**2. Endogenous Ouabain (EO) and Na+/K+-ATPase Interaction**

The hypothesis of the existence of an endogenous cardiac glycoside became solid, when several animals [5–10] and human [11,12] studies showed that, in the context of a volume-expanded condition, it was possible to find an endogenous humoral substance that could counterbalance the increased renal reabsorption of sodium and water by the inhibition of Na<sup>+</sup> transport through vascular and tubular cell membranes. The volume expansion might be a stimulus for the release of this substance, called natriuretic hormone, which could control sodium homeostasis through the inhibition of the key enzyme in the process of its tubular reabsorption, the Na+/K+-ATPase [13,14]. The effect of the endogenous cardiac glycoside is not limited to the kidney, but also involves the Na+/K+ pumps in other regions, such as the neuro-vascular system. The consequence of this enzyme's inhibition is the increase of the intracellular sodium concentration, which is exchanged for calcium through the Na+/Ca+ exchanger, particularly active in cardiac mussels and smooth vascular muscles [15]. The increase of the intracellular concentration of these two ions, promoted by elevated levels of this endogenous inhibitor, might augment vascular tone determining peripheral vasoconstriction and might lead to a rise in blood pressure [16,17]. Notably, increased levels of cardiotonic glycosides were found in low renin (i.e., volume expanded) hypertension [18]. The vasopressor effect of the cardiotonic steroids has acute and chronic aspects. The acute pressor effect is mediated by the increase in the calcium concentration that causes vasoconstriction, when the short-term cardio-vascular reflexes are blocked. In the case of sustained and chronic elevation of circulating cardiotonic steroids, the pressor effect is maintained by the activation of a signaling pathway that up-regulates the expression of several ion transports in arterial myocytes [19]. It was initially hard to demonstrate that an endogenous digitalis actually exists, and during past years, many research groups have tried to identify it, particularly under physiological and pathological conditions, such as hypertension, pregnancy and neonatal period. Consistent findings obtained over the years suggested that it was possible to isolate different candidate inhibitors of the Na+-pump in mammalian tissues, urine and plasma, and several natriuretic substances able to inhibit sodium pumps were identified [4]. Among this compound, it was isolated and characterized the one presenting the majority of the functional properties of the plant-derived cardiac glycoside. Hamlyn's and Haupert's groups were the first to describe the presence of a cardiotonic steroid indistinguishable from ouabain in human plasma in 1991 [20]. The endogenous ouabain (EO) was then isolated from bovine adrenal glands [21], human adrenal glands [22], bovine hypothalamus [23], rat adrenomedullary cells [24] and biological fluids [25,26] by using the high performance liquid chromatography (HPLC) and immunoassay methods. The presence of EO was finally demonstrated with mass spectrometry, nuclear magnetic resonance (NMR) and chromatography, confirming its existence and its identicalness to the plant-derived ouabain [27,28]. Furthermore, it was possible to identify the most important production site of EO in the adrenal cortex [29,30]. All this evidence led to the identification in mammals of numerous endogenous cardiotonic steroids as cardenolides and bufodienolides (as marinobufagenine) [31]. However, these endogenous compounds are different from each other and it is fair to assume that they could play a distinct patho-physiological role, acting as tissue-specific regulators of different isoforms of the Na+/K+-pump [19].
