RESUMEN
The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Biotina , Plaquetas/efectos de los fármacos , Quimotripsinógeno/metabolismo , Electroforesis en Gel de Poliacrilamida , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Immunoblotting , Octoxinol , Trombina/farmacologíaRESUMEN
Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-alpha and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-alpha failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.
Asunto(s)
Apoptosis/inmunología , Enfermedad de Crohn/inmunología , Inmunoconjugados , Mucosa Intestinal/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas/inmunología , Subgrupos de Linfocitos T/inmunología , Abatacept , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno B7-1/fisiología , Antígenos CD28/fisiología , Antígeno CTLA-4 , División Celular/inmunología , Línea Celular , Niño , Enfermedad de Crohn/patología , Medios de Cultivo , Femenino , Humanos , Inmunidad Innata , Inmunofenotipificación , Interleucina-10/farmacología , Interleucina-2/biosíntesis , Interleucina-2/deficiencia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Óxido Nítrico/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Factor de Necrosis Tumoral alfa/fisiología , Proteína X Asociada a bcl-2 , Receptor fas/fisiologíaRESUMEN
Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de IgG/metabolismo , Tirosina/metabolismo , Factor de von Willebrand/metabolismo , Plaquetas/metabolismo , Humanos , Cinética , Fosforilación , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas de Unión al GTP rapRESUMEN
S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.
Asunto(s)
Glutatión/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Óxido Nítrico/farmacología , Animales , Boratos/farmacología , Bovinos , Endotelio Vascular/metabolismo , Etilmaleimida/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Compuestos Nitrosos/farmacología , S-Nitrosoglutatión , Compuestos de Sulfhidrilo/metabolismoRESUMEN
A major mechanism of injury associated with the production of nitric oxide (NO*) in vivo is due to its diffusion-limited reaction with superoxide to form peroxynitrite, which in turn may cause nitration of protein tyrosine residues. To assess the physiological role of tyrosine nitration, it is crucial to identify the proteins that become nitrated. Therefore, we treated lysates from RAW 264.7 cells with 1 mM peroxynitrite and immunoprecipitated tyrosine nitrated proteins. This treatment resulted in the nitration of several proteins, with molecular weights ranging from 60-250 kD. One of these proteins was immunologically identified as the p85 regulatory subunit of the phosphatidylinositol 3-kinase, a key enzyme involved in the signal transduction cascade initiated by many agonists including growth factors. Treatment of RAW 264.7 macrophages with the NO* donor spermine NONOate also induced a nitration of the p85 subunit, demonstrating that this covalent modification also occurs in intact cells. Immunoprecipitation of the p110 catalytic subunit of the phosphatidylinositol 3-kinase co-immunoprecipitated p85 in control lysates. However, p85 could not be detected in the same immunoprecipitates when the lysates had been preincubated with 1 mM peroxynitrite, indicating that the nitration of the p85 subunit may abrogate its interaction with the p110 subunit.
Asunto(s)
Nitratos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Animales , Línea Celular , Ratones , Peso Molecular , Nitratos/química , Nitratos/farmacología , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Fosfatidilinositol 3-Quinasas/química , Conformación Proteica , Proteínas/química , Espermina/análogos & derivados , Espermina/farmacología , Tirosina/químicaRESUMEN
In platelets and megakaryoblasts Gs, the trimeric G-protein that stimulates adenylyl cyclase, is present in a short, 45 kDa, and a long, 52 kDa isoform termed G(s)alpha-S and G(s)alpha-L, respectively. To assess the relative contribution of these isoforms in the cellular synthesis of cAMP, the ratio G(s)alpha-S/G(s)alpha-L was changed in the megakaryoblastic cell line DAMI by inducing cell maturation with recombinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cytometric analysis confirmed that this treatment induced a moderate (TPO) and extensive (PMA) increase in nuclear ploidy and expression of the glycoproteins-IIIa and -Ib. Northern blot analysis revealed downregulation of total Gs-mRNA after treatment of DAMI-cells with TPO and PMA. Western blot analysis showed significant (P < 0.05) upregulation of Gs-L with respective amounts of 27 +/- 4% of total Gs in untreated cells, 35 +/- 1% in TPO- and 41 +/- 3% in PMA-treated DAMI cells (n = 3-4). DAMI cells contained 6 +/- 1 pmol cAMP/10(6) cells, which was not changed by treatment with TPO or PMA. In untreated cells this level increased to 70 +/- 9 pmol cAMP/10(6) cells after 10 min stimulation with 1 micromol/l of the stable prostacyclin analog iloprost. The same stimulation with iloprost resulted in 165 +/- 32 pmol cAMP/10(6) in TPO-treated cells and in 588 +/- 100 pmol cAMP/10(6) in cells treated with PMA. Thus, a shift from G(s)alpha-S to G(s)alpha-L during megakaryoblast maturation strongly potentiates the production of cAMP. A similar shift may occur during normal megakaryocyte maturation and may explain the extreme sensitivity to prostacyclin of platelets, which contain G(s)alpha-S and G(s)alpha-L in approximately equal amounts.
Asunto(s)
Plaquetas/citología , Plaquetas/fisiología , AMP Cíclico/fisiología , Proteínas de Unión al GTP/fisiología , Megacariocitos/citología , Megacariocitos/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Regulación hacia ArribaRESUMEN
Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the macrophage cell line RAW 267.4. RES cells that have been derived from RAW 267.4 cells by repeated exposure to lipopolysaccharide and interferon-gamma (LPS/INF-gamma), followed by outgrowth of viable cells, are resistant to apoptosis and caspase-3 activation upon exposure to SpNO. In this study we have determined that RES cells have lower levels of glutathione (GSH) and a higher oxidative state than RAW cells. Subsequently, RAW and RES cells were depleted of GSH by using l-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. GSH depleted cells did not undergo apoptosis nor demonstrate caspase-3 activity when they were exposed to SpNO. These results suggest that the redox status of the cell is one of the key factors mediating the apoptotic pathway in which glutathione plays a critical role in mediating apoptosis via NO* and reactive oxygen species (ROS).
Asunto(s)
Apoptosis/fisiología , Caspasas , Glutatión/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Línea Celular , Cisteína Endopeptidasas/biosíntesis , Inducción Enzimática/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Espermina/análogos & derivados , Espermina/farmacologíaRESUMEN
Ligation of integrins to an extracellular matrix activates signal transduction systems which produce multiple responses in different cell types. Adhesion often provides a survival signal to cells; disruption of adhesion frequently results in apoptosis. Our laboratory has utilized apoptosis-sensitive and -resistant cell lines to investigate the role of integrin expression and function in regulation of apoptosis in macrophages. Chronic exposure of murine macrophage-like RAW264.7 cells to apoptosis-inducing agents (bacterial lipopolysaccharide and interferon-gamma) resulted in the generation of a derivative cell line (RES) resistant to apoptosis. Observation of RAW and RES cultures indicated a difference in adhesion between the two cell types. The two cell lines also exhibit significant differences in expression of integrins previously characterized to be important in apoptosis.
Asunto(s)
Apoptosis/fisiología , Integrinas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Animales , Antígenos CD/metabolismo , Adhesión Celular/fisiología , Línea Celular , Matriz Extracelular/metabolismo , Integrina alfaV , Integrina beta1/metabolismo , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Transducción de SeñalRESUMEN
In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II's effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Angiotensina II/metabolismo , Ácido Araquidónico/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Proteína Adaptadora GRB2 , Factores de Intercambio de Guanina Nucleótido , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Imidazoles/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Fosforilación , Piridinas/farmacología , Quinacrina/farmacología , Conejos , Proteínas Adaptadoras de la Señalización Shc , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donor S-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.
Asunto(s)
Apoptosis , Caspasas , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Macrófagos/citología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Óxido Nítrico/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Caspasa 3 , Ciclo Celular/efectos de los fármacos , Línea Celular , Cisteína Endopeptidasas/fisiología , Activación Enzimática/efectos de los fármacos , Glutatión/análogos & derivados , Glutatión/farmacología , MAP Quinasa Quinasa 1 , Macrófagos/efectos de los fármacos , Ratones , Compuestos Nitrosos/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , S-NitrosoglutatiónAsunto(s)
Apoptosis/efectos de los fármacos , Arteriosclerosis/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Animales , Apoptosis/genética , Arteriosclerosis/patología , Fragmentación del ADN/efectos de los fármacos , Evaluación de Medicamentos , Humanos , Hipolipemiantes/administración & dosificaciónRESUMEN
Apoptotic signaling cascades converge in the activation of caspases (interleukin-1beta converting enzyme like proteases). Treatment of the human promyelocytic leukaemia cell line U937 with actinomycin D resulted in the activation of caspase-3 also known as CPP32. Protease activity was measured in cytosolic extracts by fluorometric analysis of the time-dependent cleavage of acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC), a caspase-3 substrate. Caspase activity was inhibited by thiol modifying agents such as N-ethylmaleimide or iodoacetamide and NO donors such as S-nitrosoglutathione (GSNO), BF4NO, and spermine-NO. NO-mediated enzyme inhibition was fully reversible upon the addition of DTT (dithiothreitol). NO. itself was not primarily responsible for downregulation of caspase-3, as we found no correlation between rates of NO* release and the magnitude of enzyme inhibition. It is likely that S-nitrosation accounts for enzyme inhibition by various NO donors. SIN-1 and peroxynitrite were inhibitory as well. In this case, however, enzyme activity was not restored upon DTT addition, suggesting oxidation as an additional thiol modification mechanism. Our studies provide evidence that caspases are targeted by NO via S-nitrosation and oxidation of critical thiol groups.
Asunto(s)
Apoptosis , Caspasas , Cisteína Endopeptidasas/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal , Caspasa 3 , Humanos , Nitrosación , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
Following subcellular fractionation of platelet homogenates and Western blotting, two groups of alpha-subunits of trimeric G-proteins could be distinguished. Group 1 consisted of alpha(i)-2, alpha(i)-3, alpha(z), alpha(s), and alpha(q) and was predominantly localized in membranes. Group 2 consisted of alpha16 and alph12 and was predominantly localized in the cytosol. Plasma membranes and dense tubular system (DTS)-membranes showed the same distribution of Group 1 alpha-subunits. An exception was alpha(q), which was virtually absent in the DTS as were Group 2 subunits. In addition, this compartment showed a doublet for alpha(z). Group 1 alpha-subunits were also found in fractions with the combined secretory granules and in separate dense granules. In addition, alpha16 was found in these granule fractions, but secretion granules were devoid of alpha12. These data reveal a heterogeneous distribution of alpha-subunits in platelet compartments and may indicate that G12 and G16 play different roles in platelets than members of the G(i) and G(s) classes and other members of the G(q) class.
Asunto(s)
Plaquetas/metabolismo , Proteínas de Unión al GTP/sangre , Proteínas de Unión al GTP/química , Compartimento Celular , Membrana Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Conformación Proteica , Fracciones Subcelulares/metabolismoRESUMEN
Oxygen plays such a critical role in the central nervous system that a specialized mechanism of oxygen delivery to neurons is required. Reduced oxygen tension, or hypoxia, may have severe detrimental effects on neuronal cells. Several studies suggest that hypoxia can induce cellular adaptive responses that overcome apoptotic signals in order to minimize hypoxic injury or damage. Adaptive responses of neuronal cells to hypoxia may involve activation of various ion channels, as well as induction of specific gene expression. For example, ATP sensitive K+ channels are activated by hypoxia in selective neuronal cells, and may play a role in cell survival during hypoxia/anoxia. Additionally, hypoxia-induced c-Jun, bFGF and NGF expression appear to be associated with prevention (or delay) of neuronal cell apoptosis. In this paper, these adaptive responses to hypoxia in neuronal cells are discussed to examine the possible role of hypoxia in pathophysiology of diseases.
Asunto(s)
Adaptación Fisiológica , Apoptosis , Hipoxia de la Célula/fisiología , Animales , Vasos Sanguíneos , Sistema Nervioso Central , Humanos , Macrófagos/fisiología , Neuronas/fisiologíaRESUMEN
RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 microg/ml) and interferon-gamma (100 units/ml), respond with endogenous NO. formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO.-derived apoptosis was blocked by the NO.-synthase inhibitor NG-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-gamma, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO., released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO.-mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/patología , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animales , Línea Celular , Resistencia a Medicamentos , Macrófagos/metabolismo , RatonesRESUMEN
Peroxynitrite, formed by the interaction of superoxide with nitric oxide, has previously been implicated mostly as a cytotoxic agent. In contrast, its physiological and, possibly, beneficial effects are largely unknown. We have previously shown [Journal of Biological Chemistry, 1997, 272, 7253] that RAW 264.7 macrophages can be selected to be resistant toward lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced cytotoxicity. Resistant cells produced comparable amount of nitric oxide, but showed increased formation of superoxide, which might lead to increased production of peroxynitrite. We utilized this well characterized cell model to seek evidence that peroxynitrite might cause protection of RAW cells from cytokine toxicity. Exogenous peroxynitrite (30-50 microM), applied to RAW cells before cytokine stimulation, dramatically reduced LPS/IFN-gamma toxicity. Measurement of cell viability after overnight incubation with a mixture of LPS (10 microg/ml) and IFN-gamma (100 U/ml), showed that pretreatment with 40 microM peroxynitrite completely reverted LPS/IFN-gamma cytotoxicity. Differently, pretreatment of RAW cells with peroxynitrite (10-60 microM) did not prevent cytotoxicity induced by the nitric oxide-donors S-Nitroso-L-glutathione (0.2-1 mM), or spermine NONOate (0.2-2 mM), and by Actimomycin D (0.5-1 microg/ml), suggesting that the protective effect is specific for the LPS/IFN-gamma pathway. These results were confirmed through extensive controlled studies aimed to optimize cell exposure to peroxynitrite, and showed that peroxynitrite protects macrophages from cytokine-induced cytotoxicity.
Asunto(s)
Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Interferón gamma/toxicidad , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Nitratos/farmacología , Animales , Línea Celular , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Glutatión/análogos & derivados , Glutatión/farmacología , Macrófagos/citología , Ratones , Óxido Nítrico/toxicidad , Óxidos de Nitrógeno , Compuestos Nitrosos/farmacología , S-Nitrosoglutatión , Espermina/análogos & derivados , Espermina/farmacologíaRESUMEN
A hypoxic/anoxic microenvironment has been proposed to exist within a vascular lesion due to intimal or medial cell proliferation in vascular diseases. Here, we examined whether hypoxia alters macrophage function by exposing murine macrophage-like RAW 264.7 (RAW) cells to hypoxia (2% O2). When cells were exposed to hypoxia, a significant number of RAW cells underwent apoptosis. Additionally, small subpopulations of RAW cells were resistant to hypoxia-induced apoptosis. Through repeated cycles of hypoxia exposure, hypoxia-induced apoptosis-resistant macrophages (HARMs) were selected; HARM cells demonstrate >70% resistance to hypoxia-induced apoptosis, as compared with the parental RAW cells. When heat shock protein (HSP) expression was examined after hypoxia, we observed a significant decrease in constitutive heat shock protein 70 (HSC 70) in RAW cells, but not in HARMs, as compared with the control normoxic condition (21% O2). In contrast, the expression level of glucose-regulated protein 78 (GRP 78) in RAW and HARM cells after hypoxia treatment was not altered, suggesting that HSC 70 and not GRP 78 may play a role in protection against hypoxia-induced apoptosis. When tumor necrosis factor alpha (TNF-alpha) production was examined after hypoxic treatment, a significant increase in TNF-alpha production in HARM but decrease in RAW was observed, as compared with cells cultured in normoxic conditions. HARM cells also exhibit a much lower level of modified-LDL uptake than do RAW cells, suggesting that HARMs may not transform into foam cells. These results suggest that a selective population of macrophages may adapt to potentially pathological hypoxic conditions by overcoming the apoptotic signal.
Asunto(s)
Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Proteínas de Choque Térmico , Mediadores de Inflamación/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Microscopía Confocal , Chaperonas Moleculares/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas ras/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Homología de Secuencia de Aminoácido , Factor de Intercambio de Guanina Nucleótido ral , Proteínas de Unión al GTP rap , Proteínas ras/antagonistas & inhibidoresRESUMEN
Nitric oxide (NO) functions as a pathophysiological mediator in mammalian tissues. Activated macrophages produce NO as a non-specific immune response directed against invading bacteria or micro-organisms. The same macrophages that initiate the production of NO also can be toxically affected by NO. Incubation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and/or interferon-gamma (INF-gamma) induced the formation of NO by the activation of a cytokine-inducible NO synthase (NOS). The viability of these macrophages was inversely correlated with the formation of nitrite, a final NO-oxidation product measurable in the incubation medium. The addition of an NOS inhibitor, NG-monomethyl-L-arginine, diminished NO formation and preserved cell viability in a dose- and time-dependent fashion. Treatment of macrophages with ten cycles of non-lethal doses of LPS and INF-gamma, each followed by subculturing of the surviving cells, resulted in cell resistance to the NO toxic insult induced by LPS and INF-gamma. These resistant macrophages showed a 2-fold increase in the expression of the constitutive heat shock protein (HSC 70) which is known to be involved in protecting cells against the action of various metabolic insults. Our results establish a link between cell resistance to the toxic effects of NO, and the expression of heat shock proteins in RAW 264.7 macrophages.
Asunto(s)
Proteínas de Choque Térmico/biosíntesis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Óxido Nítrico/toxicidad , Animales , Línea Celular , Resistencia a Medicamentos , Interferón gamma/toxicidad , Lipopolisacáridos/toxicidad , Activación de Macrófagos , Nitroprusiato/farmacología , Estrés Oxidativo , Ratas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Phosphorylation of rap 1b in human platelets correlates with both an upward shift of the protein on sodium dodecyl sulfate polyacrylamide gels and the translocation of the phosphorylated protein to the cytosolic fraction of platelets. We reported that this phenomenon occurs in platelets in response to agents that stimulate adenylate cyclase and thereby activate the cyclic AMP-dependent protein kinase. We now have evidence that phosphorylation of rap1b in platelets is also induced by nitric oxide generating compounds through stimulation of guanylate cyclase and activation of the cyclic GMP-dependent protein kinase. We observed time-dependent phosphorylation of rap1b and dose-dependent inhibition of collagen-stimulated aggregation in washed platelets incubated with S-nitroso serum albumin. In the presence of a combination of iloprost and 3-morpholinosydnonimine, when both PKA and PKG are activated, phosphorylation of rap1b increased synergistically to a level three times higher than the sum of their individual actions.