ABSTRACT
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.
Subject(s)
Intracellular Signaling Peptides and Proteins , Receptors, Neuropeptide/biosynthesis , Receptors, Neuropeptide/chemistry , Animals , Calcium/metabolism , Carrier Proteins/metabolism , Cell Line , Cholecystokinin/metabolism , Cyclic AMP/metabolism , DNA Primers/chemistry , DNA, Complementary/metabolism , Diltiazem/pharmacology , Dose-Response Relationship, Drug , Inositol Phosphates/metabolism , Mice , Neuropeptides/metabolism , Orexin Receptors , Orexins , Patch-Clamp Techniques , Peptides/chemistry , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Elapid Venoms/metabolism , Receptors, Muscarinic/metabolism , Amino Acid Sequence , Animals , Carbachol/pharmacology , Cell Line , Chelating Agents/metabolism , Cholinergic Agonists/pharmacology , Chromatography, Ion Exchange , DNA, Complementary/metabolism , Elapid Venoms/chemistry , Fura-2/metabolism , Humans , Insecta , Molecular Sequence Data , Receptor, Muscarinic M1 , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Alkaline Phosphatase/metabolism , Osteoblasts/drug effects , Osteosarcoma/enzymology , Tetradecanoylphorbol Acetate/pharmacology , Calcium/metabolism , Cell Division , DNA/analysis , Dose-Response Relationship, Drug , Humans , Microscopy, Electron , Osteoblasts/cytology , Osteoblasts/enzymology , Osteosarcoma/ultrastructure , Phosphorus/metabolism , Tumor Cells, Cultured , UltracentrifugationABSTRACT
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.