RESUMEN
The Na(+)/H(+) exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with b-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated (22)Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 microM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO(3) medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.
Asunto(s)
Amilorida/análogos & derivados , Movimiento Celular , Transformación Celular Neoplásica , Seudópodos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Amilorida/farmacología , Animales , Adhesión Celular , Línea Celular , Línea Celular Transformada , Tamaño de la Célula , Perros , Técnica del Anticuerpo Fluorescente Indirecta , Concentración de Iones de Hidrógeno , Microscopía por Video , Nocodazol/farmacología , Fenotipo , Seudópodos/fisiología , Seudópodos/ultraestructura , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Fibras de Estrés/metabolismo , Fibras de Estrés/ultraestructuraRESUMEN
Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+ -H+ exchanger isoform NHE-1 and the Na+ -K+ ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37 degrees C did not further reduce this parameter. On the other hand, expression of Na+ -K+ ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins.
Asunto(s)
Criopreservación , Hígado/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Membrana Celular/enzimología , Membrana Celular/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Transportador de Glucosa de Tipo 2 , Hígado/enzimología , Hígado/ultraestructura , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Recalentamiento , Factores de Tiempo , TransfecciónRESUMEN
The Apn1 protein of the budding yeast Saccharomyces cerevisiae is a DNA repair enzyme that hydrolyzes apurinic/apyrimidinic (AP) sites and removes 3'-blocking groups present at single strand breaks of damaged DNA. Yeast cells lacking Apn1 are hypersensitive to DNA damaging agents that produce AP sites and DNA strand breaks with blocked 3'-termini. In this study, we showed that the fission yeast Schizosaccharomyces pombe bears a homologue, Spapn1, that is 45% identical to S. cerevisiae Apn1. However, the Spapn1 gene is apparently not expressed. Active expression of S. cerevisiae Apn1 in S. pombe conferred no additional resistance to DNA damaging agents. These data suggest that the pathway by which S. pombe repairs AP sites is independent of a functional Apn1-like AP endonuclease.
Asunto(s)
Liasas de Carbono-Oxígeno/genética , Reparación del ADN , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Familia de Multigenes , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Clonación Molecular , Enzimas Reparadoras del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimologíaRESUMEN
The Qm family of proteins, which are found in a wide variety of species such as budding yeast, plants and humans, are believed to play a role in gene expression. Here, we report the isolation ofaa gene, spqM, from the fission yeast Schizosaccharomyces pombe, whose deduced amino-acid sequence shared 71.6 to 61.36% identity with members of the Qm family. The high degree of conservation of the Qm members suggest that they were selectively conserved, because of an important biological role.