RESUMO
To characterize the sugar translocation pathway of Na(+)/glucose cotransporter type 1 (SGLT1), a chimera was made by substituting the extracellular loop between transmembrane domain (TM) 12 and TM13 of Xenopus SGLT1-like protein (xSGLT1L) with the homologous region of rabbit SGLT1. The chimera was expressed in Xenopus oocytes and its transport activity was measured by the two-microelectrode voltage-clamp method. The substrate specificity of the chimera was different from those of xSGLT1L and SGLT1. In addition the chimera's apparent Michaelis-Menten constant (K(m)) for myo-inositol, 0.06 mM, was about one fourth of that of xSGLT1L, 0.25 mM, while the chimera's apparent K(m) for d-glucose, 0.8 mM, was about one eighth of that of xSGLT1L, 6.3 mM. Our results suggest that the extracellular loop between TM12 and TM13 participates in the sugar transport of SGLT1.
Assuntos
Transportador 1 de Glucose-Sódio/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Coelhos , Homologia de Sequência de Aminoácidos , Transportador 1 de Glucose-Sódio/química , Transportador 1 de Glucose-Sódio/genética , Especificidade por Substrato , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMO
Plasma membrane proteins of the solute carrier family 5 (SLC5) are responsible for sodium-coupled uptake of ions, sugars and nutrients in the vertebrate body. Mutations in SLC5 genes are the cause of several inherited human disorders. We have recently reported the cloning and transport properties of SGLT-1L, a Xenopus homologue of the sodium-dependent glucose cotransporter 1 (SGLT-1) [Nagata et al. (1999) Am. J. Physiol. 276: G1251 -G 1259]. Here, we describe the phylogenetic relationship of SGLT-1L with other members of the SLC5 family and characterize its expression during Xenopus embryogenesis and in organ cultures. Sequence comparisons and phylogenetic analyses of all known vertebrate SLC5 sequences indicated that Xenopus SGLT-1L encodes a novel SLC5 member, which shares highest amino acid identity with mammalian ST-1 proteins. Temporal and spatial expression of SGLT-1L during Xenopus embryogenesis was examined by whole mount in situ hybridization. Initiation of SGLT-1L expression occurred in the late tailbud embryo. Remarkably, expression was restricted to the developing pronephric kidney. SGLT-1L was highly expressed in tubular epithelia, but completely absent from the epithelia of the duct. Analysis of growth factor-treated animal caps indicated that expression of SGLT-1L could also be induced in organ cultures. Taken together, our findings indicate that the expression of sodium-dependent solute cotransporter genes in early segments of the excretory system appears to be conserved between pronephric and metanephric kidneys. Furthermore, we establish SGLT-1L as a novel, highly specific molecular marker for pronephric tubule epithelia undergoing maturation and terminal differentiation in Xenopus.
Assuntos
Rim/patologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Xenopus/biossíntese , Animais , Membrana Celular/metabolismo , Hibridização In Situ , Ligantes , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Técnicas de Cultura de Órgãos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportador 1 de Glucose-Sódio , Fatores de Tempo , Transcrição Gênica , Xenopus , Proteínas de Xenopus/genéticaRESUMO
We have previously reported results that led us to speculate that ANG II is involved in the LPS-induced production of proinflammatory cytokines, especially under dehydrated conditions. To test this possibility, in this study we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and an antagonist of the type-1 ANG II receptor (AT(1) receptor) on the LPS-induced production of the proinflammatory cytokines IL-1 and IL-6 in dehydrated rats. A single intravenous injection of LPS induced a marked increase in the expression of IL-1beta mRNA in the liver, an effect that was significantly attenuated by pretreatment with the ACE inhibitor. Furthermore, the ACE inhibitor reduced the LPS-induced increase in the hepatic concentration of IL-1beta protein. When the AT(1)-receptor antagonist was given intravenously before the LPS, the increase in the hepatic concentration of IL-1beta was significantly reduced. Finally, the ACE inhibitor reduced the LPS-induced increase in the plasma concentration of IL-6. These results represent the first in vivo evidence that ANG II and its AT(1) receptor play important roles in the production of proinflammatory cytokines that is induced by LPS under dehydrated conditions.