RESUMO
We studied the roles of glycolysis and glutaminolysis following an acute reduction in mitochondrial membrane potential (Ψ(m)) induced by the thiazolidinedione troglitazone (TRO) and compared the responses with CCCP-induced depolarization in breast cancer derived MCF-7 and MDA-MB-231 cells as well as in the MCF-10A normal breast cell line. TRO and CCCP both acutely reduced Ψ(m) but after 24 h TRO-treated cells had restored Ψ(m) associated with both increased glycolysis and glutaminolysis. In contrast, CCCP-treated cells exhibited only a partial restoration of Ψ(m) associated with increased glycolysis but decreased glutaminolysis. TRO-induced glutaminolysis was coupled to increased ammonium (GDH flux) and decreased alanine production (ALT flux) in all three cell lines. Both cancer cell lines exhibited a higher spontaneous GDH/ALT flux than the normal breast cell line associated with a reduced keto-acid pool. TRO's effect on GDH/ALT fluxes and mitochondrial keto-acid pool homeostasis was additive with glucose withdrawal suggesting limited intramitochondrial pyruvate availability. The TRO-induced acceleration in GDH flux supplies substrate for Complex I contributing to the restoration of Ψ(m) as well as Krebs cycle intermediates for biosynthesis. Inhibiting mitochondrial proton ATPase with oligomycin or nullifying the proton gradient with CCCP prevented both the TRO-induced recovery of Ψ(m) and accelerated GDH flux but restored ALT flux consonant with important roles for proton pumping in regulating GDH flux and Ψ(m) recovery. Blocking enhanced GDH flux reduced DNA synthesis consistent with glutaminolysis via GDH playing an important biosynthetic role in tumorigenesis.
Assuntos
Neoplasias da Mama/metabolismo , Cromanos/farmacologia , Glutamina/metabolismo , Glicólise/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ciclo do Ácido Cítrico/fisiologia , Feminino , Glucose/metabolismo , Humanos , TroglitazonaRESUMO
Mitochondrial Krebs cycle keto acid pool depends upon input from pyruvate and glutamate to maintain homeostasis. We studied the affect of glucose-derived pyruvate removal on compensatory input from glutamine-derived glutamate by accelerated glutamate metabolism via glutamate dehydrogenase (GDH). In glutamine minus glucose media (Gln-Glc), NH(4)(+) production increased 41% without an increase in glutamine uptake consistent with accelerated glutamate metabolism via GDH. Alanine production dropped 40% consistent with a shift of glutamate from alanine aminotransferase (ALT) to GDH. Troglitazone (TRO) added to the Gln-Glc media further enhanced glutamate metabolism via GDH at the expense of glutamate metabolism via ALT since alanine production dropped an additional 70%. TRO reduced cell glutamate content 30% while increasing lactate production 5-fold consistent with blocking of cytosolic pyruvate formed from mitochondrial malate from reentering the cycle and maintaining keto acid pool homeostasis. Consequently mitochondrial keto acid pool deficit pulls glutamate via GDH into the cycle. Additionally TRO reduced cytosolic pH which effectively pushes glutamate via GDH, rather than merely shifting glutamate from ALT to GDH. Providing intramitochondrial pyruvate in the form of methyl pyruvate reduced glutamate metabolism via GDH and elevated glutamate metabolism via ALT to control levels restoring acid-base balance. Our findings are consistent with TRO regulation of anaplerosis dependent upon dual pull (cycle keto-acid deficit)/push (cytosolic acidosis) mechanisms.
Assuntos
Cromanos/farmacologia , Células Epiteliais/metabolismo , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Hipoglicemiantes/farmacologia , Tiazolidinedionas/farmacologia , Alanina Transaminase/metabolismo , Animais , Linhagem Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/fisiologia , Desaminação , Rim/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Suínos , TroglitazonaRESUMO
Hepatocyte growth factor (HGF) is found in tumor microenvironments, and interaction with its tyrosine kinase receptor Met triggers cell invasion and metastasis. It was previously shown that acidic extracellular pH stimulated peripheral lysosome trafficking, resulting in increased cathepsin B secretion and tumor cell invasion, which was dependent upon sodium-proton exchanger (NHE) activity. We now demonstrate that HGF induced the trafficking of lysosomes to the cell periphery, independent of HGF-induced epithelial-mesenchymal transition. HGF-induced anterograde lysosome trafficking depended upon the PI3K pathway, microtubules and RhoA, resulting in increased cathepsin B secretion and invasion by the cells. HGF-induced NHE activity via increased net acid production, and inhibition of NHE activity with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or a combination of the NHE1-specific drug cariporide and the NHE3-specific drug s3226 prevented HGF-induced anterograde trafficking and induced retrograde trafficking in HGF-overexpressing cells. EIPA treatment reduced cathepsin B secretion and HGF-induced invasion by the tumor cells. Lysosomes were located more peripherally in Rab7-shRNA-expressing cells and these cells were more invasive than control cells. Overexpression of the Rab7 effector protein, RILP, resulted in a juxtanuclear location of lysosomes and reduced HGF-induced invasion. Together, these results suggest that the location of lysosomes is an inherently important aspect of invasion by tumor cells.
Assuntos
Corrente Citoplasmática , Fator de Crescimento de Hepatócito/metabolismo , Lisossomos/metabolismo , Invasividade Neoplásica , Neoplasias da Próstata/metabolismo , Catepsina B/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Clonagem Molecular , Corrente Citoplasmática/efeitos dos fármacos , Corrente Citoplasmática/genética , Fator de Crescimento de Hepatócito/genética , Humanos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/patologia , Inibidores da Bomba de Prótons/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Transgenes/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion.Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking,suggesting compensation by other NHE family members.Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.
Assuntos
Lisossomos/metabolismo , Neoplasias da Próstata/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Masculino , Neoplasias da Próstata/patologia , Transporte ProteicoRESUMO
PURPOSE: To determined the mechanism(s) through which troglitazone induces cytosolic acidification and glutamine-dependent ammoniagenesis in pig kidney derived LLC-PK1 cells. EXPERIMENTAL DESIGN: Acute experiments measured acid extrusion, acid production and simultaneous Extracellular Signal-Regulated Kinase activation. TRO-enhanced acid production was correlated with mitochondrial membrane potential and rotenone and 5-(N-ethyl-N-isopropyl) amiloride, were employed to test specifically the role of Complex I proton pumping. Chronic experiments correlated inhibitors of Complex I with prevention of TRO-increased ammoniagenesis and affects on glutamine metabolism. RESULTS: Exposure to TRO acutely activated Extracellular Signal-Regulated Kinase in a dose dependent manner associated with a fall in spontaneous cytosolic pH. Cytosolic acidosis was associated with both an increase in acid production and inhibition of sodium/hydrogen ion exchanger -mediated acid extrusion. Preventing TRO-induced Extracellular Signal-Regulated Kinase activation with Mitogen Activated Protein Kinase Kinase inhibitors blocked the increase in acid production, restored sodium/hydrogen ion exchanger-activity and prevented cytosolic acidification. Mechanistically, increased acid production was associated with a rapid mitochondrial depolarization and Complex I proton pumping. Blocking Extracellular Signal-Regulated Kinase activation prevented both the fall in Psim and the increased acid production suggesting that the former underlies the accelerated mitochondrial 'acid production'. Mitochondrial Complex I inhibitors EIPA and rotenone prevented increased acid production despite Extracellular Response Kinase activation and reduced sodium/hydrogen ion activity. Inhibition of Complex I prevented TRO's effects on glutamine metabolism. CONCLUSION: TRO induces cellular acidosis through Extracellular Signal-Regulated Kinase activation-associated acid production and impaired acid extrusion. Acutely, increased acid production reflects mitochondrial Complex I proton pumping into the cytosol while chronically Complex I activity appears coupled to mitochondrial glutamate uptake and oxidation to ammonium at the expense of cytosolic transamination and alanine formation in these proximal tubule-like cells.
Assuntos
Amônia/metabolismo , Cromanos/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Túbulos Renais Proximais/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Bombas de Próton/metabolismo , Tiazolidinedionas/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Western Blotting , Linhagem Celular , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Suínos , TroglitazonaRESUMO
PURPOSE: We previously showed that troglitazone (TRO) induces a profound cellular acidosis in MCF-7 cells as a result of inhibiting Na(+)/H(+) exchanger (NHE)1-mediated acid extrusion and this was associated with a marked reduction in cellular proliferation. The present study focuses on TRO-activated signaling pathways versus TRO-mediated NHE1-inhibition in reducing DNA synthesis. EXPERIMENTAL DESIGN: TRO activation of the signaling pathway involving epidermal growth factor receptor (EGFR)/MAPK/ERK kinase (MEK) 1/2/extracellular signal-regulated kinase (ERK) 1/2 was studied by Western blotting and phospho-specific antibodies. TRO induction of cellular acidosis and inhibition of NHE1 activity were measured using (2, 7)-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) assay and NH4(+)/NH(3) pulsing. Cellular proliferation was assessed as DNA synthesis by (3)H-thymidine incorporation. RESULTS: TRO simultaneously reduces pH(i) and elevates phosphorylated-extracellular signal-regulated kinase (p-ERK). These responses reflected inhibition of acid extrusion and EGFR activation respectively and were sustained over 18h associated with a large decrease in DNA synthesis. Preventing TRO-induced ERK activation did not restore DNA synthesis or cellular pH. CONCLUSIONS: TRO activates two parallel pathways: I] EGFR/MEK1/2/ERK1/2 and II] NHE1 inhibition/cellular acidosis. Elimination of I] did not prevent the inhibition of DNA synthesis consistent with TRO-induced growth arrest dependent upon II] in tumorigenic non-metastatic breast cancer derived MCF-7 cells.
Assuntos
Acidose/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Cromanos/farmacologia , Receptores ErbB/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Acidose/enzimologia , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Feminino , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio , Ativação Transcricional/efeitos dos fármacos , TroglitazonaRESUMO
BACKGROUND: We studied the RNA expression of the genes in response to glucose from 5 mM (condition of normoglycemia) to 20 mM (condition of hyperglycemia/diabetes) by microarray analysis in breast cancer derived cell line MDA-MB-231. We identified the thioredoxin-interacting protein (TXNIP), whose RNA level increased as a gene product particularly sensitive to the variation of the level of glucose in culture media. We investigated the kinesis of the TXNIP RNA and protein in response to glucose and the relationship between this protein and the related thioredoxin (TRX) in regulating the level of reactive oxygen species (ROS) in MDA-MB-231 cells. METHODS: MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Cells were analyzed with Affymetrix Human U133A microarray chip and gene expression profile was obtained. Semi-quantitative RT-PCR and Western blot was used to validate the expression of TXNIP RNA and protein in response to glucose, respectively. ROS were detected by CM-H2DCFDA (5-6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) and measured for mean fluorescence intensity with flow cytometry. TRX activity was assayed by the insulin disulfide reducing assay. RESULTS: We found that the regulation of TXNIP gene expression by glucose in MDA-MB-231 cells occurs rapidly within 6 h of its increased level (20 mM glucose) and persists through the duration of the conditions of hyperglycemia. The increased level of TXNIP RNA is followed by increased level of protein that is associated with increasing levels of ROS and reduced TRX activity. The inhibition of the glucose transporter GLUT1 by phloretin notably reduces TXNIP RNA level and the inhibition of the p38 MAP kinase activity by SB203580 reverses the effects of TXNIP on ROS-TRX activity. CONCLUSION: In this study we show that TXNIP is an oxidative stress responsive gene and its expression is exquisitely regulated by glucose level in highly metastatic MDA-MB-231 cells.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/biossíntese , Glucose/metabolismo , Hiperglicemia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular Tumoral , Feminino , Glucose/farmacologia , Humanos , Hiperglicemia/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , RNA Neoplásico/biossíntese , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genéticaRESUMO
In this study we investigated the relevance of the oncogenic protein NPM-ALK in regulating cellular pH (pHi) through the modulation of Na+/H+ exchanger 1 (NHE1)-activity and the consequences of pHi pharmacological manipulation in cells expressing NPM-ALK.
Assuntos
Transformação Celular Neoplásica/metabolismo , Cromanos/farmacologia , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Linfoma Difuso de Grandes Células B/patologia , Proteínas Tirosina Quinases/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Tiazolidinedionas/farmacologia , Alanina/análise , Amônia/análise , Animais , Divisão Celular , Linhagem Celular Tumoral/metabolismo , Meios de Cultivo Condicionados/química , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Células NIH 3T3/efeitos dos fármacos , Células NIH 3T3/ultraestrutura , Proteínas Tirosina Quinases/genética , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Transfecção , TroglitazonaRESUMO
The distribution of glutamate (Glu), the Glu transporter GLAST-1, and glutamine synthetase (GS) in human and monkey anterior uveal tissue, as well as serum (S) to aqueous humor (AH) Glu and glutamine (Gln) gradients were investigated. Cross-linked Glu (xGlu), GLAST-1, and GS were detected using the immunofluorescent antibody technique. S/AH Glu, Gln, and alanine (Ala) concentrations were quantified by high performance liquid chromatography. xGlu immunoreactivity was detected in melanocytes, posterior pigmented epithelial/dilator muscle cells, vascular endothelial cells, and lymphocytes of the iris, as well as the pigmented (PE) and nonpigmented epithelial (NPE) cells and muscle cells of ciliary body. xGlu immunoreactivity was highly concentrated at the apices of GLAST-1, GS positive ciliary body NPE cells, and in GLAST-1 positive iris melanocytes and iris dilator muscle cells. AH Glu concentrations were lower (p < 0.001), while Gln was higher in monkey (p = 0.01) and human cataractous (p = 0.15) AH than serum. The results indicate that Glu is concentrated within GLAST-1, GS positive NPE cells and are consistent with the suggestion that Glu and Gln concentrations in AH may be due in part to GLAST-1 and GS activity in iris and ciliary body epithelial cells.
RESUMO
Troglitazone (Tro) and pioglitazone (Pio) activation of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-gamma-independent pathways was studied in cell lines derived from porcine renal tubules. PPAR-gamma-dependent activation of PPAR response element-driven luciferase gene expression was observed with Pio at 1 microM but not Tro at 1 microM. On the other hand, PPAR-gamma-independent P-ERK activation was observed with 5 microM Tro but not with Pio (5-20 microM). In addition, Pio (1-10 microM) increased metabolic acid production and activated AMP-activated protein kinase (AMPK) associated with decreased mitochondrial membrane potential, whereas Tro (1-20 microM) did not. These results are consistent with three pathways through which glitazones may act in effecting metabolic processes (ammoniagenesis and gluconeogenesis) as well as cellular growth: 1) PPAR-gamma-dependent and PPAR-gamma-independent pathways, 2) P-ERK activation, and 3) mitochondrial AMPK activation. The pathways influence cellular acidosis and glucose and glutamine metabolism in a manner favoring reduced plasma glucose in vivo. In addition, significant interactions can be demonstrated that enhance some physiological processes (ammoniagenesis) and suppress others (ligand-mediated PPAR-gamma gene expression). Our findings provide a model both for understanding seemingly opposite biological effects and for enhancing therapeutic potency of these agents.
Assuntos
Cromanos/administração & dosagem , Túbulos Renais/química , Túbulos Renais/metabolismo , PPAR gama/metabolismo , Transdução de Sinais/fisiologia , Tiazolidinedionas/administração & dosagem , Animais , Linhagem Celular , Combinação de Medicamentos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Pioglitazona , Transdução de Sinais/efeitos dos fármacos , Suínos , TroglitazonaAssuntos
Antineoplásicos/farmacologia , Cromanos/farmacologia , Neoplasias Hematológicas/metabolismo , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Troglitazona , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We studied the signal pathway through which troglitazone (TRO) acts in inducing cellular acidosis in LLC-PK1-F+ cells in relation to ammoniagenesis and DNA synthesis. Cells were grown to confluent monolayers in 30-mm chambers and monitored for intracellular pH (pHi) by the BCECF assay and activated ERK by phospo-ERK1/2 antibodies. TRO induces a severe cellular acidosis (pHi 6.68 +/- 0.10 vs. 7.28 +/- 0.07 time control at 4 min, P < 0.01), whereas phospho-ERK1/2 to total ERK1/2 ratio increases 3.4-fold (P < 0.01). To determine whether ERK1/2 was activated by cellular acidosis or TRO was acting via MEK1/2 to activate ERK1/2, cells were pretreated with specific inhibitors of MEK1/2 activity, PD-098059 and U-0126, followed by the addition of TRO or vehicle. With MEK1/2 activity inhibited, TRO treatment failed to activate ERK1/2. Preventing ERK1/2 activation abrogated the TRO-induced cellular acidosis and maintained the pHi within the low normal range (7.06 +/- 0.11). To determine whether blocking ERK activation prevents TRO's inhibitory effect on NHE activity, cells were acid-loaded and the recovery response was monitored as DeltapHi/t over a 4-min recovery period. TRO inhibited NHE activity by 85% (P < 0.01), whereas blocking ERK activation restored the response. We measured activated ERK levels and pHi after 3- and 18-h exposure to TRO or extracellular acidosis (pHe = 6.95) to determine whether ERK activation was sustained. Whereas both TRO and extracellular acidosis increased activated ERK and decreased pHi after 3 h, only TRO sustained this response at 18 h. Furthermore, both enhanced ammoniagenesis and decreased DNA synthesis reflected the effect of TRO to induce and sustain a cellular acidosis.
Assuntos
Acidose/induzido quimicamente , Cromanos/farmacologia , Hipoglicemiantes/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Tiazolidinedionas/farmacologia , Equilíbrio Ácido-Base/efeitos dos fármacos , Acidose/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Suínos , TroglitazonaRESUMO
BACKGROUND: Cytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1) expression is associated with the onset and progression of inflammatory bowel disease (IBD). RESULTS: Using western blotting and cell-based ELISA, we show in this study that troglitazone, an activator of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), widely used in the treatment of diabetes, has as well recently been highlighted as protective in models of inflammation and cancer. We found that troglitazone (10-40 microM), significantly reduced the TNF-alpha (1 ng/ml) mediated induction of endothelial MAdCAM-1 in a dose-dependent manner, achieving a 34.7% to 98.4% reduction in induced MAdCAM-1. Trogliazone (20 microM) reduced TNF-alpha induced VCAM-1, ICAM-1 and E-selectin expression. Moreover, troglitazone significantly reduced alpha4beta7-integrin dependent lymphocyte adhesion to TNF-alpha cultured endothelial cells. CONCLUSIONS: These results suggest that PPAR-gamma agonists like troglitazone may be useful in the clinical treatment of IBD.
Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Cromanos/farmacologia , Células Endoteliais/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Selectina E/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Camundongos , Mucoproteínas , NF-kappa B/metabolismo , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Troglitazona , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Alanine and glutamine constitute the two most important nitrogen carriers released from the muscle. We studied the intracellular amino acid transport kinetics and protein turnover in nine end-stage renal disease (ESRD) patients and eight controls by use of stable isotopes of phenylalanine, alanine, and glutamine. The amino acid transport kinetics and protein turnover were calculated with a three-pool model from the amino acid concentrations and enrichment in the artery, vein, and muscle compartments. Muscle protein breakdown was more than synthesis (nmol.min(-1).100 ml leg(-1)) during hemodialysis (HD) (169.8 +/- 20.0 vs. 125.9 +/- 21.8, P < 0.05) and in controls (126.9 +/- 6.9 vs. 98.4 +/- 7.5, P < 0.05), but synthesis and catabolism were comparable pre-HD (100.7 +/- 15.7 vs. 103.4 +/- 14.8). Whole body protein catabolism decreased by 15% during HD. The intracellular appearance of alanine (399.0 +/- 47.1 vs. 243.0 +/- 34.689) and glutamine (369.7 +/- 40.6 vs. 235.6 +/- 27.5) from muscle protein breakdown increased during dialysis (nmol.min(-1).100 ml leg(-1), P < 0.01). However, the de novo synthesis of alanine (3,468.9 +/- 572.2 vs. 3,140.5 +/- 467.7) and glutamine (1,751.4 +/- 82.6 vs. 1,782.2 +/- 86.4) did not change significantly intradialysis (nmol.min(-1).100 ml leg(-1)). Branched-chain amino acid catabolism (191.8 +/- 63.4 vs. -59.1 +/- 42.9) and nonprotein glutamate disposal (347.0 +/- 46.3 vs. 222.3 +/- 43.6) increased intradialysis compared with pre-HD (nmol.min(-1).100 ml leg(-1), P < 0.01). The mRNA levels of glutamine synthase (1.45 +/- 0.14 vs. 0.33 +/- 0.08, P < 0.001) and branched-chain keto acid dehydrogenase-E2 (3.86 +/- 0.48 vs. 2.14 +/- 0.27, P < 0.05) in the muscle increased during HD. Thus intracellular concentrations of alanine and glutamine are maintained during HD by augmented release of the amino acids from muscle protein catabolism. Although muscle protein breakdown increased intradialysis, the whole body protein catabolism decreased, suggesting central utilization of amino acids released from skeletal muscle.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ácido Glutâmico/metabolismo , Falência Renal Crônica/metabolismo , Proteínas/metabolismo , Adulto , Alanina/metabolismo , Artérias , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Músculo Esquelético/metabolismo , Fenilalanina/metabolismo , Diálise Renal , VeiasRESUMO
PURPOSE: The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na(+)/H(+) exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation. EXPERIMENTAL DESIGN: Intracellular pH (pH(i)) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) gamma involvement was assessed using an antagonist and PPARgamma(-/-) NIH3T3 cells. RESULTS: TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 +/- 0.23 to 6.77 +/- 0.06; P < 0.001) and MDA-MB-231 cells (7.38 +/- 0.18 to 6.89 +/- 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH(4)Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 micromol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TRO-mediated inhibition of proliferation was not reversed by the presence of the PPARgamma inhibitor GW9662 and was demonstrable in PPARgamma(-/-) NIH3T3 cells, consistent with a PPARgamma-independent mechanism. CONCLUSIONS: TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPARgamma antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPARgamma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Cromanos/farmacologia , DNA/biossíntese , PPAR gama/farmacologia , Tiazolidinedionas/farmacologia , Acidose , Animais , Proliferação de Células , Feminino , Fibroblastos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Troglitazona , Células Tumorais CultivadasRESUMO
Troglitazone was studied in pH-sensitive LLC-PK1-F+ cells to determine the effect on pHi and glutamine metabolism as well as the role of peroxisome proliferator-activated receptor (PPARgamma)-dependent and PPARgamma-independent signaling pathways. Troglitazone induces a dose-dependent cellular acidosis that occurs within 4 min and persists over 18 h as a result of inhibiting Na+/H+ exchanger-mediated acid extrusion. Cellular acidosis was associated with glutamine-dependent augmented [15N]ammonium production and decreased [15N]alanine formation from 15N-labeled glutamine. The shift in glutamine metabolism from alanine to ammoniagenesis appears within 3 h and is associated after 18 h with both a reduction in assayable alanine aminotransferase (ALT) activity as well as cellular acidosis. The relative contribution of troglitazone-induced cellular acidosis vs. the decrease in assayable ALT activity to alanine production could be demonstrated. The PPARgamma antagonist bisphenol A diglycide ether (BADGE) reversed both the troglitazone-induced cellular acidosis and ammoniagenesis but enhanced the troglitazone reduction of assayable ALT activity; BADGE also blocked troglitazone induction of peroxisome proliferator response element-driven firefly luciferase activity. The protein kinase C (PKC) inhibitor chelerythrine mimics troglitazone effects, whereas phorbol ester reverses the effects on ammoniagenesis consistent with troglitazone negatively regulating the DAG/PKC/ERK pathway. Although functional PPARgamma signaling occurs in this cell line, the major troglitazone-induced acid-base responses appear to be mediated by pathway(s) involving PKC/ERK.
Assuntos
Equilíbrio Ácido-Base/efeitos dos fármacos , Antineoplásicos/farmacologia , Cromanos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiazolidinedionas/farmacologia , Fatores de Transcrição/metabolismo , Equilíbrio Ácido-Base/fisiologia , Acidose/induzido quimicamente , Alanina/metabolismo , Alcaloides , Animais , Compostos Benzidrílicos , Benzofenantridinas , Carcinógenos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Genes Reporter , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Células LLC-PK1 , Fenantridinas/farmacologia , Plasmídeos , Proteína Quinase C/antagonistas & inibidores , Compostos de Amônio Quaternário/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Suínos , TroglitazonaRESUMO
We studied the effect of the antihyperglycemic glitazones, ciglitazone, troglitazone, and rosiglitazone, on glutamine metabolism in renal tubule-derived Madin-Darby canine kidney (MDCK) cells. Troglitazone (25 microM) enhanced glucose uptake and lactate production by 108 and 92% (both P < 0.001). Glutamine utilization was not inhibited, but alanine formation decreased and ammonium formation increased (both P < 0.005). The decrease in net alanine formation occurred with a change in alanine aminotransferase (ALT) reactants, from close to equilibrium to away from equilibrium, consistent with inhibition of ALT activity. A shift of glutamine's amino nitrogen from alanine into ammonium was confirmed by using L-[2-(15)N]glutamine and measuring the [(15)N]alanine and [(15)N]ammonium production. The glitazone-induced shift from alanine to ammonium in glutamate metabolism was dose dependent, with troglitazone being twofold more potent than rosiglitazone and ciglitazone. All three glitazones induced a spontaneous cellular acidosis, reflecting impaired acid extrusion in responding to both an exogenous (NH) and an endogenous (lactic acid) load. Our findings are consistent with glitazones inducing a spontaneous cellular acidosis associated with a shift in glutamine amino nitrogen metabolism from predominantly anabolic into a catabolic pathway.
Assuntos
Acidose/metabolismo , Células Epiteliais/metabolismo , Glutamina/farmacocinética , Hipoglicemiantes/farmacologia , Tiazóis/farmacologia , Tiazolidinedionas , Alanina Transaminase/metabolismo , Animais , Linhagem Celular , Cromanos/farmacologia , Células Epiteliais/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Rim/citologia , L-Lactato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Isótopos de Nitrogênio , Rosiglitazona , TroglitazonaRESUMO
The cytotoxicity of bacterial cell wall components, muramyl dipeptide (synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine; L,D-MDP) and lipopolysaccharide (LPS), was investigated in several kidney cell lines. MDP and LPS were toxic to rabbit and monkey kidney cells, MDP was toxic to canine kidney cells, but not to human or porcine kidney cells. Notably, L,D-MDP was >100-fold more cytotoxic/microg than the D,D-MDP and L,L-MDP, as well as LPS. L,D-MDP and analogs containing L,D-MDP were the most widely cytotoxic of the MDP tested. The MDP-induced cytotoxicity was characterized as apoptosis by DAPI staining and DNA laddering. The acute rabbit kidney (RK13) cell apoptosis (cell death in < 5 h) induced by apical or basal application of MDP was associated with glutamate (Glu) release, decreased gamma-glutamyltranspeptidase (GGT) and acidosis and was suppressed by Indomethacin, Naproxen and Curcumin. The cytotoxic activity of L,D-MDP was decreased significantly by 24 h incubation in human sera. Aged (> 2 year-old) rabbits that apparently failed to quickly clear and excrete a uveitogenic dose of MDP within 24 h died in I week. The results indicate that minute amounts (5 ng/ml) of MDP containing L-alanyl-D-isoglutamine can induce renal cell apoptosis in vitro and support MDP-induced kidney cytotoxicity in rabbits. Also, the results indicate that MDP in sera can be detected utilizing the RK13 cell bioassay and that failure to rapidly clear and excrete L,D-MDP is associated with uveitis and death in aged rabbits.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Apoptose , Rim/citologia , Rim/patologia , Acetilmuramil-Alanil-Isoglutamina/química , Adjuvantes Imunológicos/farmacologia , Animais , Ácido Araquidônico/farmacologia , Linhagem Celular , Inibidores de Ciclo-Oxigenase/farmacologia , DNA/metabolismo , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Coelhos , Estereoisomerismo , Suínos , Fatores de Tempo , gama-Glutamiltransferase/metabolismoRESUMO
We studied the effect of troglitazone on cellular acid-base balance and alanine formation in isolated rat mesangial cells. Mesangial cells were grown to confluency in RPMI 1640 media on 30-mm chambers used to monitor both cellular pH using the pH-sensitive dye 2'7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein and metabolic acid production as well as glutamine metabolism. Troglitazone (10 microM) induced a spontaneous cellular acidosis (6.95 +/- 0.02 vs. 7.47 +/- 0.04, respectively; P < 0.0001) but without an increase in lactic acid production. Alanine production was reduced 64% (P < 0.01) consistent with inhibition of the glutamate transamination. These findings pointed to a decrease in acid extrusion rather than an increase in acid production as the underlying mechanism leading to the cellular acidosis. To test their acid extrusion capabilities, mesangial cells were acid loaded with NH and then allowed to recover in Krebs-Henseleit media or in Krebs-Henseleit media minus bicarbonate (HEPES substituted), and the recovery response (Delta pH(i)/min) was monitored. In the presence of 10 microM troglitazone, the recovery response to the NH acid load was virtually eliminated in the bicarbonate-buffered media (0.00 +/- 0.001 vs. 0.06 +/- 0.02 pH(i)/min, P < 0.0001 vs. control) and reduced 75% in HEPES-buffered media (0.01 +/- 0.01 vs. 0.04 +/- 0.02 pH(i)/min, P < 0.002 vs. control). These results show that troglitazone induces a spontaneous cellular acidosis resulting from a reduction in cellular acid extrusion.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Ácidos/metabolismo , Cromanos/farmacologia , Mesângio Glomerular/metabolismo , Hipoglicemiantes/farmacologia , Tiazóis/farmacologia , Tiazolidinedionas , Equilíbrio Ácido-Base/efeitos dos fármacos , Alanina/biossíntese , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Líquido Intracelular/metabolismo , Ácido Láctico/metabolismo , Ratos , Ratos Sprague-Dawley , TroglitazonaRESUMO
BACKGROUND: Etiology of dialysis induced hypotension and hypertension remains speculative. There is mounting evidence that nitric oxide (NO) and endothelin (ET-1) may play a vital role in these hemodynamic changes. We examined the intradialytic dynamic changes in NO and ET-1 levels and their role in the pathogenesis of hypotension and rebound hypertension during hemodialysis (HD). METHODS: The serum nitrate + nitrite (NT), fractional exhaled NO concentration (FENO), L-arginine (L-Arg), NGNG-dimethyl-L-arginine (ADMA) and endothelin (ET-1) profiles were studied in 27 end-stage renal disease (ESRD) patients on HD and 6 matched controls. The ESRD patients were grouped according to their hemodynamic profile; Group I patients had stable BP throughout HD, Group II had dialysis-induced hypotension, and Group III had intradialytic rebound hypertension. RESULTS: Pre-dialysis FENO was significantly lower in the dialysis patients compared to controls (19.3 +/- 6.3 vs. 28.6 +/- 3.4 ppb, P < 0.002). Between the experimental groups, pre-dialysis FENO was significantly higher in Group II (24.1 +/- 6.7 ppb) compared to Group I (17.8 +/- 5.6 ppb) and Group III (16.1 +/- 4.2 ppb; P < 0.05). Post-dialysis, FENO increased significantly from the pre-dialysis values (19.3 +/- 6.3 vs. 22.6 +/- 7.9 ppb; P=0.001). Pre-dialysis NT (34.4 +/- 28.2 micromol/L/L) level was not significantly different from that of controls (30.2 +/- 12.3 micromol/L/L). Serum NT decreased from 34.4 +/- 28.2 micromol/L/L at initiation of dialysis to 10.0 +/- 7.4 micormol/L/L at end of dialysis (P < 0.001). NT concentration was comparable in all the three groups at all time points. Pre-dialysis L-Arg (105.3 +/- 25.2 vs. 93.7 +/- 6.0 micromol/L/L; P < 0.05) and ADMA levels were significantly higher in ESRD patients (4.0 +/- 1.8 vs. 0.9 +/- 0.2 micromol/L/L; P < 0.001) compared to controls. Dialysis resulted in significant reduction in L-Arg (105.3 +/- 25.2 vs. 86.8 +/- 19.8 micromol/L/L; P < 0.005) and ADMA (4.0 +/- 1.8 vs. 1.6 +/- 0.7 micromol/L/L; P < 0.001) concentrations. Pre-dialysis ET-1 levels were significantly higher in ESRD patients compared to the controls (8.0 +/- 1.9 vs. 12.7 +/- 4.1 pg/mL; P < 0.002), but were comparable in the three study groups. Post-dialysis ET-1 levels did not change significantly in Group I compared to pre-dialysis values (14.3 +/- 4.3 vs.15.0 +/- 2.4 pg/mL, P=NS). However, while the ET-1 concentration decreased significantly in Group II (12.0 +/- 4.0 vs. 8.7 +/- 1.8 pg/mL, P < 0.05), it increased in Group III from pre-dialysis levels (12.8 +/- 3.8 vs. 16.7 +/- 4.5 pg/mL, P=0.06). CONCLUSION: Pre-dialysis FENO is elevated in patients with dialysis-induced hypotension and may be a more reliable than NT as a marker for endogenous NO activity in dialysis patients. Altered NO/ET-1 balance may be involved in the pathogenesis of rebound hypertension and hypotension during dialysis.