burst/immunologyABSTRACT
The ability of formyl peptide receptors (FPRs) stably expressed in undifferentiated HL-60 cells to undergo ligand-induced desensitization was compared with their ability in normal and vector-transfected HL-60 cells following granulocyte differentiation with DMSO. fMet-Leu-Phe failed to induce uncoupling of FPRs from G-proteins in FPR-transfected cells, whereas uncoupling was induced in differentiated HL-60 cells and differentiated vector-transfected HL-60 cells, as determined by ligand-stimulated guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding and GTPgammaS inhibition of fMet-Leu-Phe binding to isolated membranes. Immunoprecipitation of Galpha(i2) from solubilized, azidoanalide (AA-gammaGTP) photolabeled membranes showed that receptors in desensitized FPR-transfected HL-60 cells remained coupled to Galpha(i2), whereas desensitized receptors in differentiated HL-60 cell membranes were uncoupled from Galpha(i2). As determined by immunoblotting, Galpha(i2) expression was similar in undifferentiated and differentiated HL-60 cells and FPR-transfected cells. Ligand-stimulated receptor internalization and desensitization of calcium redistribution were similar in all three groups of cells. Immunoblotting also indicated that G-protein-coupled receptor kinases (GRKs) 2 and 4 were present in undifferentiated FPR-transfected HL-60 cells at 50% of the level seen in differentiated HL-60 cells. However, differentiation did not increase GRK2 or GRK4 expression, indicating that differences in GRK expression do not explain deficient desensitization. The data indicated that undifferentiated HL-60 cells are unable to induce homologous desensitization of FPRs.
Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Cell Differentiation/physiology , GTP-Binding Proteins/metabolism , Granulocytes/cytology , Granulocytes/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , HL-60 Cells , Humans , Receptors, Formyl Peptide , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/genetics , Receptors, Peptide/biosynthesis , Receptors, Peptide/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.
Subject(s)
Apoptosis , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Mitogen-Activated Protein Kinases/physiology , Neutrophils/cytology , Neutrophils/enzymology , Phosphatidylinositol 3-Kinases/physiology , Protein Serine-Threonine Kinases , Signal Transduction , Apoptosis/drug effects , Carrier Proteins/metabolism , Cells, Cultured , Chromones/pharmacology , Drug Synergism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-8/physiology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Morpholines/pharmacology , Neutrophils/drug effects , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Recombinant Proteins , Signal Transduction/drug effects , Time Factors , bcl-Associated Death ProteinABSTRACT
The oxidative burst of neutrophils from patients with renal failure before the initiation of dialysis is primed for an enhanced response after stimulation by phagocytosis or chemoattractants. This study shows that phagocytosis-stimulated oxidative burst activity remains primed in patients treated with both high-efficiency hemodialysis and continuous ambulatory peritoneal dialysis (CAPD), but it is normal in patients with a functioning renal transplant. Incubation of normal neutrophils or HL-60 granulocytes in azotemic plasma results in increased resting and phagocytosis-stimulated H2O2 production, which is rapidly reversible on removal of the plasma. Priming of the oxidative burst by azotemic plasma is independent of changes in opsonization and phagocytosis and does not require protein synthesis. These results suggest that azotemic plasma contains a substance or substances capable of reversibly priming oxidative burst activity in neutrophils and neutrophil-like cell lines. The Inability of high-efficiency hemodialysis and CAPD to normalize oxidative burst activity suggests that this substance is of higher molecular weight.
Subject(s)
Kidney Transplantation , Neutrophils/metabolism , Renal Dialysis , Respiratory Burst , Uremia/blood , Uremia/surgery , Adult , Aged , Female , Granulocytes/metabolism , Humans , Hydrogen Peroxide/metabolism , Male , Middle Aged , Peritoneal Dialysis, Continuous Ambulatory , Phagocytosis , Uremia/metabolismABSTRACT
The hypothesis that bacterial phagocytosis by human polymorphonuclear neutrophils (PMNs) stimulates MAPK cascades that regulate respiratory burst activation was tested. Extracellular response kinase (ERK) and p38 kinase, but not c-Jun NH2-terminal kinase, activities were increased within 5 min of phagocytosis of plasma-opsonized Staphylococcus aureus (S-SA), reached maximum at 20-30 min, and remained elevated through 60 min. The role of Fcy receptors was examined using gamma globulin-opsonized SA (IgG-SA), whereas CR3 receptors were activated by particulate beta-glucan. IgG-SA stimulated a maximal ERK activity at 30 min, whereas p38 activity was maximal at 5 min. Beta-glucan stimulated maximal ERK activity at 5 min and maximal p38 activity at 2 min. Non-opsonized bacteria were ingested at 10% of the level of S-SA and stimulated a minimal increase in ERK and p38 activity at 60 min. S-SA stimulation of ERK was inhibited by wortmannin, LY294002, and genistein, but not calphostin C; whereas p38 stimulation was inhibited by calphostin C and genistein, but not wortmannin and LY294002. Simultaneous measurement of phagocytosis and H2O2 production by flow cytometry was used to assess the role of ERKs and p38 kinase in phagocytosis. The MEK inhibitor PD098059 had no significant effect on phagocytosis or H2O2 production. The p38 kinase inhibitor SB203580 significantly attenuated H2O2 production, whereas phagocytosis was unaffected. In conclusion, bacterial phagocytosis stimulates ERK and p38 activation by distinct signal transduction pathways. Phagocytosis-stimulated p38 kinase activity is necessary for optimal H2O2 production.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Neutrophils/enzymology , Neutrophils/microbiology , Phagocytosis/physiology , Signal Transduction/physiology , Staphylococcus aureus/physiology , Antigens, CD/physiology , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Enzyme Activation , Humans , JNK Mitogen-Activated Protein Kinases , Macrophage-1 Antigen/physiology , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/physiology , Neutrophils/physiology , Receptors, IgG/physiology , Respiratory Burst/physiology , p38 Mitogen-Activated Protein KinasesABSTRACT
The signal transduction pathways activated by tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF-alpha stimulated a dose-dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF-alpha or GM-CSF. GM-CSF stimulated ERK activity comparable to that of TNF-alpha, but GM-CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM-CSF, but not TNF-alpha. GM-CSF, but not TNF-alpha, stimulated wortmannin-sensitive activation of Raf-1. TNF-alpha and GM-CSF priming of superoxide release stimulated by N-formyl-methionyl-leucyl-phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF-alpha and GM-CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF-alpha and GM-CSF to prime the respiratory burst response in human PMNs.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Neutrophils/physiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Sequence , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , Kinetics , MAP Kinase Kinase 1 , Molecular Sequence Data , Neutrophils/drug effects , Neutrophils/enzymology , Protein Serine-Threonine Kinases/blood , Protein Serine-Threonine Kinases/chemistry , Protein-Tyrosine Kinases/blood , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins c-raf/blood , Respiratory Burst , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/physiology , p38 Mitogen-Activated Protein KinasesABSTRACT
Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in membranes from FPRwt but not FPRdel cells. GTPgammaS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alphai2 and alphaq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alphai2 but not to alphaq/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cytoplasm/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Amino Acid Sequence , Calcium/metabolism , Cell Differentiation , Enzyme Activation , GTP-Binding Proteins/metabolism , HL-60 Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Receptors, Formyl Peptide , Receptors, Immunologic/genetics , Receptors, Peptide/genetics , TransfectionABSTRACT
The purpose of this study was to determine the mechanisms of dopamine regulation of phosphate uptake in opossum kidney (OK) cells, a model of proximal renal tubules. Dopamine stimulated cAMP generation and inhibited radiolabeled phosphate uptake into OK cell monolayers by 14.4 +/- 1.8%. The effect of dopamine was transient, as phosphate uptake returned toward control level by 3 h despite the continued presence of dopamine. Pretreatment with pertussis toxin increased dopamine inhibition of phosphate uptake to 25 +/- 3%, increased the duration of the dopamine effect to at least 3 h, and enhanced cAMP generation. In an OK cell clone that overexpressed cAMP phosphodiesterase, dopamine did not inhibit phosphate uptake, but pharmacologic inhibition of protein kinase A activation did not prevent dopamine inhibition of phosphate uptake. A DA1 receptor agonist inhibited phosphate uptake more potently than dopamine (29.5 +/- 1.1%) or a DA2 receptor agonist (7.9 +/- 2%). However, both DA1 and DA2 receptor antagonists completely blocked dopamine inhibition of phosphate uptake. DA1, but not the DA2, antagonists blocked dopamine-stimulated cAMP generation. Treatment with alpha-adrenergic receptor antagonists potentiated dopamine inhibition of phosphate uptake to the same extent as pertussis toxin and was not additive with pertussis toxin. It is concluded that dopamine inhibits phosphate uptake through DA1 and DA2 receptor stimulation by cAMP-dependent and -independent pathways and activates a pertussis toxin-sensitive counter-regulatory pathway that attenuates this response through alpha-adrenergic receptor stimulation.
Subject(s)
Dopamine/physiology , Kidney/metabolism , Phosphates/pharmacokinetics , Receptors, Adrenergic, alpha/physiology , Receptors, Dopamine D1/physiology , Receptors, Dopamine D2/physiology , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Adrenergic alpha-Antagonists/pharmacology , Animals , Cells, Cultured , Cyclic AMP/biosynthesis , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dopamine/pharmacology , Dopamine Antagonists/pharmacology , Enzyme Inhibitors/pharmacology , Kidney/cytology , Kidney/drug effects , Opossums , Pertussis Toxin , Receptors, Dopamine D1/agonists , Receptors, Dopamine D2/agonists , Virulence Factors, Bordetella/pharmacologyABSTRACT
A baculovirus expression system was used to determine the contribution of carboxyl methylation of specific G protein gamma subunits to the interaction between alpha and beta gamma subunits. beta gamma subunits were carboxyl methylated by a membrane bound methyltransferase in Sf9 cells, and periodate-oxidized adenosine inhibited this methylation by 90%. Carboxyl methylation of beta(1) gamma(2), beta(2) gamma(3), and beta(2) gamma(7) enhanced pertussis toxin-catalyzed ADP-ribosylation of alpha(i2) and alpha(i3) by about 2-fold. On the other hand, methylation did not enhance membrane attachment of beta gamma subunits. These results suggest that methylation of isoprenylated gamma subunits is required for optimal G protein-mediated signal transduction, but not membrane attachment.
Subject(s)
GTP-Binding Proteins/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Catalysis , Cell Line , Cell Membrane/metabolism , GTP-Binding Proteins/genetics , Methylation , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Pertussis Toxin , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Spodoptera , Virulence Factors, Bordetella/pharmacologyABSTRACT
Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , N-Formylmethionine Leucyl-Phenylalanine/metabolism , NADPH Oxidases/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Signal Transduction/immunology , Enzyme Activation/immunology , HL-60 Cells , Humans , JNK Mitogen-Activated Protein Kinases , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , NADPH Oxidases/physiology , Neutrophil Activation/immunology , Neutrophils/enzymology , Neutrophils/immunology , Neutrophils/metabolism , Oxidation-Reduction , Receptors, Formyl Peptide , p38 Mitogen-Activated Protein KinasesABSTRACT
Priming of polymorphonuclear leukocyte responses to chemoattractants by TNF plays an important role in host defenses and inflammatory responses. TNF-induced priming is associated with an 80% increase in the membrane density of G alpha(i2) protein that is coupled to chemoattractant receptors. The present study examines the hypothesis that TNF stimulates increased synthesis of alpha(i2). Within 10 min of addition, TNF stimulated a significant increase in total cellular G alpha(i2), as determined by pertussis toxin-catalyzed ADP ribosylation, which was blocked by the translation inhibitor cycloheximide. Immunoprecipitation of biosynthetically labeled alpha(i2) showed that TNF increased alpha(i2) synthesis by about 20% at 10 min. Nuclear run-ons showed no change in alpha(i2) mRNA synthesis in TNF-treated cells; however, steady state alpha(i2) mRNA levels were reduced following a 10-min exposure to TNF. Pretreatment with cycloheximide prevented the TNF-induced reduction in steady state alpha(i2) mRNA levels. Therefore, TNF stimulates alpha(i2) protein synthesis and mRNA degradation in the same time frame as priming. The increased alpha(i2) synthesis results from increased translation, not transcription, of alpha(i2) mRNA. Simultaneous G alpha(i2) protein synthesis and mRNA degradation provide a mechanism by which TNF priming is associated with a rapid, self-limiting increase in G protein expression.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Proteins/biosynthesis , Neutrophils/metabolism , Protein Biosynthesis/immunology , Proto-Oncogene Proteins/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Biological Transport , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Proteins/drug effects , Humans , Neutrophils/drug effects , Protein Biosynthesis/drug effects , Proto-Oncogene Proteins/drug effects , Up-RegulationABSTRACT
The oxidative burst of neutrophils from azotemic patients (AzoPMNs) is primed for an enhanced response compared to neutrophils from normal subjects (NorPMNs). The mechanism for this priming is unknown, although TNF alpha does not further prime AzoPMNs. The present study examines the hypothesis that azotemia and TNF alpha prime neutrophils by the same mechanism. Formyl peptide receptor expression and degranulation were not primed in AzoPMNs, but were primed by both LPS and TNF alpha. LPS was also able to prime the AzoPMN oxidative burst. Guanine nucleotide exchange by multiple guanine nucleotide binding proteins, including heterotrimeric G-proteins and low molecular weight GTP-binding proteins (LMWGs), was increased in AzoPMNs, as demonstrated by GTP gamma S binding and azidoanilide GTP photoaffinity labeling. The plasma membrane density of G-protein alpha i2, alpha i3, and alpha s subunits and the density in the cytosol of the LMWG, Rap1A, was present in significantly greater amounts on plasma membranes from AzoPMNs. FMet-Leu-Phe-stimulated phospholipase D activity, but not basal activity, was significantly greater in AzoPMNs. Finally, incubation of NorPMNs in plasma from azotemic patients resulted in a significant increase in basal GTP gamma S binding. These results demonstrate that priming of AzoPMNs is restricted to oxidative burst activity and that it occurs by a mechanism distinct from that utilized by TNF alpha and LPS. While the exact mechanism remains unknown, it appears to involve a plasma factor and changes in LMWG expression or activity.
Subject(s)
Lipopolysaccharides/pharmacology , Neutrophils/drug effects , Neutrophils/metabolism , Respiratory Burst/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Uremia/metabolism , Adult , Aged , Cell Degranulation/drug effects , GTP-Binding Proteins/metabolism , Humans , In Vitro Techniques , Middle Aged , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phagocytosis/drug effectsABSTRACT
The oxidative burst of neutrophils from azotemic patients is refractory to priming by tumor necrosis factor-alpha (TNF alpha). Soluble TNF alpha binding protiens (sTNFR) accumulate in the plasma of azotemic patients. To test the hypothesis that these increased sTNFR concentrations inhibit TNF alpha priming of oxidative burst activity, we measured plasma sTNFR concentrations in nondialyzed azotemic patients, hemodialysis patients, and normal subjects, and determined TNF alpha priming of fMet-Leu-Phe-stimulated superoxide production in neutrophils incubated in plasma with differing levels of sT-NFR. These sTNFR concentrations increased significantly as creatinine clearance decreased and were significantly greater in hemodialysis patients than could be accounted for by loss of renal function alone. TNF alpha primed superoxide production by normal neutrophils in normal plasma, but this effect was significantly reduced in plasma with increased concentrations of sTNFR. Neutrophils from azotemic and hemodialysis patients were refractory to priming by TNF alpha in autologous plasma, and incubation in normal plasma only partially corrected this defect. We conclude that sTNFR accumulate as a result of the loss of renal function and hemodialysis and inhibit TNF alpha priming of neutrophils in azotemic and hemodialysis patients, but that these cells also have an intrinsic functional defect.
Subject(s)
Antigens, CD/metabolism , Kidney Failure, Chronic/metabolism , Neutrophils/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Renal Dialysis , Tumor Necrosis Factor-alpha/metabolism , Adult , Aged , Aged, 80 and over , Antigens, CD/blood , Blood Proteins/metabolism , Female , Humans , Male , Middle Aged , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Protein Binding , Receptors, Tumor Necrosis Factor/blood , Receptors, Tumor Necrosis Factor, Type I , Regression Analysis , Respiratory Burst , Superoxides/metabolism , Uremia/blood , Uremia/therapyABSTRACT
Purinergic P2 receptors are present on proximal renal tubules, but their function is unknown. Because P2 agonists antagonize vasopressin-stimulated water transport in the distal tubule by inhibiting activation of adenylyl cyclase, we postulated that P2 receptor activation blocks parathyroid hormone (PTH) inhibition of phosphate uptake in proximal tubule by preventing PTH-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation. PTH inhibition of sodium-dependent phosphate uptake was attenuated by alpha,beta-methylene-ATP (AMP-CPP), a P2x receptor agonist, but not by 2-methyl-thio-ATP, a P2y receptor agonist, in a dose-dependent manner. AMP-CPP did not attenuate inhibition of phosphate uptake produced by direct activation of adenylyl cyclase with forskolin, by addition of the cAMP analogue 8-bromo-cAMP, or by inhibition of cAMP phosphodiesterase with RO-20-1724. Additionally, AMP-CPP had no effect on basal or PTH-stimulated cAMP production. As PTH also stimulates protein kinase C activation, the effect of AMP-CPP on inhibition of phosphate uptake stimulated by phorbol 12-myristate 13-acetate (PMA) was tested. AMP-CPP had no effect on PMA-induced inhibition of phosphate uptake. Pretreatment with pertussis toxin abolished the attenuating effect of AMP-CPP on PTH inhibition of sodium-dependent phosphate uptake. We conclude that activation of purinergic P2 receptors attenuates the inhibitory effect of PTH on sodium-dependent phosphate uptake by a G protein-dependent mechanism that is independent of cAMP generation protein kinase A activation, or protein kinase C activation.
Subject(s)
GTP-Binding Proteins/physiology , Kidney/metabolism , Parathyroid Hormone/pharmacology , Phosphates/antagonists & inhibitors , Receptors, Purinergic/physiology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Adenylate Cyclase Toxin , Animals , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation , Kidney/cytology , Opossums , Pertussis Toxin , Phosphates/pharmacokinetics , Protein Kinase C/metabolism , Purinergic Agonists , Sodium/physiology , Virulence Factors, Bordetella/pharmacologyABSTRACT
Hemodialysis with cellulose membranes causes a complement-mediated neutropenia. Changes in neutrophil function have also been reported; however, it is unclear if these changes indicate a direct effect of the membrane on neutrophils or if they are a consequence of the neutropenia. We tested the hypothesis that neutrophil oxidative burst activity is enhanced during dialysis with cellulose membranes. Resting and Staphylococcus aureus-stimulated H2O2 production were determined predialysis and in blood entering and leaving the dialyzer during the first 30 min of dialysis and in blood leaving the membrane module in a single-pass on-line model of hemodialysis. Resting H2O2 production increased slightly but significantly during the first 30 min of dialysis. Transit of neutrophils through the dialyzer caused a marked increase in stimulated H2O2 production, indicating priming of the oxidative burst. However, priming was limited to the first 5 min of dialysis before the onset of neutropenia. In contrast, stimulation and priming of H2O2 production persisted throughout 30 min of single-pass on-line perfusion. These results indicate that cellulose membranes both stimulate and prime neutrophil oxidative burst activity but that these effects are partially obscured by neutropenia.