RESUMO
OBJECTIVE: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. METHODS: For contractility studies, a total of 57 trabeculae were isolated from two non-failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three alpha isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. RESULTS: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p << 0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol.g-1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three alpha isoforms were expressed at the mRNA level in both normal and failing hearts. CONCLUSIONS: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.
Assuntos
Cardiomiopatias/enzimologia , Isoenzimas/metabolismo , Contração Miocárdica/efeitos dos fármacos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Northern Blotting , Humanos , Isoenzimas/análise , Miocárdio/enzimologia , RNA Mensageiro/análise , ATPase Trocadora de Sódio-Potássio/análise , ATPase Trocadora de Sódio-Potássio/genética , Estimulação QuímicaRESUMO
Differential display technique was used to identify mRNAs that are differentially expressed in malignant versus benign brain tumors. Using this method, a novel 1.4-kb long cDNA (MM1) clone was isolated and sequenced. The nucleotide and the translated amino acid sequence of MM1 cDNA clone did not show significant homology to any known sequence in the Genebank. The expression of MM1 appears to be almost eightfold higher in glioblastomas compared to low grade astrocytomas and slightly higher in malignant meningiomas than in benign meningiomas. The size of mRNA from northern analysis appears to be 7 kb, which is much higher than the size of the isolated MM1 cDNA clone. Expression of MM1 was also seen in various cell lines derived from human tumors including glioblastomas. Whereas low level expression was seen in kidney, esophagus, liver, lymph node, ovary and testis, none of the other tissues from a total of 18 different human organs showed any MM1 expression.
Assuntos
Neoplasias Encefálicas/genética , Sequência de Aminoácidos , Astrocitoma/genética , Sequência de Bases , Northern Blotting , DNA Complementar , Glioblastoma/genética , Humanos , Meningioma/genética , Dados de Sequência Molecular , RNA Mensageiro/análise , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The expression of complement regulatory proteins (CRP) on the surface of neoplastic cells has been proposed as a mechanism by which these cells evade immune surveillance. We have examined the RNA expression of the genes that encode 5 kinds of CRP in various human brain tumors to determine whether CRP expression might play a role in the malignant progression of these tumors. The benign and atypical meningiomas, and the astrocytomas showed high expression of SP-40,40, low expression of CD59, and barely detectable expression of CD46, CD55 and S-protein. The benign and atypical menigiomas showed significantly greater expression of SP-40,40 at the RNA level when compared to malignant meningiomas. This study describes the mRNA expression of meningiomas, astrocytomas, tumor cell lines and normal human tissues.
Assuntos
Neoplasias Encefálicas/genética , Proteínas Inativadoras do Complemento/genética , Chaperonas Moleculares , RNA/análise , Antígenos CD/genética , Astrocitoma/genética , Sequência de Bases , Antígenos CD55 , Antígenos CD59 , Clusterina , Glicoproteínas , Humanos , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Meningioma/genética , Dados de Sequência Molecular , Células Tumorais Cultivadas , VitronectinaRESUMO
The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Testículo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Genes , Humanos , Isoenzimas/genética , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The expression pattern of the multiple isoforms of Na,K-ATPase was examined in the human heart. Isoform specific oligonucleotide probes for the alpha 1, alpha 2, alpha 3 and beta 1 subunits were used to probe Northern blots. The adult human ventricle expresses mRNAs for all three alpha subunit isoforms in addition to beta 1 subunit mRNA.
Assuntos
Isoenzimas/genética , Miocárdio/enzimologia , ATPase Trocadora de Sódio-Potássio/genética , Adulto , Sequência de Bases , Northern Blotting , Criança , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificaçãoRESUMO
Recent studies of the Na,K-ATPase have demonstrated that multiple isoforms of both the alpha and beta subunits exist and that these are expressed in a tissue and developmental specific manner. In the case of the alpha subunit, there are three known isoforms, alpha 1, alpha 2 and alpha 3. We have examined adult human heart for the presence of these isoforms and found that all three exist in approximately equal amounts. This is in contrast to the adult rat heart which contains only alpha 1 and alpha 2 isoforms. The difference in abundance of various isoforms in various tissues could result from the necessity to express Na,K-ATPase with different properties at various developmental stages or in specific cell types. For example, enzymes with differences in Na+ or K+ affinity or the ability to respond to various effector molecules may be required. Alternatively, the presence of three isoforms may simply result from the triplication of the alpha subunit gene and the divergence of expression of these genes during evolution. In this case the isozymes would not confer a specific function to the Na,K-ATPase. In order to provide information with respect to these two alternatives, cell lines producing rat alpha 1, alpha 2 and alpha 3 were developed and the enzymatic properties of the resulting enzyme determined. The results indicate that Na,K-ATPase carrying the alpha 1 are alpha 2 isoforms are fairly similar while enzyme with the alpha 3 isoform differs in its apparent affinity for sodium. The K0.5 for Na+ is approximately three fold lower for this isoform.(ABSTRACT TRUNCATED AT 250 WORDS)