RESUMEN
Hyperlipidemia may complicate the use of HIV protease inhibitors (PIs) in AIDS therapy. To determine the cause of hyperlipidemia, the effect of PIs on lipid metabolism was examined with HepG2 liver cells and AKR/J mice. In HepG2 cells, the PIs ABT-378, nelfinavir, ritonavir, and saquinavir stimulated triglyceride synthesis; ritonavir increased cholesterol synthesis; and amprenavir and indinavir had no effect. Moreover, nelfinavir increased mRNA expression of diacylglycerol acyltransferase and fatty acid synthase. The retinoid X receptor agonist LG100268, but not the antagonist LG100754, further increased PI-stimulated triglyceride synthesis and mRNA expression of fatty acid synthase in vitro. In fed mice, nelfinavir or ritonavir did not affect serum glucose and cholesterol, whereas triglyceride and fatty acids increased 57% to 108%. In fasted mice, ritonavir increased serum glucose by 29%, cholesterol by 40%, and triglyceride by 99%, whereas nelfinavir had no effect, suggesting these PIs have different effects on metabolism. Consistent with the in vitro results, nelfinavir and ritonavir increased triglyceride 2- to 3-fold in fasted mice injected with Triton WR-1339, an inhibitor of triglyceride clearance. We propose that PI-associated hyperlipidemia is due to increased hepatic triglyceride synthesis and suggest that retinoids or meal restriction influences the effects of select PIs on lipid metabolism.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Hiperlipidemias/inducido químicamente , Hígado/efectos de los fármacos , Triglicéridos/biosíntesis , Aciltransferasas/biosíntesis , Animales , Glucemia/análisis , Radioisótopos de Carbono , Colesterol/biosíntesis , Colesterol/sangre , Diacilglicerol O-Acetiltransferasa , Ayuno , Ácido Graso Sintasas/biosíntesis , Ácidos Grasos/sangre , Inhibidores de la Proteasa del VIH/toxicidad , Hiperlipidemias/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos AKR , Nelfinavir/farmacología , Ácidos Nicotínicos/farmacología , Ácidos Nicotínicos/toxicidad , Periodo Posprandial , ARN Mensajero/biosíntesis , Receptores de Ácido Retinoico/agonistas , Receptores X Retinoide , Ritonavir/farmacología , Saquinavir/farmacología , Tetrahidronaftalenos/farmacología , Tetrahidronaftalenos/toxicidad , Factores de Transcripción/agonistas , Triglicéridos/sangre , Células Tumorales CultivadasRESUMEN
AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.
Asunto(s)
Adipocitos/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Lipólisis/efectos de los fármacos , Proteínas de Neoplasias , Tiazolidinedionas , Triglicéridos/metabolismo , Adipocitos/citología , Animales , Compuestos Azo/farmacología , Bexaroteno , Carbamatos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colorantes/farmacología , Proteínas de Unión a Ácidos Grasos , Furanos , Indinavir/farmacología , Insulina/farmacología , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Nelfinavir/farmacología , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Ritonavir/farmacología , Rosiglitazona , Saquinavir/farmacología , Células Madre , Sulfonamidas/farmacología , Tetrahidronaftalenos/farmacología , Tiazoles/farmacología , Factores de Transcripción/agonistas , Factores de Transcripción/metabolismoRESUMEN
Human immunodeficiency virus (HIV) protease inhibitors (PI) may alter lipid metabolism in patients with acquired immunodeficiency syndrome (AIDS). However, the influence of dietary fat on the metabolic effects of PI therapy remains unknown. AKR/J mice were fed high or low fat diets and treated with the PI indinavir (IDV), nelfinavir (NFV), saquinavir (SQV) or amprenavir (APV) by subcutaneous delivery for 2 wk. Serum concentrations of glucose, insulin, triglyceride, free fatty acid, glycerol, pancreatic lipase, bilirubin, alkaline phosphatase, blood urea nitrogen and PI, and interscapular and epididymal fat weights were determined. Some metabolic effects of PI were dependent on diet. IDV- and NFV-treated mice had greater serum glucose concentration and body weight; IDV-treated mice had lower serum insulin; NFV-treated mice had greater interscapular fat mass; and SQV treated mice had lower serum triglyceride concentration than control mice fed the low but not the high fat diet. In contrast, NFV- and IDV-treated mice had greater triglyceride concentration and blood urea nitrogen, and SQV treated mice had greater serum cholesterol than control mice fed the high but not the low fat diet. The serum concentration of SQV was lower in mice fed the high fat compared with the low fat diet. Other effects were not dependent on diet. IDV- and NFV-treated mice had greater fatty acids, and IDV-treated mice had greater pancreatic lipase, bilirubin and alkaline phosphatase than control mice fed either diet. APV treatment had little effect on these serum measurements. Thus, changes in dietary fat can influence some but not all of the effects of PI on metabolism. Furthermore, each PI produces different effects in vivo, indicating that various PI affect distinct metabolic pathways.
Asunto(s)
Glucemia/efectos de los fármacos , Grasas de la Dieta/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Hígado/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Carbamatos , Grasas de la Dieta/administración & dosificación , Interacciones Farmacológicas , Furanos , Inhibidores de la Proteasa del VIH/administración & dosificación , Indinavir/administración & dosificación , Indinavir/sangre , Indinavir/farmacología , Inyecciones Subcutáneas , Insulina/sangre , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos AKR , Nelfinavir/administración & dosificación , Nelfinavir/sangre , Nelfinavir/farmacología , Saquinavir/administración & dosificación , Saquinavir/sangre , Saquinavir/farmacología , Sulfonamidas/administración & dosificación , Sulfonamidas/sangre , Sulfonamidas/farmacologíaRESUMEN
HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A(1) acid (all-trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5, 5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di-tert-butylchalcone 4'-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Indinavir/farmacología , Transducción de Señal/efectos de los fármacos , Vitamina A/metabolismo , Animales , Antineoplásicos/farmacología , Células Cultivadas , Inhibidores de la Proteasa del VIH/efectos adversos , Indinavir/efectos adversos , Ratones , Tretinoina/farmacologíaRESUMEN
AIMS/HYPOTHESIS: Although retinoid X receptor (RXR) and peroxisome proliferator activated receptor-gamma (PPARgamma) agonists have antidiabetic effects in hyperinsulinaemic animals, little information exists on their effects after pancreatic beta-cell failure. Thus, we examined if RXR and PPARgamma agonists alter distinct metabolic pathways in animals suffering from impaired insulin secretion. METHODS: Adverse side effects and antidiabetic responses were measured in db/db mice treated from 14-16 weeks of age with the RXR agonist, LG100268, and/or the PPARgamma agonists, BRL49653 or GW1929. RESULTS: In animals treated with LG100268 or BRL49653, serum glucose, glycohaemoglobin and the cardiovascular risk factor, fibrinogen, decreased to the same extent. Both of these agonists were equally effective at increasing insulin accumulation in beta cells, although neither agent had an effect on serum insulin concentrations. In contrast, the RXR agonist was less effective than the PPARgamma agonists at lowering serum triglycerides and non-esterified fatty acids and increasing interscapular brown fat and body weight. Further, LG100268 increased serum alkaline phosphatase and liver mass, hepatic fat accumulation, lauric acid hydroxylase activity, catalase-immunostaining and peroxisomal number more than the PPARgamma agonists. Moreover, co-treatment with the RXR and PPARgamma agonists reduced glucose, triglycerides, non-esterified fatty acids and cholesterol more than either agent alone. CONCLUSION/INTERPRETATION: These data suggest 1) RXR and PPARgamma agonists decrease islet degeneration, cardiovascular risk and cachexia during later stages of diabetes, 2) RXR agonists are less effective than PPARgamma agonists at decreasing serum lipids and causing weight gain and 3) RXR agonists have a more pronounced effect on liver metabolism (e.g. peroxisome accumulation and hepatomegaly) than PPARgamma agonists.
Asunto(s)
Glucemia/metabolismo , Enfermedades Cardiovasculares/prevención & control , Diabetes Mellitus/tratamiento farmacológico , Hepatomegalia/inducido químicamente , Ácidos Nicotínicos/uso terapéutico , Receptores de Ácido Retinoico/agonistas , Tetrahidronaftalenos/uso terapéutico , Factores de Transcripción/agonistas , Animales , Caquexia/prevención & control , Complicaciones de la Diabetes , Diabetes Mellitus/fisiopatología , Fibrinógeno/metabolismo , Hemoglobina Glucada/metabolismo , Hiperinsulinismo/etiología , Islotes Pancreáticos/fisiopatología , Lípidos/sangre , Hígado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Nicotínicos/toxicidad , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores X Retinoide , Factores de Riesgo , Tetrahidronaftalenos/toxicidadRESUMEN
PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.
Asunto(s)
Adipocitos/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Cromanos/farmacología , Metabolismo Energético , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/farmacología , Espectrofotometría Infrarroja/métodos , Tiazoles/farmacología , Tiazolidinedionas , Adipocitos/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Canales Iónicos , Proteínas/genética , Receptores Citoplasmáticos y Nucleares , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Factores de Transcripción , Troglitazona , Proteína Desacopladora 2RESUMEN
Troglitazone and metformin are antidiabetic agents that belong to the thiazolidinedione and biguanide classes of drugs, respectively. To evaluate how these drugs influence fuel utilization, we compared their effects on several pathways regulating carbohydrate and lipid metabolism in vitro. Both drugs stimulated glucose transport and utilization in C3H10T1/2 cells, a cell line capable of differentiating into adipocytes when treated with thiazolidinediones. However, we observed that these drugs had a number of different in vitro effects. Unlike metformin, troglitazone stimulated beta3-adrenergic receptor-mediated lipolysis, lipogenesis, and transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Further, by using a mitochondrial-specific fluorescent dye, we found troglitazone to be more effective than metformin at increasing mitochondrial mass. In contrast to troglitazone, metformin was more effective at increasing mitochondrial fatty acid beta-oxidation, peroxisomal fatty acid beta-oxidation, and anaerobic respiration (i.e. lactate production). Additionally, metformin stimulated and troglitazone inhibited both aerobic respiration and basal lipolysis. Insulin enhanced the effects of troglitazone, but not those of metformin, on these cells. Taken together, the data show that troglitazone and metformin affect two distinct metabolic pathways: one that is anabolic (i.e. troglitazone) and the other that is catabolic (i.e. metformin). Further, these observations suggest that the metabolic activity of mitochondria may be lower in cells treated with troglitazone than with metformin.
Asunto(s)
Cromanos/farmacología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Metabolismo de los Lípidos , Metformina/farmacología , Tiazoles/farmacología , Tiazolidinedionas , Animales , Transporte Biológico/efectos de los fármacos , Ácido Láctico/metabolismo , Ratones , Ratones Endogámicos C3H , Mitocondrias/efectos de los fármacos , Palmitoil Coenzima A/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , TroglitazonaAsunto(s)
Regulación Enzimológica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Quinasas/aislamiento & purificación , Proteína S6 Ribosómica , Proteínas Ribosómicas/metabolismoRESUMEN
Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.
Asunto(s)
Tejido Adiposo/enzimología , Éteres Cíclicos/farmacología , Insulina/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Células Cultivadas , Glucógeno Sintasa Quinasa 3 , Humanos , Ionóforos/farmacología , Cinética , Masculino , Datos de Secuencia Molecular , Peso Molecular , Ácido Ocadaico , Fosfoproteínas/aislamiento & purificación , Proteínas Quinasas/aislamiento & purificación , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Especificidad por SustratoRESUMEN
Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cromatografía en Gel , Citosol/enzimología , Cinética , Ratones , Datos de Secuencia Molecular , Péptidos/síntesis química , Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas , Especificidad por SustratoRESUMEN
A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.
Asunto(s)
Insulina/farmacología , Oocitos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Femenino , Técnicas In Vitro , Cinética , Microinyecciones , Oocitos/efectos de los fármacos , Fosfoproteínas Fosfatasas/administración & dosificación , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas , Proteínas Quinasas S6 Ribosómicas , Tirosina , Xenopus laevisRESUMEN
Mitogenic stimulation of mammalian cells results in increased serine phosphorylation of ribosomal protein S6. Phorbol esters, which stimulate protein kinase C activity, can also increase S6 phosphorylation. In order to further investigate the role of protein kinase C in the activation S6 kinase, we studied the stimulation of an S6 kinase activity in response to phorbol ester and epinephrine in a renal epithelial cell line, Madin-Darby canine kidney cells (MDCK). In these cells, S6 phosphorylating activity in cytosolic extracts was increased following the addition of phorbol ester to the intact cells. S6 kinase and protein kinase C activities were measured in separate fractions prepared by DEAE-Sephacel fractionation of cytosolic extracts prepared from the same cells. The time course and dose-response curves for the effects of phorbol 12-myristate 13-acetate (PMA) on S6 kinase activity were similar to those for its effects on protein kinase C binding to the membrane fraction, indicating that S6 kinase activation was correlated with protein kinase C activation. Epinephrine, acting via alpha1-adrenergic receptors, also stimulated S6 kinase activity in MDCK cells; the magnitude of this effect was similar to that of PMA. However, epinephrine causes only a slight and transient association of protein kinase C with the membrane. The effect of epinephrine on S6 kinase activity, unlike that of PMA, was dependent on the presence of extracellular calcium. A23187, a calcium ionophore, could also stimulate S6 kinase activity. These results suggest that S6 kinase can be activated through more than one signaling pathway in MDCK cells. The properties of the PMA-stimulated S6 kinase were further investigated following partial purification of the enzyme. The S6 kinase was distinct from protein kinase C by several criteria. Noteably, the S6 kinase was highly specific for S6 as substrate. These results show that phorbol esters, acting through protein kinase C, stimulate the activity of a unique S6 kinase. This S6 kinase can also be activated through a signaling pathway that appears to be dependent on increased intracellular calcium.
Asunto(s)
Epinefrina/farmacología , Riñón/enzimología , Proteínas Quinasas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Aminoácidos , Animales , Calcimicina/farmacología , Línea Celular , Cromatografía por Intercambio Iónico , Citosol/enzimología , Perros , Activación Enzimática/efectos de los fármacos , Epitelio/enzimología , Cinética , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/aislamiento & purificación , Ratas , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Especificidad por SustratoRESUMEN
In the present article, we have reviewed several promising areas of research, the results of which support the existence of enzymatic cascades linking extracellular growth factor receptor/tyrosine kinases with intracellular serine/threonine kinases. Proceeding either "upstream" from select serine/threonine kinases or "downstream" from the receptors, characterization of both the distal and proximal elements of these cascades is yielding insights into the multiple pathways of signal transduction. It is anticipated that future work will define the intermediate effectors in these cascades and provide us with a clear understanding of the biochemical mechanisms underlying intracellular responses to extracellular signals.
Asunto(s)
Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Sistemas de Mensajero Secundario , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Glucógeno Sintasa Quinasa 3 , Sustancias de Crecimiento/farmacología , Ratones , Fosforilación , Ratas , Receptores de Superficie Celular/metabolismo , Proteína S6 Ribosómica , Proteínas Ribosómicas/metabolismo , Xenopus laevis/metabolismoRESUMEN
Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.
Asunto(s)
Macrófagos/fisiología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/análisis , Receptores Acoplados a Proteínas G , Animales , Unión Competitiva , Calcio/análisis , Membrana Celular/metabolismo , Células Cultivadas , Quimiotaxis , Cromatografía Líquida de Alta Presión , Fosfatos de Inositol/análisis , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Fosforilación , Factor de Activación Plaquetaria/fisiología , Biosíntesis de Proteínas , Organismos Libres de Patógenos EspecíficosRESUMEN
LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.
Asunto(s)
Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/fisiología , Fosfatidilinositoles/metabolismo , Animales , Calcio/fisiología , Citoplasma/fisiología , Endotoxinas/farmacología , Hidrólisis , Lípido A/farmacología , Ratones , Peso Molecular , Fosfoproteínas/fisiologíaRESUMEN
Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-gamma (IFN-gamma). To explore the biochemical transductional events initiated by IFN-gamma, peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-gamma to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca++ was sufficient for priming, whereas the addition of Mg++ was much less efficient. Priming by IFN-gamma, however, was not blocked by EGTA. Efflux of 45Ca++ from preloaded cells was significantly increased by A23187 and by IFN-gamma. Quin-2/AM, an intracellular chelator of Ca++, blocked priming by IFN-gamma. In summary, the data suggest that priming of macrophages for tumoricidal function by IFN-gamma involves, at least in part, alterations in protein kinase C and in levels of intracellular Ca++.
Asunto(s)
Calcimicina/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Sarcoma de Mastocitos/inmunología , Forboles/farmacología , Acetato de Tetradecanoilforbol/farmacología , Animales , Calcio/metabolismo , Calcio/fisiología , Ácido Egtácico/farmacología , Interferón gamma/farmacología , Canales Iónicos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Cavidad PeritonealRESUMEN
Covalent modification of proteins via phosphorylation is a well-documented mechanism whereby intracellular events are controlled by external stimuli. Treatment of thioglycollate-elicited, C57Bl/6 murine peritoneal macrophages with nanogram quantities of bacterial lipopolysaccharide (LPS) consistently results in altered 32Pi labeling of a specific set of proteins (e.g., proteins of 67, 37, 33, and 28 kD), as measured by autoradiography after SDS-polyacrylamide gel electrophoresis. Induction of this pattern of phosphorylation is duplicated by the lipid A moiety of LPS. The LPS-stimulated changes in phosphate labeling are both dose- and time-dependent. Of various pharmacologic agents tested, the phosphorylation pattern induced in macrophages by the tumor promoter phorbol myristic acetate shows similarity to the pattern induced by LPS. Analysis of pp 28 and pp 37 from both LPS- and PMA-treated macrophages by limited proteolysis demonstrates that these phosphoproteins are structurally related and that the sites of phosphorylation are similar for both treatment conditions. Macrophages from the genetically LPS-unresponsive C3H/HeJ strain show no alteration in their pattern of phosphorylation after treatment with LPS. Control macrophages, from C3H/HeN mice, respond to LPS in a fashion identical to that seen in C57Bl/6 macrophages. Pretreatment of macrophages with IFN-gamma potentiates the effect of LPS (i.e., yields a level of altered phosphate labeling greater than that observed with LPS or PMA alone). Together, the data indicate that LPS causes altered phosphate labeling of a defined set of proteins, and that the circumstances of this response are consistent with a possible role in coupling LPS-initiated signals to the induction of functional competence in macrophages.
Asunto(s)
Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Animales , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Interferón gamma/farmacología , Cinética , Activación de Macrófagos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Cavidad Peritoneal , Fosfoproteínas/aislamiento & purificación , FosforilaciónRESUMEN
The number of transferrin receptors in thioglycollate-elicited murine peritoneal macrophages is markedly depressed after exposure to murine gamma-interferon (IFN gamma) in vitro. This change has been used as a model system to study the molecular and cellular mechanisms of IFN gamma signal transduction. We observed that the downshift of the transferrin receptor could be mimicked by exposure to the calcium ionophore (A23187) or the potent tumor promoter, phorbol 12-myristate 13-acetate (PMA). Saturation binding studies on thioglycollate (TG)-elicited peritoneal macrophages after exposure to A23187 or PMA showed the reduced expression of transferrin binding activity attributable to a decrease in the total number of cellular transferrin receptors and not an alteration in receptor-ligand affinity, in agreement with previous results obtained after exposure to IFN gamma. The loss of transferrin receptors in response to A23187 or PMA was dose dependent, and the kinetics of the change were identical to those observed with IFN gamma treatment. Phorbol 12,13-dibutyrate or 4-beta-phorbol 12,13-didecanoate, both biologically active phorbol esters, also induced reduced expression of transferrin receptors, whereas nonesterified phorbol or 4-alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, had no effect on transferrin receptor expression. Finally, PMA and A23187, when used together, acted cooperatively to modulate transferrin receptor expression when both agents were present at subthreshold concentrations. These results, taken together, suggest that elevation of intracellular Ca++ levels and/or stimulation of protein kinase C are involved in the response of macrophages to IFN gamma.
Asunto(s)
Calcimicina/farmacología , Interferón gamma/farmacología , Macrófagos/metabolismo , Forboles/farmacología , Receptores de Superficie Celular/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transferrina/metabolismo , Animales , Células Cultivadas , Cinética , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ésteres del Forbol/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Receptores de TransferrinaRESUMEN
Nonionic detergent extracts of murine monocytes contain a specific, high-affinity binding site for the serum glycoprotein transferrin. The binding activity was saturable and specific for 125I-labeled transferrin. The transferrin-receptor content of monocytes was compared with that of resident peritoneal macrophages and thioglycollate-elicited macrophages. Whereas resident cells showed no detectable activity, inflammatory macrophages and monocytes both bound transferrin to a similar degree. The calculated dissociation constant (2.3 X 10(-10)) and the number of sites per monocyte (11,400) compared favorably with those reported for transferrin receptors on inflammatory macrophages. Thus, based on the expression of the transferrin receptor, murine monocytes resemble murine inflammatory peritoneal macrophages rather than resident tissue macrophages.