RESUMO
The kallikrein-kinin system is involved in the inflammatory process, in blood pressure regulation, and in renal homeostasis. The presence of kallikreins, kininogens, and kinins in renal tissues and fluids is well established; however, the occurrence and distribution of the bradykinin (B2) receptor in the kidney are unknown. Using chemically cross-linked conjugates of bovine serum albumin and the B2 agonist bradykinin or the potent B2 antagonist HOE140, followed by antibodies to the respective ligand and the peroxidase-anti-peroxidase system, we were able to detect the B2 receptor. The receptor has been found in straight portions of the proximal tubules, in distal straight tubules, in connecting tubules, and in collecting ducts of rat kidney. The staining patterns produced by the ligand conjugate-antiligand approach are in agreement with those obtained by conventional autoradiography using [125I]-Tyr0-bradykinin. Immunocytochemical localization of B2 receptor by antipeptide antibodies to the receptor confirmed these findings and demonstrated the presence of B2 receptor in the basal infoldings and luminal membranes of the tubule cells, and in smooth muscle cells of the cortical radial artery and of afferent arterioles. Co-localization of the B2 receptor with kallikrein and kininogens in connecting tubule cell and collecting duct cell layers, respectively, provides a structural basis for the hypothesized physiological functions of the kallikrein-kinin system in the kidney.
Assuntos
Túbulos Renais/química , Rim/química , Receptores da Bradicinina/análise , Animais , Autorradiografia , Bradicinina/análogos & derivados , Feminino , Técnicas Imunoenzimáticas , Calicreínas/análise , Rim/irrigação sanguínea , Rim/ultraestrutura , Túbulos Renais/ultraestrutura , Cininogênios/análise , Ligantes , Masculino , Microscopia Imunoeletrônica , Músculo Liso Vascular/química , Ratos , Receptor B2 da Bradicinina , Receptores da Bradicinina/imunologia , Soroalbumina BovinaRESUMO
Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.
Assuntos
Cininas/fisiologia , Receptores da Bradicinina/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Sítios de Ligação de Anticorpos , Células Cultivadas , Técnicas de Cultura , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Rim/metabolismo , Dados de Sequência Molecular , Neutrófilos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor B2 da Bradicinina , Homologia de Sequência , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
Spectrophotometric evidence for the formation of hypomanganate(V), [CAR-Mn(V)O43-], and manganate(VI), [CAR-Mn(VI)O42-], intermediate complexes has been confirmed during the oxidation of iota- and lambda-carrageenan-sulfated polysaccharides (CAR) by alkaline permanganate at pHs 12 using a conventional spectrophotometer. These short-lived intermediate complexes were identified and characterized. A reaction mechanism in good consistence with the experimental results is suggested.
Assuntos
Compostos de Manganês/análise , Compostos de Manganês/química , Óxidos/análise , Óxidos/química , Polissacarídeos/química , Ácidos Sulfúricos/química , Absorção , Carragenina/química , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , EspectrofotometriaRESUMO
BACKGROUND: From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma-crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma-crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. METHODS: The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. RESULTS: The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. CONCLUSION: The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice.
Assuntos
Catarata/genética , Catarata/metabolismo , Cristalinas/metabolismo , Cristalino/embriologia , Cristalino/metabolismo , Mutação , Transcrição Gênica , Alelos , Animais , Catarata/embriologia , Catarata/patologia , Cristalinas/genética , Desenvolvimento Embrionário e Fetal , Hibridização In Situ , Cristalino/patologia , Camundongos , Camundongos Mutantes , Fenótipo , Sondas RNA , RNA Mensageiro/metabolismoAssuntos
Receptores da Bradicinina/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Códon de Iniciação/genética , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Receptor B2 da Bradicinina , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro , SpodopteraRESUMO
Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Apllication of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue