RESUMO
cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925-Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.
Assuntos
Encéfalo/metabolismo , Genoma Humano , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Humanos , Isomerismo , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oócitos/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/fisiologia , Proteínas Recombinantes , Ribonucleases , Distribuição Tecidual , XenopusRESUMO
Due to calcium and phosphorus solubility problems in parenteral nutrition solutions, it is difficult to provide the premature infant with enough of these two minerals for adequate bone mineralization. In order to determine the maximum amounts of both Ca and P soluble in neonatal parenteral nutrition solutions, we employed the following procedure: (1) using concentrations of dextrose 10 to 25% and amino acid 0.5 to 4.0% with standard electrolyte and vitamin concentrations, Ca and P additions were sequentially made to determine the critical concentrations at which precipitates formed; (2) the pH of each test solution was determined; (3) all test solutions were incubated for 30 hr at room temperature; (4) following incubation, all tests were visually observed for calcium-phosphate crystals; (5) the solutions not obviously precipitated were filtered using black Millipore filters to determine the presence of any microprecipitates. Multiple graphs of Ca and P solubility in various dextrose/amino acid solutions were prepared from data generated by the study. The Ca and P interaction is primarily pH sensitive. Factors affecting the solution pH include both dextrose and amino acid concentrations. Our study showed that increases in amino acid concentrations enabled us to increase both Ca and P in the solutions.