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Isolation of marinobufotoxin from the supernatant of cultured PC12 cells
Author(s) -
Yoshika Masamichi,
Komiyama Yutaka,
Takahashi Hakuo
Publication year - 2011
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.2011.05512.x
Subject(s) - high performance liquid chromatography , chemistry , cell culture , sodium , ouabain , metabolism , secretion , microbiology and biotechnology , chromatography , biochemistry , biology , genetics , organic chemistry
Summary 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.