RESUMO
PURPOSE: The cellular and molecular mechanisms that regulate the organization of bladder urothelium into basal, intermediate and superficial cell layers remain poorly understood. We tested the hypothesis that fibroblast growth factor (FGF)-7 is essential for generating a multilayered stratified bladder epithelium. MATERIALS AND METHODS: The morphological and molecular characteristics of bladder urothelium in age and sex matched FGF-7 +/+ wild-type and -/- null mice were evaluated. In addition, the effect of exogenous FGF-7 on the growth and differentiation of primary murine urothelial cells was assessed. RESULTS: Morphometric analyses demonstrate that FGF-7 null urothelium is markedly thinned compared with wild-type urothelium. Electron microscopy revealed that null urothelium lacks the intermediate cell layers and molecular marker analyses confirmed this observation. In vitro cell culture experiments indicated that FGF-7 regulates urothelial cell growth, differentiation and stratification. Primary urothelial cultures maintained without FGF-7 ceased to divide and expressed proteins characteristic of terminally differentiated umbrella cells. In contrast, cultures maintained with exogenous FGF-7 contained proliferating epithelial cells with protein expression patterns consistent with those of intermediate cells in addition to terminally differentiated, post-mitotic umbrella cells. Importantly, isolated urothelial cells maintained with exogenous FGF-7 formed a multilayered epithelium in vitro. CONCLUSIONS: Collectively these data indicate that FGF-7 is essential for normal bladder urothelial stratification, specifically the formation of the intermediate cell layers. Fibroblast growth factor-7 stimulates urothelial proliferation and delays the differentiation of these cells into post-mitotic umbrella cells.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Queratinócitos/fisiologia , Bexiga Urinária/citologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Fator 7 de Crescimento de Fibroblastos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Urotélio/citologiaRESUMO
The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the in vivo role of the Notch2 gene, we constructed a targeted mutation, Notch2(del1). Unexpectedly, we found that alternative splicing of the Notch2(del1) mutant allele leads to the production of two different in-frame transcripts that delete either one or two EGF repeats of the Notch2 protein, suggesting that this allele is a hypomorphic Notch2 mutation. Mice homozygous for the Notch2(del1) mutation died perinatally from defects in glomerular development in the kidney. Notch2(del1)/Notch2(del1 )mutant kidneys were hypoplastic and mutant glomeruli lacked a normal capillary tuft. The Notch ligand encoded by the Jag1 gene was expressed in developing glomeruli in cells adjacent to Notch2-expressing cells. We show that mice heterozygous for both the Notch2(del1) and Jag1(dDSL) mutations exhibit a glomerular defect similar to, but less severe than, that of Notch2(del1)/Notch2(del1 )homozygotes. The co-localization and genetic interaction of Jag1 and Notch2 imply that this ligand and receptor physically interact, forming part of the signal transduction pathway required for glomerular differentiation and patterning. Notch2(del1)/Notch2(del1 )homozygotes also display myocardial hypoplasia, edema and hyperplasia of cells associated with the hyaloid vasculature of the eye. These data identify novel developmental roles for Notch2 in kidney, heart and eye development.
Assuntos
Vasos Coronários/embriologia , Olho/irrigação sanguínea , Olho/embriologia , Rim/irrigação sanguínea , Rim/embriologia , Receptores de Superfície Celular/metabolismo , Deleção de Sequência/genética , Alelos , Processamento Alternativo/genética , Animais , Biomarcadores , Proteínas de Ligação ao Cálcio , Morte Celular , Diferenciação Celular , Divisão Celular , Vasos Coronários/patologia , Desenvolvimento Embrionário e Fetal/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genótipo , Cardiopatias Congênitas/patologia , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Rim/anormalidades , Rim/patologia , Glomérulos Renais/anormalidades , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/embriologia , Glomérulos Renais/patologia , Ligantes , Proteínas de Membrana , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Morfogênese , Proteínas/genética , Proteínas/metabolismo , Receptor Notch2 , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Proteínas Serrate-JaggedRESUMO
WT1 encodes a zinc finger transcription factor implicated in kidney differentiation and tumorigenesis. In reporter assays, WT1 represses transcription from GC- and TC-rich promoters, but its physiological targets remain uncertain. We used hybridization to high-density oligonucleotide arrays to search for native genes whose expression is altered following inducible expression of WT1. The major target of WT1 was amphiregulin, a member of the epidermal growth factor family. The WT1(-KTS) isoform binds directly to the amphiregulin promoter, resulting in potent transcriptional activation. The in vivo expression profile of amphiregulin during fetal kidney development mirrors the highly specific pattern of WT1 itself, and recombinant Amphiregulin stimulates epithelial branching in organ cultures of embryonic mouse kidney. These observations suggest a model for WT1 as a transcriptional regulator during kidney differentiation.
Assuntos
Proteínas de Ligação a DNA/genética , Glicoproteínas/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fatores de Transcrição/genética , Ativação Transcricional/genética , Anfirregulina , Animais , Sequência de Bases , Pegada de DNA , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta a Droga , Família de Proteínas EGF , Glicoproteínas/farmacologia , Glicoproteínas/fisiologia , Substâncias de Crescimento/farmacologia , Substâncias de Crescimento/fisiologia , Humanos , Rim/anatomia & histologia , Rim/embriologia , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Elementos de Resposta/genética , Fatores de Tempo , Fatores de Transcrição/fisiologia , Células Tumorais Cultivadas , Ureter/embriologia , Proteínas WT1RESUMO
The nephric duct is the first epithelial tubule to differentiate from intermediate mesoderm that is essential for all further urogenital development. In this study we identify the domain of intermediate mesoderm that gives rise to the nephric duct and demonstrate that the surface ectoderm is required for its differentiation. Removal of the surface ectoderm resulted in decreased levels of Sim-1 and Pax-2 mRNA expression in mesenchymal nephric duct progenitors, and caused inhibition of nephric duct formation and subsequent kidney development. The surface ectoderm expresses BMP-4 and we show that it is required for the maintenance of high-level BMP-4 expression in lateral plate mesoderm. Addition of a BMP-4-coated bead to embryos lacking the surface ectoderm restored normal levels of Sim-1 and Pax-2 mRNA expression in nephric duct progenitors, nephric duct formation and the initiation of nephrogenesis. Thus, BMP-4 signaling can substitute for the surface ectoderm in supporting nephric duct morphogenesis. Collectively, these data suggest that inductive interactions between the surface ectoderm, lateral mesoderm and intermediate mesoderm are essential for nephric duct formation and the initiation of urogenital development.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma , Indução Embrionária , Túbulos Renais/embriologia , Mesoderma , Animais , Proteína Morfogenética Óssea 4 , Linhagem da Célula , Embrião de Galinha , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Hibridização In Situ , Fator de Transcrição PAX2 , RNA Mensageiro/isolamento & purificação , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The importance of proportioning kidney size to body volume was established by clinical studies which demonstrated that in-born defecits of nephron number predispose the kidney to disease. As the kidney develops, the expanding ureteric bud or renal collecting system induces surrounding metanephric mesenchyme to proliferate and differentiate into nephrons. Thus, it is likely that nephron number is related to ureteric bud growth. The expression patterns of mRNAs encoding Fibroblast Growth Factor-7 (FGF-7) and its high affinity receptor suggested that FGF-7 signaling may play a role in regulating ureteric bud growth. To test this hypothesis we examined kidneys from FGF-7-null and wild-type mice. Results of these studies demonstrate that the developing ureteric bud and mature collecting system of FGF-7-null kidneys is markedly smaller than wild type. Furthermore, morphometric analyses indicate that mature FGF-7-null kidneys have 30+/-6% fewer nephrons than wild-type kidneys. In vitro experiments demonstrate that elevated levels of FGF-7 augment ureteric bud growth and increase the number of nephrons that form in rodent metanephric kidney organ cultures. Collectively, these results demonstrate that FGF-7 levels modulate the extent of ureteric bud growth during development and the number of nephrons that eventually form in the kidney.
Assuntos
Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/fisiologia , Rim/embriologia , Ureter/embriologia , Animais , Diferenciação Celular , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Substâncias de Crescimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Néfrons/embriologia , Técnicas de Cultura de Órgãos , Ratos , Transdução de SinaisRESUMO
Kidney development requires reciprocal interactions between the ureteric bud and the metanephrogenic mesenchyme. Whereas survival of mesenchyme and development of nephrons from mesenchymal cells depends on signals from the invading ureteric bud, growth of the ureteric bud depends on signals from the mesenchyme. This codependency makes it difficult to identify molecules expressed by the ureteric bud that regulate mesenchymal growth. To determine how the ureteric bud signals the mesenchyme, we previously isolated ureteric bud cell lines (UB cells). These cells secrete soluble factors which rescue the mesenchyme from apoptosis. We now report that four heparin binding factors mediate this growth activity. One of these is basic fibroblast growth factor (bFGF), which is synthesized by the ureteric bud when penetrating the mesenchyme. bFGF rescues three types of progenitors found in the mesenchyme: precursors of tubular epithelia, precursors of capillaries, and cells that regulate growth of the ureteric bud. These data suggest that the ureteric bud regulates the number of epithelia and vascular precursors that generate nephrons by secreting bFGF and other soluble factors.
Assuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/farmacologia , Rim/embriologia , Mesoderma/citologia , Células-Tronco/fisiologia , Ureter/embriologia , Animais , Antígenos Virais de Tumores/biossíntese , Antígenos Virais de Tumores/genética , Apoptose , Linhagem Celular , Sobrevivência Celular , Meios de Cultivo Condicionados , Fator 1 de Crescimento de Fibroblastos/biossíntese , Fator 10 de Crescimento de Fibroblastos , Fator 5 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Fator 9 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/biossíntese , Substâncias de Crescimento/biossíntese , Rim/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/fisiologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Transdução de Sinais , Vírus 40 dos Símios/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Ureter/fisiologiaRESUMO
Metanephric mesenchyme gives rise to both the epithelial cells of the nephron and the stromal cells of the mature kidney. The function of the stroma. in kidney morphogenesis is poorly understood. We have generated mice with a null mutation in the Winged Helix (WH) transcription factor BF-2 to examine its function during development. BF-2 expression within the developing kidney is restricted to the stromal cell lineage. Homozygotes die within the first 24 hr after birth with abnormal kidneys. Mutant kidneys are small, fused longitudinally, and rotated 90 degrees ventrally. Histological examination reveals a smaller collecting system, numerous large condensations of mesenchyme, and a decrease in the number of nephrons. Using molecular markers we show that induction and condensation of the nephrogenic mesenchyme occurs normally in mutant. The disruption of BF-2 reduces the rate of differentiation of the condensed mesenchyme into tubular epithelium, as well as the rate of growth and branching of the ureter and collecting system. Our findings demonstrate that BF-2 and stromal cells have essential functions during kidney morphogenesis. Furthermore, they suggest that BF-2 controls the production, by the stroma, of signals or factors that are required for the normal transition of induced mesenchyme into tubular epithelium and full growth and branching of the collecting system.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Mesoderma/fisiologia , Proteínas do Tecido Nervoso/genética , Células Estromais/fisiologia , Animais , Sequência de Bases , Indução Embrionária , Epitélio , Fatores de Transcrição Forkhead , Homozigoto , Túbulos Renais Coletores/crescimento & desenvolvimento , Túbulos Renais Coletores/patologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Dados de Sequência Molecular , Morfogênese , Mutação , Néfrons , Ureter/embriologiaRESUMO
The kidney forms from two tissue populations derived from intermediate mesoderm, the ureteric bud and metanephric mesenchyme. It is currently accepted that metanephric mesenchyme is committed to differentiating into nephrons while the ureteric bud is restricted to forming the renal collecting system. To test this hypothesis, we transferred lacZ into pure metanephric mesenchyme isolated from gestation day 13 rat embryos. The fate of tagged mesenchymal cells and their progeny was characterized after co-culture with isolated ureteric buds. When induced to differentiate by the native inducer of kidney morphogenesis, lineage-tagged mesenchymal cells exhibit the potential to differentiate into collecting system epithelia, in addition to nephrons. The fate of cells deriving from isolated ureteric buds was also examined and results of these lacZ gene transfer experiments indicate that the majority of ureteric bud cells differentiate into the renal collecting system. These cell fate studies combined with in situ morphological observations raise the possibility that collecting system morphogenesis in vivo occurs by growth of the ureteric bud and recruitment of mesenchymal cells from the metanephric blastema. Thus, metanephric mesenchyme may be a pluripotent renal stem population.
Assuntos
Rim/embriologia , Mesoderma/fisiologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Epiteliais , Epitélio/embriologia , Técnicas de Transferência de Genes , Rim/citologia , Mesoderma/citologia , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Ureter/citologia , Ureter/embriologiaRESUMO
The conversion of intermediate mesoderm into renal tubular structures occurs throughout embryonic development from the formation of the nephric duct through to the generation of metanephric nephrons. Studies using an in vitro model system of renal epithelial morphogenesis indicate that the signals mediating tubulogenesis rescue intermediate mesoderm from programmed cell death, trigger spindle-shaped mesodermal cells to differentiate into epithelia, and lastly promote epithelial cell diversification and segmented tubule assembly. Recent data indicate that members of the Wnt family of developmental regulatory genes play an important role in the signaling pathway mediating renal epithelial morphogenesis.
Assuntos
Rim/embriologia , Animais , Epitélio/embriologia , Genes , MorfogêneseRESUMO
During kidney development, unknown signals derived from the ureteric bud induce metanephric mesenchymal cells to differentiate into nephron epithelia. In addition to the ureteric bud, a number of other tissues can act as heterologous inducers of this process in vitro, including embryonic spinal cord. In this report we demonstrate that Wnt-1, a gene that encodes a secreted glycoprotein expressed in embryonic spinal cord, is capable of conferring nephron-inducing activity to fibroblast cell lines. When cocultured with cells expressing exogenous Wnt-1, metanephric mesenchyme differentiated into glomerular and renal tubular epithelia. No such effect was observed using control cells. These data imply that the ability of embryonic spinal cord to act as an inducer of nephrogenesis may result from its production of Wnt-1 protein and suggest that a member of the Wnt gene family may be a mediator of renal epithelial morphogenesis in vivo.
Assuntos
Rim/embriologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Peixe-Zebra , Animais , Indução Embrionária , Células Epiteliais , Ratos , Medula Espinal/embriologia , Proteínas Wnt , Proteína Wnt1RESUMO
The transporting epithelia of the kidney are derived from an embryonic rudiment containing two distinct cell populations: ureteric bud epithelia and mesenchymal cells of the metanephric blastema. The ureteric bud is a caudal outgrowth of the Wolffian Duct and gives rise to the renal collecting system by branching morphogenesis. The metanephric blastema gives rise to diverse cells of the nephron after receiving an inductive stimulus. It has been proposed that mesenchymal progenitors of the metanephric blastema derive directly from intermediate mesoderm, although this hypothesis has never been tested directly. Utilizing direct lineage analysis techniques we demonstrate, in an organ culture system, that mesenchymal nephron progenitors are immediate descendants of ureteric bud epithelia. Ureteric bud epithelia can give rise to mesenchymal nephron progenitors that populate the metanephric blastema by undergoing an epithelial-to-mesenchymal transition followed by delamination. If this process occurs in vivo, renal morphogenesis can be characterized by two phenotypic conversions: an epithelial-to-mesenchymal transition leading to the generation of mesenchymal-nephron progenitors, followed by a mesenchymal-to-epithelial transition leading to the generation of diverse nephron epithelial cell types. We have immortalized an embryonic renal mesenchymal cell line and demonstrate that the clonal cell line, RSTEM-1, undergoes phenotypic conversions in vitro, providing a suitable model to study the regulation of the epithelial phenotype.
Assuntos
Rim/embriologia , Animais , Diferenciação Celular , Linhagem Celular , Polaridade Celular , Células Epiteliais , Epitélio/embriologia , Epitélio/metabolismo , Imuno-Histoquímica , Queratinas/metabolismo , Rim/citologia , Rim/metabolismo , Mesoderma/citologia , Técnicas de Cultura de Órgãos , Ratos , Células-Tronco/citologia , Vimentina/metabolismoRESUMO
During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.
Assuntos
Apoptose , Indução Embrionária , Epiderme/embriologia , Mesoderma/fisiologia , Néfrons/embriologia , Alcaloides/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Indução Embrionária/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Epiderme/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Néfrons/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Ratos/embriologia , Medula Espinal/embriologia , Estaurosporina , Ureter/embriologiaRESUMO
We have derived an immortalized cell line from primary cultures of bicarbonate-secreting intercalated cells from rabbit. Cells were transfected with a plasmid encoding a temperature-sensitive large T antigen of SV40 plus the neomycin resistance gene under the control of an SV40 promoter. Transfectants were selected for resistance to G418. One stably transfected clone, designated IC250, was subcloned to ensure clonality, and a subclone (clone C) was characterized in detail. The cells divide continuously at permissive temperature. At restrictive temperature, they cease dividing and assume morphological and transport properties of true bicarbonate-secreting intercalated cells. They express appropriate ultrastructural features, bind peanut lectin in an apical pattern, are rich in carbonic anhydrase, stain for proton-adenosinetriphosphatase in a basolateral pattern, and do not stain with antibodies to erythrocyte band 3. Most monolayers of transformed type B intercalated cells do not achieve a significant transepithelial resistance; those monolayers that are sufficiently electrically tight for electrophysiological studies are capable of chloride-dependent bicarbonate transport.
Assuntos
Bicarbonatos/metabolismo , Linhagem Celular Transformada , Rim/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Divisão Celular , Cloretos/fisiologia , Rim/citologia , Rim/ultraestrutura , Coelhos , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
At least fourteen epithelial cell types of the mammalian nephron develop from the metanephric mesenchyme. To distinguish whether this single embryological primordium contains a heterogenous population of committed renal cell lines or a multipotent stem cell, the lac-Z gene was introduced into individual renal progenitors by retroviral mediated gene transfer. The differentiated fate of lac-Z-tagged daughters derived from single metanephric mesenchymal cells was characterized after cytodifferentiation. We found that the metanephric mesenchyme contains multipotent stem cells that can generate at least three distinct cell types; glomerular, proximal and distal epithelia. After induction the fate of this multipotent cell becomes restricted to populate a single nephron segment.
Assuntos
Indução Embrionária/fisiologia , Rim/embriologia , Mesoderma/citologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Epitélio/embriologia , Técnicas Histológicas , Óperon Lac , Néfrons/embriologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos , TransfecçãoRESUMO
We developed a procedure to introduce and stably express foreign genes into the kidney. The Lac Z reporter gene encoding the bacterial protein beta-galactosidase was introduced by retrovirus-mediated gene transfer into rat nephrogenic mesenchymal cells, which were induced for 24 h with embryonic spinal cord in vitro. The Lac Z-tagged mesenchymal cells were subsequently transplanted underneath the capsule of the neonatal kidney. Two weeks after transplantation, the Lac Z-tagged cells derived from transplants were identified by their beta-galactosidase expression. Well-differentiated Lac Z positive cells were observed in glomerulus and proximal and distal nephron segments. To determine if the tagged mesenchymal cells developed into functional nephrons, fluorescein isothiocyanate-labeled dextran was infused into transplanted animals before death. We observed that fluorescent apical vesicles were colocalized to beta-galactosidase positive proximal tubular cells, indicating that the transplanted mesenchymal cells were integrated into reabsorbing nephrons. These results show the feasibility of introducing foreign genes into epithelia of functioning nephron segments.
Assuntos
Fluoresceína-5-Isotiocianato/análogos & derivados , Rim/embriologia , Transfecção , beta-Galactosidase/genética , Animais , Animais Recém-Nascidos , Quimera , Dextranos , Feminino , Fluoresceínas , Corantes Fluorescentes , Histocitoquímica , Rim/enzimologia , Rim/crescimento & desenvolvimento , Gravidez , Ratos , Ratos Endogâmicos , beta-Galactosidase/análiseRESUMO
Intercalated cells of the distal nephron secrete either H+ or HCO3-. We have succeeded in isolating HCO3- -secreting intercalated cells from the rabbit kidney. When seeded onto collagen-coated permeable supports, these cells form monolayers with a resistance of 595 +/- 75 omega.cm2. The monolayers maintain the characteristics of an epithelium, with apical microvillae and tight junctions. They display the same polarity as do HCO3- -secreting intercalated cells in vivo, namely apical peanut lectin binding and apical Cl- -HCO3- exchange. The monolayers are capable of transepithelial HCO3- transport via this exchanger. The rate of Cl- -dependent transepithelial HCO3- transport is 4 +/- 0.4 nmol.min-1.cm-2. Transepithelial HCO3- transport is completely abolished by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid applied to the apical side of the monolayer. These cultured HCO3- -secreting intercalated cells should prove useful for defining the cellular regulation of HCO3- secretion.
Assuntos
Bicarbonatos/metabolismo , Separação Celular/métodos , Túbulos Renais Coletores/citologia , Túbulos Renais/citologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Células Cultivadas , Cloretos/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/ultraestrutura , Coelhos , Canais de Sódio/fisiologiaAssuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Resinas de Troca Aniônica/análise , Eritrócitos/citologia , Regulação da Expressão Gênica , Resinas de Troca Iônica/análise , Rim/citologia , Especificidade de Órgãos , Animais , Sequência de Bases , DNA/análise , DNA/genética , Eritrócitos/metabolismo , Humanos , Rim/metabolismo , Dados de Sequência Molecular , Estrutura MolecularAssuntos
Anticorpos Monoclonais , Anticorpos , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Transporte de Monossacarídeos , Simportadores , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo , Técnicas Imunoenzimáticas , Proteínas de Membrana Transportadoras/análise , Oligopeptídeos/síntese química , RadioimunoensaioRESUMO
Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant "uncoupled" for beta-galactoside/H+ symport [Wong, P. T. S., Kashket, E. R., & Wilson, T. H. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 63], are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H+ electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H+ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide [Viitanen, P., Garcia, M. L., Foster, D. L., Kaczorowski, G. J., & Kaback, H. R. (1983) Biochemistry 22, 2531]. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent Km. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)