RESUMEN
It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas del Citoesqueleto/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Mutación , Nefronas/citología , Nefronas/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/fisiología , Transfección , Xenopus laevisRESUMEN
Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2-methylthioadenosine diphosphate (2-MeSADP) on Cl- transport in A6 cells, a commonly used model of the distal section of the Xenopus laevis nephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of the Xenopus P2Y1 receptor. 2-MeSADP increased intracellular [Ca2+] and cAMP and Cl- efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl- efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2-MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADP-dependent stimulation of Cl- efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl- efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl- efflux, respectively. These data suggest that the X laevis P2Y1 receptor in A6 cells can increase both cAMP/PKA and Ca2+/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Fosfoproteínas/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Cloruros/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/citología , Gliburida/metabolismo , Indometacina/metabolismo , Isoquinolinas/metabolismo , Túbulos Renales/citología , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Intercambiadores de Sodio-Hidrógeno , Sulfonamidas/metabolismo , Tionucleótidos/metabolismo , Xenopus laevisRESUMEN
Tumour angiogenesis and cellular pH regulation, mainly represented by Na(+)/H(+) antiporter exchange, have been heretofore considered unrelated subfields of cancer research. In this short review, the available experimental evidence relating these areas of modern cancer research is introduced. This perspective also helps to design a new approach that facilitates the opening and development of novel research lines oriented towards a rational incorporation of anticancer drugs into more selective and less toxic therapeutic protocols. The final aim of these efforts is to control cancer progression and dissemination through the control of tumour angiogenesis. Finally, different antiangiogenic drugs that can already be clinically used to this effect are briefly presented.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Transformación Celular Neoplásica , Neovascularización Patológica , Intercambiadores de Sodio-Hidrógeno/fisiología , Equilibrio Ácido-Base , División Celular , Movimiento Celular , Progresión de la Enfermedad , Homeostasis , Humanos , Concentración de Iones de HidrógenoRESUMEN
Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.
Asunto(s)
Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Oocitos/efectos de los fármacos , betaendorfina/farmacología , Animales , Bovinos , Quelantes/farmacología , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Flavonoides/farmacología , Células de la Granulosa/efectos de los fármacos , Técnicas In Vitro , Meiosis/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Oocitos/enzimología , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/genéticaRESUMEN
As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Calcio/metabolismo , AMP Cíclico/biosíntesis , Células Epiteliales/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Tionucleótidos/metabolismo , Adenosina/farmacología , Adenosina Difosfato/farmacología , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Concentración de Iones de Hidrógeno , Ratas/genética , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Tionucleótidos/farmacología , Transfección , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The opioid receptor antagonist, naloxone, has been shown to have beneficial effects in the kidney and to be implicated in renal salt and water balance. In the present study the signal transduction pathways utilized by naloxone were studied in an epithelial cell line model of the cortical collecting duct, A6 cells. We found that naloxone has a dual effect depending on the concentration used: at a low concentration (10(-6) M) it antagonized the beta-endorphin-dependent increase in cytoplasmic calcium [Ca(2+)](i), while at higher concentrations (>10(-5) M) it increased [Ca(2+)](i) and intracellular inositol phosphate levels. While naloxone-induced increases in [Ca(2+)](i) occurred in the absence of external calcium, it was significantly stimulated by increasing the external calcium concentration, suggesting that naloxone increases [Ca(2+)](i) via both calcium release and calcium influx. In polarized A6 cell monolayers naloxone inhibited the activity of the Na(+)/H(+) exchanger (NHE) only when added to the basolateral cell surface. This inhibition of the NHE was prevented by pretreatment of the cells with either the intracellular calcium chelator, BAPTA or with the protein kinase C inhibitor, calphostin C. These findings demonstrate that naloxone induces a rapid increase in intracellular calcium which inhibits the NHE via the calcium-dependent protein kinase C regulatory pathway.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Naloxona/farmacología , Proteína Quinasa C/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Línea Celular , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Naftalenos/farmacología , Antagonistas de Narcóticos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Espectrometría de Fluorescencia , Sulfonamidas/farmacología , betaendorfina/metabolismoRESUMEN
We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in [Ca(2+)](i) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases [Ca(2+)](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca(2+)](i) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry.
Asunto(s)
Adenosina/análogos & derivados , Calcio/metabolismo , Cloruros/metabolismo , Células Epiteliales/fisiología , Riñón/fisiología , Receptores Purinérgicos P1/fisiología , Adenosina/farmacología , Animales , Señalización del Calcio , Línea Celular , Colforsina/farmacología , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Dihidropiridinas/farmacología , Conductividad Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Piridinas/farmacología , Receptor de Adenosina A3 , Rolipram/farmacología , Transducción de SeñalRESUMEN
In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE-1 proton regulatory site. Annulment of the E7-induced cytoplasmic alkalinization by specific inhibition of the NHE-1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum-independent growth, anchorage-independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F-mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformation-dependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delayed the development of HPV16-keratinocyte tumors. Our data confirm that activation of the NHE-1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.
Asunto(s)
Amilorida/análogos & derivados , Transformación Celular Neoplásica , Intercambiadores de Sodio-Hidrógeno/fisiología , Células 3T3 , Amilorida/farmacología , Animales , Sitios de Unión , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Medio de Cultivo Libre de Suero/farmacología , Ciclina E/efectos de los fármacos , Ciclina E/metabolismo , Glucólisis , Humanos , Concentración de Iones de Hidrógeno , Queratinocitos/citología , Queratinocitos/virología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/fisiología , Proteínas E7 de Papillomavirus , Fenotipo , Fase S , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The present study was undertaken to evaluate the presence of GnRH receptors (GnRH-R) in breast cancer and not-involved breast tissue, and the relationships between GnRH-R and receptors for estrogen (ER) and progesterone (PgR) in the same tissues. Utilizing a tritiated natural GnRH in order to assay the native receptor binding we analyzed the level of binding sites for GnRH in membranes derived from 90 breast tumors and in 40 cases from neighboring, not-involved breast tissue. GnRH-R was found both in cancer and normal tissues. The prevalence for GnRH-R was higher in tumor than in not-tumor tissue (45% vs 39%, respectively), but the overall levels were not significantly different (15.9+/-24 fmol/mg protein vs 18.2+/-39 fmol/mg protein, respectively). The only statistically different content of GnRH-R we found concerned PgR negative vs PgR positive tumor tissues (mean content: 23 vs 11 fmol/mg protein, respectively in PgR- and PgR+ tumors, p=0.03 by t test); furthermore the proportion of GnRH-R positive cases in the tumor resulted significantly higher in premenopausal patients vs postmenopausal (56% vs 32%, by Chi square test, p<0.05). The GnRH receptors status of primary tumor and contiguous not-involved breast tissue resulted associated (overall agreement: 63%, p<0.05) but no specific steroid patterns for GnRH-R positivity was observed.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/metabolismo , Receptores de Somatotropina/metabolismo , Sitios de Unión , Mama/patología , Mama/ultraestructura , Neoplasias de la Mama/patología , División Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Femenino , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Comunicación Paracrina/fisiología , Proteínas/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismoRESUMEN
Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.
Asunto(s)
Sangre , Neoplasias de la Mama/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adenosina Trifosfato/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Cloruro de Amonio/farmacología , Androstadienos/farmacología , Western Blotting , Línea Celular , Movimiento Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ácido Láctico/biosíntesis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba , WortmaninaRESUMEN
Transforming growth factor-beta (TGF-beta) exerts an inhibitory effect on epithelial cell proliferation while insulin-like growth factor-1 (IGF-1) is a positive regulator of proliferation and together they may participate in driving neoplastic progression. The regulation of TGF-beta1 and IGF-1 gene expression was analyzed in an in vitro model of an estrogen receptor positive (ER+), non-metastatic (MCF-7) and an (ER-), metastatic (MDA-MB-435) breast cancer cell line, respectively. Our results indicate a loss of the regulation of TGF-beta1 and the gain of the expression and upregulation of IGF-1 pathways during malignant progression. These data demonstrate that two factors, convergent on cell growth, can have divergent roles in the regulation of the expression of TGF-beta1.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Metástasis de la Neoplasia , Factor de Crecimiento Transformador beta/genética , Sangre , Neoplasias de la Mama , División Celular/fisiología , Progresión de la Enfermedad , Estradiol/fisiología , Retroalimentación , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Células Tumorales CultivadasRESUMEN
Although cytosolic expression of the protein pS2 (TFF1) is considered to be a marker of oestrogen receptor (OR) function, there exists some clinical data to suggest an inverse relationship of cytosolic pS2 to tumour proliferation. Although secreted from breast cancer cells, the relationship of pS2 secretion to tumour natural history has been little studied. The mechanisms and kinetics of pS2 release and its relation to tumour cell proliferation were studied in a human breast cancer cell line MCF-7 and verified in a preliminary clinical study. Stimulation by stripped serum or oestradiol resulted in parallel increases of proliferation and pS2 release in both time course and dose-response experiments. Direct pharmacological alterations of proliferation were followed by identical changes in pS2 release. The relationship between serum pS2 levels and tumour proliferative activity when analysed as a function of steroid status showed a slope of 0.56 in OR+ vs. 0.19 in OR- tumours. It is concluded that pS2 release from breast cancer cells is associated with their proliferation and measurement of serum pS2 levels might be a good predictor of tumour proliferative state and could permit noninvasive monitoring of this tumour parameter.
Asunto(s)
Neoplasias de la Mama/patología , Sustancias de Crecimiento/fisiología , Proteínas/fisiología , Amilorida/análogos & derivados , Amilorida/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/cirugía , División Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Desoxiadenosinas/farmacología , Femenino , Sustancias de Crecimiento/sangre , Sustancias de Crecimiento/genética , Humanos , Cinética , Persona de Mediana Edad , Proteínas/análisis , Proteínas/genética , Análisis de Regresión , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The present study describes two Na+/H+ exchanger (NHE) isoforms in an immortalized rabbit renal cortical collecting tubule cell line (RC.SV3). Na+/H+ exchange activity was assayed using fluorescence measurements of intracellular pH (pHi) in monolayers mounted in a cuvette containing two fluid compartments, making it possible to independently measure Na+/H+ exchange activity on either the apical or basolateral surface. RC.SV3 monolayers express Na+/H+ exchange activities in both the apical and basolateral membrane domains. The two exchangers have half-saturation constants (Km) for external sodium and sensitivities to dimethylamiloride, to HOE-694 and to cimetidine and clonidine consistant with the NHE-1 isoform on the basolateral cell surface and the NHE-2 isoform on the apical surface. Protein kinase A inhibition of basolateral exchanger activity was significantly higher than that of the apical exchanger. Protein kinase C significantly stimulated both exchangers equally. RT-PCR analysis found RNA for only NHE-1 and NHE-2, and immunofluorescence with an antibody against NHE-1 demonstrated a basolateral location for this isoform. The results suggest that RC.SV3 cells have two Na+/H+ exchange activities separated spatially to the two cellular membranes, with the NHE-1 and the NHE-2 isoforms located on the basolateral and the apical membranes, respectively.
Asunto(s)
Túbulos Renales Colectores/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Cimetidina/farmacología , Clonidina/farmacología , Cartilla de ADN/genética , Guanidinas/farmacología , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Cinética , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , Conejos , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/genética , Sulfonas/farmacologíaRESUMEN
Data concerning the hormone sensitivity of the release and role of the aspartyl protease cathepsin D in tumor proliferative and invasive processes have been contradictory. To clarify the mechanisms of its release and role we first studied the contribution of estradiol and stripped serum to the time course and kinetics of cathepsin D release, proliferation, and invasion in parallel in the MCF-7 in vitro breast cancer cell culture model. Both estradiol and stripped serum independently stimulated both proliferation and cathepsin D release. However, the dose-response of estradiol and stripped serum-dependent stimulated release were similar to those for invasion and differed from those for proliferation: cathepsin D release and invasion were first stimulated at a stripped serum concentration more than 10-fold lower than that which initiated proliferation and had half stimulation constants almost 10-fold lower than those for proliferation. These results demonstrate that cathepsin D release is not related in any direct way to proliferation. The effect of the reduction of cathepsin D activity or release on in vitro invasion was also measured: both the inhibition of secreted cathepsin D activity by a specific inhibitor, diazoacetyl-DL-Nle-OMe, and the reduction of cathepsin D release by antisense oligonucleotides against its translation start site reduced cellular in vitro invasion without affecting proliferation. Cathepsin D release and activity are concluded to be directly involved in the process of invasion.
Asunto(s)
Neoplasias de la Mama/enzimología , Catepsina D/fisiología , Invasividad Neoplásica/diagnóstico , Sangre , Neoplasias de la Mama/patología , División Celular , Medios de Cultivo , Estradiol/farmacología , Femenino , Humanos , Oligonucleótidos Antisentido/farmacología , Células Tumorales CultivadasRESUMEN
The effect of adenosine on Na+/H+ exchange activity was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on Na+/H+ exchange activity depending on the side of addition. Basolateral CPA induced a stimulation of Na+/H+ exchange activity that was completely prevented by preincubation with an A2A-selective antagonist, 8-(3-chlorostyryl)caffeine, whereas apical CPA induced a slight but significant inhibition of Na+/H+ exchange activity that was significantly reduced by the A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine. Protein kinase C activation may be involved in mediating the apical CPA inhibition of Na+/H+ exchange activity; this inhibition was prevented by the protein kinase C inhibitor calphostin C. Treatment with either forskolin or 8-bromo-cAMP significantly stimulated Na+/H+ exchange activity; only basolateral CPA addition induced an increase in cAMP level. These observations together with the finding that the CPA-dependent stimulation of exchange activity was prevented by the protein kinase A inhibitor H-89 support the hypothesis that basolateral CPA stimulates Na+/H+ exchange via adenylate cyclase/protein kinase A activation. Basolateral CPA also increased transepithelial Na+ transport, and this stimulation was prevented by the Na+/H+ exchange inhibitor HOE-694, suggesting that changes in pHi during hormone action can act as an intermediate in the second-messenger cascade.
Asunto(s)
Adenosina/fisiología , Concentración de Iones de Hidrógeno , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Línea Celular , AMP Cíclico/metabolismo , Hidrógeno/metabolismo , Riñón , Antagonistas de Receptores Purinérgicos P1 , Sodio/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Xenopus laevisRESUMEN
The effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (Isc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the Isc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic Isc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This Isc response to apical CPA was also strongly reduced in Cl--free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+]i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl- secretion via A1 receptor-mediated changes in [Ca2+]i.
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Adenosina/fisiología , Cloruros/metabolismo , Receptores Purinérgicos P1/análisis , Transducción de Señal/fisiología , Sodio/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Amilorida/farmacología , Bumetanida/farmacología , Calcio/análisis , Línea Celular , Membrana Celular/metabolismo , Canales de Cloruro/fisiología , AMP Cíclico/metabolismo , Diuréticos/farmacología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Riñón/citología , Receptores Purinérgicos P1/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In vitro research into hormone sensitivity and the relation to proliferation of cytokeratin release from cancer cells is scarce. Therefore, we examined the stimulation of proliferation and the release of cytokeratins in a breast cancer cell culture model. Cell growth was stimulated by 17 beta-oestradiol (10(-11) M), stripped serum (10%) and by the two together. Cytokeratin release was stimulated only by stripped serum, oestradiol having no effect. After long incubation periods (> 12 h), cytokeratin release also commenced in the control and oestradiol treatments. Release rate versus time analysis suggested that there are two different release processes. Cytokeratin release was first stimulated at a stripped serum concentration approximately 100 times lower than that which initiated proliferation. Pharmacological alteration of proliferation with cordyceptin resulted in growth changes without alterations in cytokeratin release. We conclude that cytokeratin release in these cells is unrelated to proliferation, independent of oestrogen action and probably in some way related to growth factor receptor function.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Sustancias de Crecimiento/farmacología , Queratinas/metabolismo , Antígeno Polipéptido de Tejido/metabolismo , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Cinética , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na(+)-K(+)-2Cl- cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na(+)-K(+)-2Cl- cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the beta-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (approximately 20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF(-)4 treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (approximately 6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (approximately 3-fold and approximately 6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca(2+)-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na(+)-K(+)-2Cl- cotransporter.
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Proteínas Portadoras/metabolismo , Cloruros/metabolismo , Glándula Parótida/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Animales , AMP Cíclico/fisiología , Isoproterenol/farmacología , Masculino , Mapeo Peptídico , Fosforilación , Ratas , Ratas Wistar , Simportadores de Cloruro de Sodio-Potasio , Regulación hacia ArribaRESUMEN
We demonstrate the presence of a 160 kD protein in rabbit parotid basolateral membranes that can be labeled with the irreversible sulfhydryl reagent [14C]-N-ethylmaleimide in a bumetanide-protectable fashion. The specificity of this labeling, and our previous evidence for the existence of an essential sulfhydryl group closely associated with the bumetanide-binding site on the parotid Na(+)-K(+)-Cl-cotransporter (J. Membrane Biol. 112:51-58, 1989), provide strong evidence that this protein is a part or all of the parotid bumetanide-binding site. When this protein is treated with endoglycosidase F/N-glycosidase F to remove N-linked oligosaccharides, its apparent molecular weight decreases to 135 kD. The pI of this deglycosylated protein is approximately 6.4. The bumetanide-binding protein was purified using two preparative electrophoresis steps. First, a Triton X-100 extract enriched in this protein was run on preparative electrophoresis to obtain fractions containing proteins in the 160 kD range. These were then deglycosylated with endoglycosidase F/N-glycosidase F and selected fractions were pooled and rerun on preparative electrophoresis to obtain a final 135 kD fraction. The enrichment of the bumetanide-binding protein in this final 135 kD fraction estimated from [14C]-N-ethylmaleimide labeling was approximately 48 times relative to the starting membrane extract. Since the bumetanide-binding site represents approximately 2% of the total protein in this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fraction.