RESUMEN
Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Receptores de Neuropéptido/biosíntesis , Receptores de Neuropéptido/química , Animales , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Colecistoquinina/metabolismo , AMP Cíclico/metabolismo , Cartilla de ADN/química , ADN Complementario/metabolismo , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Fosfatos de Inositol/metabolismo , Ratones , Neuropéptidos/metabolismo , Receptores de Orexina , Orexinas , Técnicas de Placa-Clamp , Péptidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de FluorescenciaRESUMEN
Mamba venoms contain peptides with high selectivity for muscarinic receptors. Due to the limited availability of the M(1) muscarinic receptor-selective MT7 or m1-toxin 1, the peptide was expressed in Sf9 cells using a synthetic cDNA and purified. The isolated peptide had over four orders of magnitude higher affinity for the M(1) compared to M(2)-M(5) muscarinic receptors. The peptide strongly inhibited Ca(2+) mobilisation through recombinant and endogenously expressed M(1) receptors, having no effect on the function of the other subtypes. The MT7 peptide provides a unique tool for identification and functional characterisation of M(1) receptors in cells and tissues.
Asunto(s)
Venenos Elapídicos/metabolismo , Receptores Muscarínicos/metabolismo , Secuencia de Aminoácidos , Animales , Carbacol/farmacología , Línea Celular , Quelantes/metabolismo , Agonistas Colinérgicos/farmacología , Cromatografía por Intercambio Iónico , ADN Complementario/metabolismo , Venenos Elapídicos/química , Fura-2/metabolismo , Humanos , Insectos , Datos de Secuencia Molecular , Receptor Muscarínico M1 , Proteínas Recombinantes/metabolismo , Células Tumorales CultivadasRESUMEN
The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS. The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium. The secretion of APase appeared after a lag period of 4-6 hours of TPA treatment, and it could also be visualized with histological staining. Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles. The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes. It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Osteoblastos/efectos de los fármacos , Osteosarcoma/enzimología , Acetato de Tetradecanoilforbol/farmacología , Calcio/metabolismo , División Celular , ADN/análisis , Relación Dosis-Respuesta a Droga , Humanos , Microscopía Electrónica , Osteoblastos/citología , Osteoblastos/enzimología , Osteosarcoma/ultraestructura , Fósforo/metabolismo , Células Tumorales Cultivadas , UltracentrifugaciónRESUMEN
Energy linked Ca2+ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca2+, we observe a respiration-linked uptake of Ca2+ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca2+ uptake. The results suggest that although plant mitochondria accumulate Ca2+ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.