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1.
Clin Exp Pharmacol Physiol ; 38(5): 334-7, 2011 May.
Article in English | MEDLINE | ID: mdl-21401694

ABSTRACT

1. Digitalis-like factors (DLFs) are believed to be involved in sodium metabolism via inhibition of Na(+) /K(+) -ATPase and may cause hypertension. Yet, the source and regulation of secretion of DLFs remain unknown. Recently, marinobufagenin (MBG) was isolated in mammals and implicated in renal sodium and water metabolism. More recently, we isolated marinobufotoxin (MBT), a suberoyl arginine ester of MBG, in Y-1 cells. We have developed an ELISA to measure MBG-like immunoreactivity (MBG-IR) and have characterized MBG-IR using chromatography. We have also identified a ouabain-like factor in cultured PC12 cells from a phaeochromocytoma cell line. In the present study, we examined whether MBT was produced in the adrenal medulla. 2. PC12 cells were cultured in serum-free medium and culture supernatants were collected over a period of 24 h. The supernatants were analysed by ELISA and HPLC to determine MBG-IR content. The HPLC fraction containing the main peak of MBG-IR was characterized by LC/MS. 3. Compared with samples collected at 0.5 h, the concentration of MBG-IR in culture supernatants increased significantly after 2 h and continued to increase until 24 h. The fraction with the highest ELISA peak for MBG-IR had the same HPLC elution time as authentic MBT. Furthermore, tandem mass spectrometry indicated that each fraction of MBT and MBG had the correct specific daughter ions. 4. The results indicate that MBT and MBG are produced and/or secreted by adrenomedullary cells.


Subject(s)
Adrenal Gland Neoplasms/metabolism , Cardanolides/isolation & purification , Culture Media, Conditioned/chemistry , Pheochromocytoma/metabolism , Adrenal Gland Neoplasms/pathology , Animals , Cardanolides/metabolism , Chromatography, High Pressure Liquid , Chromatography, Liquid , Enzyme-Linked Immunosorbent Assay , Mass Spectrometry , PC12 Cells , Pheochromocytoma/pathology , Rats , Tumor Cells, Cultured
2.
Article in English | MEDLINE | ID: mdl-181688

ABSTRACT

The binding of digitoxigenin to lecithin model membranes was investigated by application of electron spin- and nuclear magnetic resonance methods. A digitoxigenin spin-label derivative was found to bind specifically to the pseudohexagonal rigid lattice of lecithin membranes. The binding is accomplished by some of the structural features which are required for the biological activity of cardiac glycosides. Digitoxigenin has a procooperative effect on dipalmitoyl-lecithin membranes. The induction of lipid phase separation is discussed as a hypothetic molecular mechanism of action of cardiac glycosides on biological membranes.


Subject(s)
Cardanolides/metabolism , Digitoxigenin/metabolism , Membranes, Artificial , Phosphatidylcholines/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Binding Sites , Digitoxigenin/pharmacology , Electron Spin Resonance Spectroscopy , Guinea Pigs , Models, Structural , Myocardial Contraction/drug effects , Spin Labels , Temperature
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