RESUMEN
Rapid steroid effects, reported in several cell types, have pointed out the possibility of non-genomic mechanisms of action, presumably on cell surface receptors. Here we analyzed the effects of antibody-mediated aggregation of a novel type of progesterone receptor on the plasma membrane of human sperm cells. We report that aggregation of hormone-receptor complexes induces Ca2+ influx and a Ca(2+)-dependent exocytotic event in this system. These data suggest a possible mechanism for rapid steroid-induced events. Further research is warranted to examined if a similar mechanism is involved in rapid steroid effects in other cell types.
Asunto(s)
Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Espermatozoides/metabolismo , Transporte Biológico , Calcio/metabolismo , Cationes Bivalentes , Membrana Celular/metabolismo , Humanos , Inmunohistoquímica , MasculinoRESUMEN
The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.
Asunto(s)
Activación de Linfocitos/fisiología , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Antígenos CD/biosíntesis , Transporte Biológico , Agregación Celular , Cromatografía por Intercambio Iónico , Citosol/química , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-2/biosíntesis , Leucemia de Células T , Activación de Linfocitos/efectos de los fármacos , Mutagénesis , Fosforilación , Proteína Quinasa C/farmacocinética , Proteína S6 Ribosómica , Proteínas Ribosómicas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Jurkat T cells, loaded with the fluorescent calcium probe Indo 1, responded to exogenous phosphatidic acid (PA) by transiently increasing their cytosolic Ca2+ concentration. This effect was dose-dependent, remained unmodified when external Ca2+ was chelated with EGTA, and was totally inhibited when cells were first exposed to CD3 monoclonal antibodies, indicating that it was solely due to the release of an intracellular pool, which is also mobilized during a stimulation via the CD3 T-cell receptor (TcR) molecular complex. CD3- and phytohaemagglutinin (PHA)-stimulated Jurkat cells also produced PA, the dose-responses and kinetics of which were consistent with those of calcium release. Moreover, diacylglycerol (DAG) kinase inhibitors abrogated PA production and lowered calcium release by CD3-stimulated cells. PA did not induce any apparent increase in inositol triphosphates (IP3), nor did it modify the increase entailed by activation of the CD3 pathway, pointing out that IP3 can be supplemented in mobilizing calcium from intracellular stores. Conversely, a first exposure to PA only partially inhibited the CD3- or ionomycin-induced internal release of calcium, suggesting either a rapid restoration of the PA-sensitive stores, or a contribution of other mediators, such as IP3, in the CD3 activation pathway.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Calcio/metabolismo , Ácidos Fosfatidicos/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/inmunología , Complejo CD3 , Línea Celular , Diacilglicerol Quinasa , Humanos , Inositol 1,4,5-Trifosfato/biosíntesis , Leucemia de Células T/metabolismo , Ácidos Fosfatidicos/biosíntesis , Fosfotransferasas/antagonistas & inhibidores , Fitohemaglutininas/inmunología , Células Tumorales Cultivadas/metabolismoRESUMEN
Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Toxina del Pertussis , Linfocitos T/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , Adenosina Difosfato Ribosa/metabolismo , Calcio/metabolismo , Comunicación Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Interleucina-1/fisiología , Interleucina-2/biosíntesis , Monocitos/efectos de los fármacos , Monocitos/inmunología , Receptores de Interleucina-2/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Activación de Linfocitos/fisiología , Toxina del Pertussis , Linfocitos T/inmunología , Factores de Virulencia de Bordetella/farmacología , Adenosina Difosfato/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3 , Calcio/metabolismo , Regulación hacia Abajo , Proteínas de Unión al GTP/efectos de los fármacos , Interleucina-2/biosíntesis , Cinética , Receptores de Antígenos de Linfocitos T/metabolismo , Ribosa/metabolismo , Células Tumorales CultivadasRESUMEN
We have studied the effect of pertussis toxin (PT) on in vivo priming of T lymphocytes to insulin. Mice were immunized with bovine insulin in complete Freund's adjuvant and antigen-specific DNA synthesis was measured in lymphoid cell suspensions from lymph nodes and spleens. Insulin-specific response was greatly enhanced both in spleen and lymph nodes if mice were given PT at the time of immunization. Mice given PT presented 3 times more cells in spleen and 4 times less in lymph nodes. However, the major antigen-specific response was still observed in lymph nodes. PT had a strong mitogenic effect in vitro on lymph node cells but a weak effect on spleen cells indicating that the adjuvant activity of PT involves other effects besides the mitogenic activity.
Asunto(s)
Insulina/inmunología , Toxina del Pertussis , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos , Mitógenos/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/fisiología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Interleukin-2 (IL-2) production by activated Jurkat T cells was markedly delayed when these cells were treated with low concentrations of the chymotryptic-type protease inhibitor N-alpha-p-tosyl-L-phenylalanine chloromethylketone (TPCK). This increased lag time observed in the presence of TPCK directly correlates with the interaction of the inhibitor with a unique 42,000 molecular weight (MW) serine protease, which can be labelled with [3H]DFP, and was not due to an intracellular accumulation of a non-mature form of IL-2 nor to a non-specific inhibition of overall protein synthesis. The results presented in this report indicate that a 42,000 MW chymotryptic-like serine protease is required for IL-2 production by activated Jurkat T cells.
Asunto(s)
Quimotripsina/inmunología , Interleucina-2/biosíntesis , Linfocitos T/inmunología , Línea Celular , Quimotripsina/antagonistas & inhibidores , Humanos , Cinética , Clorometilcetona de Tosilfenilalanila/farmacología , Células Tumorales Cultivadas/inmunologíaRESUMEN
Activation of Jurkat T cells with phytohemagglutinin, CD3 or CD2 mAbs results in a marked inhibition of phosphatidylserine (PS) synthesis. Monitoring PS synthesis in T cells shows that: (i) after modulation of CD3 molecules the cells become refractory to further treatment with CD3 mAbs as well as to a further challenge with CD2 mAbs; and (ii) treatment of T cells with fluoride ions and cholera toxin, two known effectors of guanosine triphosphate-binding proteins, also resulted in a strong inhibition of the synthesis of this phospholipid. The inhibition of PS synthesis thus appears to be regulated similarly to the other activation events, suggesting that transmembrane signalling mechanisms leading to PS inhibition are the same as those previously proposed for increasing phosphatidylinositides turnover and subsequent rise in the intracellular calcium concn in lymphocytes.
Asunto(s)
Antígenos CD/inmunología , Guanosina Trifosfato/metabolismo , Fosfatidilserinas/biosíntesis , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Línea Celular , Toxina del Cólera/farmacología , Fluoruros/farmacología , Humanos , Activación de Linfocitos , Células Tumorales Cultivadas/inmunología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Interleukin 2 production by activated Jurkat T cells is markedly decreased by prostaglandin E2 (PGE2). The target of PGE2 action has been investigated in the present study. Among the biochemical events occurring after CD3.TCR triggering by anti-CD3 monoclonal antibody, phosphorylation of two cytosolic proteins, pp21 and pp23, was strongly inhibited by PGE2, forskolin, and 8-bromo-cAMP, whereas anti-CD3 monoclonal antibody-induced CD3.TCR modulation and Ca2+ influx were not affected. The inhibition of both pp21 and pp23 phosphorylation and interleukin 2 synthesis by PGE2 can be largely reversed by the cAMP-dependent protein kinase inhibitor, N-[2-(methylamino)-ethyl-1]-5-isoquinoline sulfonamide. Together with the demonstration of a cAMP-dependent protein kinase activity in Jurkat T cells, these results are consistent with the participation of the cAMP-dependent protein kinase mediating the inhibitory action of PGE2, probably through the inhibition of pp21 and pp23 phosphorylation. Thus, it appears that the modulation of the phosphorylation of these cytosolic proteins represents an essential step in the regulation of T lymphocyte activation.
Asunto(s)
Calcio/fisiología , Citosol/metabolismo , Activación de Linfocitos , Fosfoproteínas/metabolismo , Proteínas Quinasas/fisiología , Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3 , Calcio/metabolismo , Línea Celular , Colforsina/farmacología , AMP Cíclico/farmacología , Dinoprostona/farmacología , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/enzimologíaRESUMEN
T lymphocytes can be activated to proliferate by triggering the T-cell antigen-receptor complex (CD3-Ti) with anti-CD3 (Cluster of Differentiation 3) monoclonal antibody (mAb) or with the mitogenic lectin phytohaemagglutinin A (PHA). We have investigated the relationship between lymphocyte activation and protein phosphorylation in the human leukaemic T-cell line Jurkat. Incubation of 32P-labelled Jurkat cells with anti-CD3 mAb or PHA induced the phosphorylation of two cytosolic proteins that migrate with apparent Mr values of 21,000 (pp21) and 23,000 (pp23) and pI values of 5.1 and 5.0 respectively. Peptide mapping of the two proteins produced the same phosphopeptides pattern, suggesting that pp21 and pp23 are closely related. The phosphorylation of pp21 and pp23 induced by anti-CD3 mAb appeared to be transient, since it was already detected 2 min after the addition of the mAb, reached a maximum at 10 min and recovered its basal level after 1 h. Phosphorylation of pp21 and pp23 could also be elicited by the Ca2+ ionophore A23187 and sodium orthovanadate (Na3VO4), two agents that bypass the T-cell-receptor complex and produced an increase in cytosolic Ca2+ concentration. In addition, we found that vanadate, like the Ca2+ ionophore, induced the secretion of interleukin-2 (IL-2) when used in combination with a submitogenic concentration of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results show that the Ca2+-dependent phosphorylation of pp21 and pp23 represents an early event in the process of signal transduction through the CD3-Ti receptor complex.
Asunto(s)
Citosol/metabolismo , Activación de Linfocitos , Proteínas/metabolismo , Linfocitos T/fisiología , Calcimicina/farmacología , Línea Celular , Humanos , Interleucina-2/farmacología , Mapeo Peptídico , Fosforilación , Células Tumorales Cultivadas/efectos de los fármacos , Vanadatos/farmacologíaRESUMEN
We used a photoaffinity labeling technique to investigate whether a molecular interaction occurs between antigen and Ia molecules on antigen presenting cells (APC) in the absence of T lymphocytes. M.12.4.1 B lymphoma cells (Iad), which are able to present bovine insulin to Iad lymph node primed T cells, were given radioiodinated bovine insulin derivatized with the photoreactive group (2-nitro-4-azidophenylacetyl) at Lys 29 of the B chain of the insulin molecule. Processing of insulin was allowed by incubating the APC with antigen for increasing periods of time at 37 degrees C or 4 degrees C. The covalent coupling of the processed photoreactive antigen to any neighboring cellular protein was thereafter induced by u.v. irradiation. Immunoprecipitation of membrane proteins by monoclonal antibodies showed that under these conditions, the alpha and beta subunits of the Ia molecules were selectively photolabeled. Labeling was time- and temp-dependent as was the internalization of insulin. The apparent mol. wts of the antigen-Ia molecule complexes were not significantly different from that of native Ia molecules radioiodinated by surface labeling, indicating that only a small fragment of the antigen was covalently coupled to Ia molecules. Similar experiments performed with human B lymphoma cells (526 cells) gave similar results. These observations therefore indicate: (1) that Ia molecules expressed by intact APC are able to bind antigens in the absence of T lymphocyte antigen receptor; and (2) that this association, at least for insulin, requires uptake and a proteolytic fragmentation of the antigen by the APC.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Marcadores de Afinidad , Animales , Línea Celular , Insulina/metabolismo , Receptores de Antígenos de Linfocitos T , Linfocitos T , Células Tumorales CultivadasRESUMEN
While calcium ions are known to play a prominent role in signal transduction in the activation of T lymphocytes, its mechanism of action, target and function have not been elucidated. One crucial event in the calcium-dependent process is the activation of the CD3 complex, and this, too, is not understood. While studying the release of Ca2+ from intracellular stores by several monoclonal antibodies (mAb) against CD3, we found that one of them, mAb 141, was ineffective unless free Ca2+ was present in the external medium. By flow cytometric analysis of the binding of this mAb to Jurkat T cells and peripheral blood lymphocytes we showed that 141 does not recognize the CD3 complex when external Ca2+ is chelated by EGTA. The binding was restored by addition of Ca2+ but not Mg2+. Finally at least one subunit of the CD3 complex displayed a modified electrophoretic migration rate when immunoprecipitated by Leu-4 in the absence of external free Ca2+. These results suggest that the conformation of the CD3 complex depends on Ca2+, the epitope recognized by 141 being concealed at low Ca2+ concentration.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T , Calcio/fisiología , Conformación Proteica , Receptores de Antígenos de Linfocitos T , Linfocitos T/metabolismo , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3 , Línea Celular , Citoplasma/metabolismo , Humanos , Pruebas de Precipitina , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de SeñalRESUMEN
Several chymotryptic-type protease inhibitors were found to inhibit both anti-CD3 mAb- and PHA-induced rise in Ca2+ and IL-2 production in Jurkat T cells. The magnitude of inhibition was a function of the effectors used to stimulate Ca2+ entry and depended on the concentration of the inhibitors. Neither tryptic-type protease inhibitors nor an elastase substrate prevented anti-CD3 mAb- or PHA-induced Ca2+ rise in Jurkat cells. The inhibitory effect of N-alpha-p-tosyl-L-phenylalanine chloromethyl-ketone on anti-CD3 mAb- and PHA-induced rise in Ca2+ resulted from a rapid increase in Ca2+ efflux. The inhibitors which were effective on Ca2+ mobilization also inhibited IL-2 production initiated by an anti-CD3 mAb in the presence of 12-O-tetradecanoylphorbol-13-acetate, and to a lesser extent by PHA or the calcium ionophore A23187. No inhibition of IL-2 production was observed when tryptic-type protease inhibitors or the elastase inhibitor were used. In addition, membrane preparations from Jurkat cells were found to hydrolyze the chymotryptic substrate Suc-Ala-Ala-Phe-paranitroaniline, an effect markedly inhibited by N-alpha-p-tosyl-L-phenylalanine chloromethylketone. Moreover, this inhibitor protected one potential endogenous substrate (Mr 38 kDa) from proteolysis. Taken together, these observations show that chymotryptic-type protease inhibitors block the responses generated by the binding of anti-CD3 mAb to Jurkat cells, and suggest that a chymotryptic-like membrane protease contributes to T cell activation.
Asunto(s)
Calcio/biosíntesis , Quimotripsina/farmacología , Inmunosupresores/farmacología , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/metabolismo , Anticuerpos Monoclonales/fisiología , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3 , Calcimicina , Línea Celular , Membrana Celular/enzimología , Relación Dosis-Respuesta Inmunológica , Ácido Egtácico , Humanos , Hidrólisis , Proteínas de la Membrana/metabolismo , Peso Molecular , Fitohemaglutininas , Inhibidores de Proteasas/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Clorometilcetona de Tosilfenilalanila/farmacologíaRESUMEN
Activation of Jurkat T lymphocytes to produce IL2 is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. This inhibition was obtained either with the mitogenic lectin PHA, anti-CD3 monoclonal antibodies (mAb), anti-CD2 mAb or anti-Ti mAb. Bypassing membrane receptor signalling, by using a Ca2+ ionophore or a protein phosphatase inhibitor, sodium ortho-vanadate, also results in a marked inhibition of PS synthesis. Activators of phospholipid -Ca2+ dependent protein kinase C (PKC) did not significantly modify PS synthesis, suggesting that the observed changes only involve the transduction of the first activation signal. PS being a necessary cofactor for PKC, our results strongly suggest that the inhibition of PS synthesis induced by receptor triggering exerts a feed back control on PKC therefore leading to a transient activation of the enzyme upon full lymphocyte activation.
Asunto(s)
Activación de Linfocitos/fisiología , Fosfatidilserinas/biosíntesis , Linfocitos T/metabolismo , Anticuerpos Monoclonales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Complejo CD3 , Línea Celular , Humanos , Activación de Linfocitos/efectos de los fármacos , Fitohemaglutininas/farmacología , Proteína Quinasa C/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Inmunológicos/fisiología , Serina/metabolismo , TritioRESUMEN
TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethylketone), a potent inhibitor of chymotryptic-type serine proteases, was found to decrease IL2 synthesis in Jurkat T cells. Conversely, the tryptic-type protease inhibitor, TLCK (N-alpha-p-tosyl-lysine chloromethylketone), which structurally is very similar to TPCK, had no effect on IL2 synthesis. Prostaglandin synthesis, a process that is known to reduce IL2 production in T cells, was increased by TPCK but not by TLCK, suggesting that this process could be, at least in part, responsible for the inhibition of IL2 production. Our results imply that a chymotryptic-type serine protease plays an active role in the regulation of IL2 synthesis and thus in the whole process of T-lymphocyte activation.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Interleucina-2/biosíntesis , Prostaglandinas/biosíntesis , Linfocitos T/metabolismo , Clorometilcetona Tosilisina/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Ácidos Araquidónicos/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Humanos , Activación de LinfocitosRESUMEN
Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.
Asunto(s)
Activación de Linfocitos , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Tirosina/metabolismo , Especificidad de Anticuerpos , Humanos , Cinética , Leucemia , Fosforilación , Pruebas de Precipitina , Células Tumorales Cultivadas , Vanadatos/farmacologíaRESUMEN
UNLABELLED: Recently it was speculated that activation of GTP-binding proteins and of phospholipase is involved in the transmission of a signal from the insulin-receptor kinase to effector systems in the cell. To confirm this hypothesis, we have tested the effect of AlCl3, which has been recently used as an experimental tool to activate GTP-binding proteins, on glucose transport in fat-cells. We found that AlCl3 has a partial insulin-like effect on glucose transport activity (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 9.6 +/- 2, AlCl3 29.6 +/- 4, insulin 74.0 +/- 3). The AlCl3 effect is totally blocked by pertussis toxin, whereas the insulin effect was not altered. The effect starts at [AlCl3] greater than 1 fM and reaches its maximum at 0.1 nM. Addition of phospholipase C (PLC; 50 munits/ml) also stimulated glucose transport (maximal 53.0 +/- 5%). Both substances acted faster than insulin itself (maximal values within 1 min for PLC, 2 min for AlCl3 and 5-10 min for insulin). Using the cytochalasin-B-binding assay to determine the effects of AlCl3 and PLC on the distribution of glucose carrier sites in subcellular fractions, we found that their glucose-transport-stimulating effect does not occur through an increase in glucose carrier sites in the plasma-membrane fraction. When PLC was combined with the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate), which increases glucose carrier sites in the plasma membrane, an additive effect on glucose transport was found [PLC (50 munits/ml), 53.0 +/- 5%, TPA (1 nM), 17.3 +/- 2%; PLC + TPA, 68.0 +/- 3%]. IN CONCLUSION: (1) the data show that AlCl3, probably through activation of a pertussis-toxin-inhibitable G protein, and PLC are able to modulate the intrinsic glucose carrier activity; (2) as pertussis toxin did not modify the effect of insulin, it seems unlikely that the insulin signal on glucose transport involves activation of this specific G protein.
Asunto(s)
Tejido Adiposo/metabolismo , Compuestos de Aluminio , Proteínas Portadoras/metabolismo , Metilglucósidos/farmacocinética , Metilglicósidos/farmacocinética , 3-O-Metilglucosa , Aluminio/farmacología , Cloruro de Aluminio , Animales , Transporte Biológico/efectos de los fármacos , Cloruros/farmacología , Citocalasina B/metabolismo , Técnicas In Vitro , Insulina/farmacología , Masculino , Toxina del Pertussis , Ratas , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Phosphorylation of membrane proteins is one of the earliest steps in cell activation induced by growth-promoting agents. Since MHC (major histocompatibility complex) class I molecules are known to contain phosphorylation sites in their C-terminal intracellular domain, we have studied the regulation of HLA (human leucocyte antigen) phosphorylation in intact cells by two mitogens, namely TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, and insulin, which are thought to exert their mitogenic effects through the stimulation of different protein kinases (protein kinase C and a tyrosine kinase respectively). Human B lymphoblastoid cells (526 cell line) were pulsed with [32P]Pi to label the intracellular ATP pool. Cells were then stimulated for 10 min with TPA, insulin, cyclic AMP or EGF (epidermal growth factor). The reaction was stopped by cell lysis in the presence of kinase and phosphatase inhibitors, and class I HLA antigens were immunoprecipitated with monoclonal antibodies. Analysis of labelled proteins by gel electrophoresis and autoradiography revealed that TPA increased the phosphorylation of the 45 kDa class I heavy chain by 5-7-fold, and insulin increased it by 2-3-fold. Cyclic AMP and EGF had no stimulatory effect. Analysis of immunoprecipitated HLA molecules by two-dimensional gel electrophoresis showed that TPA and insulin stimulated the incorporation of 32P into different 45 kDa molecular species, suggesting that different sites were phosphorylated by two agents. Moreover, incubation of purified class I MHC antigens with partially purified insulin-receptor tyrosine kinase and [gamma-32P]ATP revealed that class I antigens could also be phosphorylated in vitro by this tyrosine kinase. Altogether, these results therefore confirm that insulin receptors and HLA class I molecules are not only structurally [Fehlmann, Peyron, Samson, Van Obberghen, Brandenburg & Brossette (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8634-8637] but also functionally associated in the membranes of intact cells.
Asunto(s)
Linfocitos B/efectos de los fármacos , Antígenos HLA , Antígenos de Histocompatibilidad Clase I , Insulina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Humanos , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
Activation of Jurkat T cells with anti-TCR, anti-CD3, anti-CD2, or PHA is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. The inhibition of the synthesis of this phospholipid could be partially reversed by IL-1. In Jurkat cells, IL-1 did not activate phosphodiesterases as demonstrated by the lack of change of inositol triphosphate and diacylglycerol levels as well as the lack of change in cytosolic Ca2+ concentration. Furthermore, IL-1 did not modify the intracellular level of cGMP and cAMP, suggesting that the observed rise of PS synthesis could play the role of mediator IL-1 action. As PS is a necessary cofactor for the activation of protein kinase C, our results suggest strongly that IL-1 modulate protein kinase C activity in the activated lymphocyte through its action on PS synthesis.
Asunto(s)
Interleucina-1/farmacología , Interleucina-2/biosíntesis , Fosfatidilserinas/biosíntesis , Linfocitos T/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
Two separate convenient sandwich enzyme immunoassay methods were developed for measuring the production of the monokines interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) from peripheral blood mononuclear cells. Polyclonal antisera raised against the recombinant proteins and selected on the basis of their ability to neutralize IL-1-induced IL-2 secretion were used for coating microtiter plates or preparing peroxidase-Fab' conjugates. Both techniques were able to accurately and specifically detect monokines from various sources in the sub-picomolar range and were not influenced by compounds currently used for cell activation. A high molecular weight form of IL-1 beta was demonstrated under certain conditions and the two enzyme immunoassays were successfully applied to the detection of IL-1 alpha and IL-1 beta present in cell supernatants following stimulation with mitogenic or chemical agents.