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Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na+ /K+ -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis-like factor, marinobufagenin (MBG) has been implicated as a caus...

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.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.