Novel Digitalis-Like Factor, Marinobufotoxin, Isolated From Cultured Y-1 Cells, and Its Hypertensive Effect in Rats

Abstract

Abstract-Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 g/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufageninlike immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135Ϯ3.0 versus 126Ϯ2.0 mm Hg and 1.41Ϯ0.286 versus 0.34Ϯ0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail

“…The molecular formula is consistent with arginine-suberoyl marinobufagenin, reported by Yoshika et al [45] (Figure 5). The MS 2 spectrum shows the expected loss of the marinobufagenin part of the molecule with charge retention on the arginine-suberoyl part (Supporting Information, Figure S10), consistent with data reported by Yoshika et al [45]. Subsequent losses involve typical small molecule losses (H 2 O, CO, NH 3 , and CH 2 N 2 ) expected for the arginine-suberoyl part.…”

Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets

“…Because in this study (Lichtstein et al, 1998) the detection of bufalin was based on an assay using antibody with very high (19.5%) cross-reactivity to the cardenolide strophanthidin, it is difficult to exclude the possibility that these results may, in fact, refer to the production of EO. Murine Y1 adrenocortical cells have been demonstrated to produce marinobufotoxin, an MBG-suberylarginine conjugate previously isolated from various amphibian tissues (Yoshika et al, 2007). At this juncture, it seems that although both bufadienelide and cardenolide CTS derive from cholesterol, only cardenolide CTS synthesis requires cholesterol side chain cleavage.…”

The central mechanism underlying hypertension: a review of the roles of sodium ions, epithelial sodium channels, the renin–angiotensin–aldosterone system, oxidative stress and endogenous digitalis in the brain

“…We originally isolated MBT, 20 telocinobufagin 121 and telocinobufotoxin 122 from human plasma and from cultured Y-1 and PC-12 cells. Some reports indicate that these bufadienolides are of adrenocortical origin.…”

“…Interestingly, there are several EDLFs in the circulating blood, even in subjects who have never ingested any digitalis glycoside either as medicine or in food containing digitalis-like substances. 16, 17, 18, 19, 20 EDLFs include ouabain, digoxin, marinobufagenin (MBG), marinobufotoxin (MBT), telocinobufagin, proscillaridin A, bufalin and others. There are two types of EDLFs: cardenolides derived from plants and bufadienolides derived from toads.…”