Báo cáo Y học: Endogenous cardiac glycosides, a new class of steroid hormones pot

MINIREVIEW Endogenous cardiac glycosides, a new class of steroid hormones Wilhelm Schoner Institut fu ¨ r Biochemie und Endokrinologie, Justus-Liebig-Universita ¨ t Giessen, Germany 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, adre- nals, 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 cor- tical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that oua- bain 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 concen- trations 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 con- centration is increased after cardiac infarction, may show natriuretic properties because it inhibits the a1 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 pos- sibilities 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. Keywords: endogenous digitalis; endogenous ouabain; ouabain antagonists; cardiotonic steroids; cardiac glyco- side binding globulin; signal transduction; congestive heart failure; hypertension. INTRODUCTION For more than 200 years, digitalis, a cardiotonic steroid, and its congeners have been used to treat congestive heart failure [1]. The beneficial result of this therapy was so impressive in the first half of the twentieth century that in 1953, Albert Szent-Gyo ¨ rgyi postulated the existence of an endogenous digitalis in mammals [2], thereby reviving a similar idea that Ringer had published in 1885 [3]. Modern understanding of digitalis therapy arose 50 years ago, when in 1953 Schatzmann discovered that cardiotonic steroids are specific inhibitors of the sodium pump [4] and that the digitalis receptor is the Na + /K + -ATPase of plasma mem- branes [5]. The discovery of the Na + /Ca 2+ exchanger in the late 1960s in mammalian cardiac muscle led to the view that the inhibition of the sodium pump by cardiotonic steroids leadstoanincreaseintheconcentrationofintracellular Ca 2+ as a secondary event, which in turn results in a positive inotropic effect on cardiac muscle [6]. This model has been recently refined: it is clear now that the a1 isoform of the sodium pump is ubiquitously distributed in plasma membranes of cardiomyocytes but that the a 2 /a 3 isoforms reside in plasma membrane areas close to the endoplasmic reticulum. Such ÔplasmerosomesÕ also contain the Na + / Ca 2+ exchanger protein. Inhibition of the a 2 and a 3 isoforms of Na + /K + -ATPase in such a restricted area leads to a change in cytosolic Na + and, indirectly, Ca 2+ concentrations. This modulates in turn the Ca 2+ content of the sarcoplasmic reticulum and Ca 2+ signaling, and leads finally to the positive inotropic effect of cardiac glycosides [7,8] and an altered gene expression of proteins [9]. The search for an endogenous digitalis-like compound was aided in the last few decades when it became apparent that volume-expanded forms of hypertension may lead to the release of a natriuretic hormone. The Dahl-deWardener- Blaustein concept [10–12] of a natriuretic hormone proposes that an enhanced production of endogenous inhibitor(s) of the sodium pump occurs with the adaptive function of decreasing the volume of circulating fluid by means of inhibition of the Na + /K + -ATPase in renal tubules. The increased production of endogenous digitalis-like com- pounds would also contribute to hypertension by means of inhibition of Na + /K + -ATPase in cardiovascular tissues [10–12]. For some time it was not possible to establish that an endogenous digitalis actually exists. Hamlyn et al. were the first to demonstrate that the concentration of a circulating factor in blood plasma inhibiting purified Na + / K + -ATPase correlated with the blood pressure of the donors [13]. This observation paved the way for the Correspondence to W. Schoner, Institut fu ¨ r Biochemie und Endokri- nologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany. Tel.: + 49 641 99 38171; Fax: + 49 641 99 38179, E-mail: wilhelm.schoner@vetmed.uni-giessen.de Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; ACE, angiotensin converting enzyme. (Received 15 October 2001, revised 25 February 2002, accepted 3 April 2002) Eur. J. Biochem. 269, 2440–2448 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02911.x identification of endogenous ÔdigitalisÕ as a group of cardenolides and bufadienolides whose physiological and pathophysiological function is only starting to be under- stood. Readers who are interested in more detailed infor- mation are referred to recent reviews [9,14–17]. OUABAIN IS A NEW STEROID HORMONE Identification of ouabain as endogenous inhibitor of the sodium pump There is now much evidence that ouabain is a new steroid hormone of the adrenal cortex and hypothalamus (Fig. 1). Ouabain-like immunoreactivity has been found in almost all tissues, including plasma, but the highest concentrations have been observed in the adrenal, hypophysis, and hypothalamus [16,18,19]. The long-standing dispute as to whether endogenous ouabain is identical to plant-derived ouabain or whether it is an isomer thereof is turning in favor of a structure identical to the plant-derived ouabain [20–22]. A substance with striking similarity to ouabain was previously isolated from human urine [23] and bovine adrenals [24]. Hamlyn and his coworkers isolated 10 lgof ouabain or its isomer from 85 L of human plasma [18,25]. The compound of 585 295 Da in fast atom bombardment mass spectrometry (FAB-MS) gave very similar results in a direct comparison with ouabain in linked tandem MS, after derivatization with acetic anhydride coupled with FAB-MS and in analytical HPLC that was able to detect an altered stereochemistry of a single sugar OH group [25]. Hence, it was concluded that the endogenous inhibitor of the sodium pump is ouabain or a closely related isomer. Schneider et al. however, were the first to show that ouabain is in fact a constituent of the adrenals [21]. They isolated 20 lgofa pure substance from 20 kg of bovine adrenals and identified the substance by ESI-MS and 1 H-NMR spectroscopy as ouabain [21]. The hypothalamic inhibitor of the sodium pump from bovine brain was considered for a long time to be an isomer of ouabain, but it was determined recently that the previous microanalysis was erroneous because of the presence of borate, which had diffused out of the borosili- cate glass used to store the minute amount of pure substance. Hence, it was demonstrated beyond doubt that the inhibitor from bovine hypothalamus is also ouabain [20]. In summary, ouabain, an arrow poison of the African Ouabaio tree and of Strophanthus gratus plants and a long- known inhibitor of the sodium pump, has been identified in blood plasma, adrenal glands, and the hypothalamus of mammals. The adrenal gland as a source of ouabain The surprising observation that a plant toxin can be purified from mammalian sources leads to the question as to whether this substance was taken up from the diet and consequently stored in the tissues where it has been identified. In fact, orally and parenterally administered ouabain is selectively taken up by the adrenal [26], although intestinal uptake of ouabain is considered to be only 3–5% of administered substance [27]. Ouabain is also taken up by cultured adrenal cells [28]. Consistent with the adrenal gland being a major place of synthesis and/or storage of ouabain, adrenalectomy leads to a lowering of ouabain plasma levels [18,29]. The adrenal cortex is likely to be the place of storage and/or synthesis, because it contains more ouabain than the medulla [19] and demedullectomized rats show no lowering of their plasma concentrations of ouabain when compared with sham-operated controls [16]. Adrenal glands of con- scious dogs release ouabain [30]. It is not known whether storage vesicles exist that release ouabain upon hormonal stimulus. Biosynthesis of cardenolides in mammals Ouabain is apparently synthesized in the adrenals and released upon hormonal stimulation. Extirpation of an adrenocortical tumor overproducing ouabain, a ouabai- noma, in a human patient reverted the elevated blood pressure to normal [31]. In another case, compression of the tumor during extirpation of a pheochromocytoma increased the patient’s plasma level of ouabain immunoreactivity and norepinephrine. The tumor had higher tissue concentrations of ouabain than the normal adrenal cortex [32]. De novo synthesis of ouabain and dihydro-ouabain has been Fig. 1. Structures of endogenous cardiotonic steroids that have been isolated in the search for ‘endogenous digitalis’. Compounds with an unsaturated five-membered lactone ring are cardenolides and those with an unsaturated six-membered lactone ring are bufadienolides. PST 2238 is a ouabain antagonist [75,76]. Ó FEBS 2002 Endogenous cardiac glycosides (Eur. J. Biochem. 269) 2441 demonstrated in tissue culture experiments [33]. Bovine adrenocortical cells in vitro secrete ouabain in amounts that exceed their cell content by up to tenfold [28,34,35]. The biosynthesis occurs in zona fasciculata cells. Bovine adreno- cortical cells in tissue culture release ouabain upon exposure to adrenocorticotropin, a1-adrenergic receptor agonists, and angiotensin II [36–38]. Human CLR7050 cells (an adrenal cortex-derived cell line) are insensitive to adreno- corticotropin and angiotensin II but sensitive to arginine vasopressin and phenylephrine [38]. The phenylephrine- dependent release of ouabain from human CRL7050 and bovine adrenocortical cells in culture is blocked by the a1-adrenergic receptor antagonist doxazosin. This was interpreted to indicate that the sympathetic nervous system is involved in regulation of the release of this hormone to the bloodstream [38]. In bovine adrenal cortical cells, angioten- sin II acts via the angiotensin type 2 (AT 2 ) receptor, because the AT 2 agonist CGP42112 stimulates the release of ouabain and the AT 2 antagonist PD123319 inhibits it [36]. However, a signaling pathway involving the brain’s O 2 chemoreceptor seems to exist as well, because hypoxia triggers a marked release of the hypothalamic inhibitory factor (ouabain) from midbrain and adrenals in rats [39,40]. More direct evidence for the existence of the biosynthetic pathway in mammals comes from the demonstration that the administration of certain precursors increases the rate of synthesis of the cardiotonic steroid or, if given in their radioactive form, leads to the formation of radioactive cardiotonic steroids. Progesterone and pregnenolone have been shown to be precursors of endogenous ouabain [16,22,35]. In addition, rhamnose could readily enter adreno- cortical cells and increase the biosynthesis of endogenous ouabain [35]. Inhibition of the 3b-hydroxysteroid dehydro- genase that forms progesterone stimulates the secretion of endogenous ouabain [16] similar to that seen with the addition of progesterone itself [35]. Hence, it seems possible that various 3b-hydroxysteroid dehydrogenase isoforms are involved in the biosynthesis of aldosterone and ouabain. When [7- 3 H]pregnenolone is added to primary rat adrenal cells, radioactivity is found in a fraction with digitalis-like activity but not with ouabain [41]. At present, it is unclear why different cell systems yield different results. There is no doubt, however, that biosynthesis of the cardenolides ouabain or digitalis occurs in adrenocortical cells. PHYSIOLOGY AND PATHOPHYSIOLOGY OF ENDOGENOUS OUABAIN Regulatory short-term effects We are only starting to obtain information on the physiology and pathophysiology of endogenous ouabain. Recently, we reported that submaximal treadmill exercise of dogs rapidly increased the concentrations of ouabain in blood by about 50- to 500-fold. Upon rest, ouabain levels fell with a half-life of 5–8 min. Pretreatment of dogs with the b-blocker atenolol as well as the angiotensin converting enzyme (ACE) inhibitor benazepril abolished the exercise-dependent rise in endo- genous ouabain levels, indicating that the release of ouabain in dogs is under the control of epinephrine and angiotensin II [42]. adrenocorticotropin did not stimulate ouabain release in man [43] or dogs [42]; but was effective in rats [44]. Similarly, ergometric training of normotensive human volunteers led to a rapid increase in endogenous ouabain concentrations that declined rapidly upon rest [45]. The observations of a rapid rise and decline of endogenous ouabain upon physical exercise are consistent with the properties of a fast-acting, circulating hormone. Regulatory long-term effects Elevated concentrations of endogenous ouabain (ouabain- like immunoreactivity) have been found under a number of conditions such as sodium imbalance, chronic renal failure, hyperaldosteronism, congestive heart failure, and pre- eclampsia [14,16,46,47]. Ouabain has been shown to produce vasoconstriction in man at low doses. The most striking finding in humans is that approximately 50% of Caucasians with uncomplicated essential hypertension show increased concentrations of endogenous ouabain, reduced heart rate, and greater left ventricular mass and stroke volume of the heart [48]. Circulating levels of endogenous ouabain corre- late directly with mean blood pressure, relative thickness of the left ventricular heart wall, and the total peripheral resistance index [49,50]. Immunization of rats against ouabain lowers arterial blood pressure [16] as does infusion of the commercially available Fab fragment of an anti- digoxin Ig (Digibind), which cross-reacts with ouabain, in humans and rats [51,52]. Exposure of rats for a long period to small (nanomolar) doses of ouabain or other cardeno- lides leads to hypertension [53–56]. The hypertensinogenic action of ouabain was also observed with ouabagenin, dihydro-ouabain, iso-ouabain (containing an oxygen bridge between C14 of the steroid and C21 in the lactone ring), and an lactone ring-opened ouabain. Unexpectedly, however, the hypertensinogenic effect of these ouabain derivatives increased with the decrease in the potency of the compounds to inhibit Na + /K + -ATPase; i.e. an inverse linear relation- ship between the rise in blood pressure and the logarithm of the IC 50 was observed [57]. Also surprising, although consistent with clinical experience, was that digoxin was unable to raise blood pressure [48,58]; on the contrary, it lowered it [57]. Moreover, digoxin and digitoxin reduce the hypertensive effect of ouabain [48,59]. The observation that the hypertensinogenic activity of cardiac glycosides is not directly related to their potency as inhibitors of Na + /K + - ATPase raises the possibility that the sodium pump may not be the initial target in the mechanism by which ouabain induces a sustained increase in blood pressure. The hyper- tensinogenic activity of ouabain and its analogs may arise from a novel mechanism linked with the steroid nucleus [57]. It has been known for some time that treatment of cells in culture with cardiac glycosides affects cell proliferation as well as the expression of isoforms of Na + /K + -ATPase. When infused for 6 weeks in rats, ouabain and digoxin differentially affected the expression of isoforms of the sodium pump in different tissues [58]. The molecular mechanism by which ouabain affects cell differentiation has been studied in heart muscle in great detail by Xie & Askari and their coworkers [60]. Therapeutic concentrations of ouabain stimulate the growth of muscle cells and protein biosynthesis, including the Ca 2+ -dependent expression of the early response genes c-fos and c-jun as well as Ras and p42/44 mitogen-activated protein kinases, which are viewed as key mediators of cardiac hypertrophy [60]. In rat renal epithelial cells, ouabain causes low-frequency intracellular 2442 W. Schoner (Eur. J. Biochem. 269) Ó FEBS 2002 calcium oscillations at concentrations that partially inhibit the sodium pump. These oscillations are caused by a concerted action of InsP 3 receptors and capacitive calcium entry via plasma membrane channels. The low-frequency intracellular oscillations elicit an activation of the transcrip- tion factor, NF-jB [61]. It is well known that changes in monovalent ion fluxes are also intimately connected to the processes of mitogenesis and differentiation. Consistent with a central role of the sodium pump in these processes, ouabain selectively inhibits interferon a-induced gene expression [62], induces mRNAs encoding the growth factors interleukin 6 and granulocyte macrophage colony- stimulating factor [63,64], and increases c-fos and c-jun expression in a variety of cultured cells [62]. Because the expression of c-fos is regulated by a number of hormones, growth factors, and mitogens associated with cell prolifer- ation and differentiation, it is not surprising that other cardiotonic steroids, namely bufalin and bufalin-like fac- tors, also affect cell differentiation [65–67]. Ouabain, a neurosteroid, mediates sympathetic hyperactivity in salt-sensitive hypertension Ouabain has been identified in the hypothalamus and it is present in the pituitary and in medullary neurons. Pituitary ouabain is considered to be released into the circulation but paracrine secretion may occur. Cold-induced brain edema in cats leads to a significant increase in ouabain-like activity in the cerebrospinal fluid and the edematous brain hemi- sphere [68]. In conscious rats, acute intracerebroventricular injection of ouabain or crude hypothalamic or pituitary extracts containing ouabain-like activity causes similar increases in sympathetic activity, blood pressure, and heart rate. These effects can be prevented by the simultaneous intracerebroventricular administration of Fab fragments of Digibind, which cross-react with ouabain [69,70]. The effects of central Na + and ouabain are attenuated in transgenic rats that are deficient in brain angiotensinogen [71]. In normal rats, sympathetic hyperactivity and hyper- tension induced by chronic ouabain and hypertonic saline treatment is prevented by angiotensin type 1 receptor blockade [72]. High salt intake also increases the expression and activity of the ACE in hypothalamus and pons of Dahl salt-sensitive rats without a parallel increase in angioten- sin II levels. Chronic blockade of brain ÔouabainÕ by intraventricular infusion of a ouabain-binding antibody lowered the NaCl-dependent rise in the amount of ACE mRNA, which may indicate that the increase in ACE mRNA is secondary to the activation by brain ÔouabainÕ [73]. Presently, it is unclear how inhibition of the sodium pump in brain cells affects ACE expression. It is conceivable that inhibition of the pump leads to an increase in intracellular Ca 2+ concentrations that in turn lead to an increase in ACE expression and activity, as has been shown for ACE expression induced by platelet activating factor and endothelin [74]. A tentative regulatory scheme based on the information available thus far is shown in Fig. 2. Anti-ouabain as an antihypertensinogenic agent Ouabain-induced hypertension is believed to be mediated via inhibition of the sodium pump. Therefore, ouabain Fig. 2. Hormonal control of the release of endogenous ouabain from zona fasciculata cells of the adrenal cortex by hypernatriaemia, hypoxia, and physical exercise. The scheme represents a compilation of the findings that ouabain levels are increased in patients with low-renin hypertension [50], in hypoxemia [39,40], and during physical exercise [42]. In adrenocortical cells, ouabain is released by phenylephrine and angiotensin II, and its action is blocked by the AT 2 inhibitor PD123319 [36–38]. The ACE inhibitor prazolol and the b-blocker atenolol abolish the exercise-dependent rise in ouabain levels in dogs [42]. Digoxin counteracts the ouabain-dependent rise in blood pressure in rats [48,57,59] and lowers renin levels in humans [86]. It has been shown that ouabain and angiotensin II stimulate the release and synthesis of catecholamines and that catecholamines stimulate the release of renin. Increased concentrations of Na + increase the synthesis of angiotensin converting enzyme (ACE) [107]. Stimulation of ouabain release by adrenocorticotropin has been observed in tissue culture [36] and in vivo in the rat [44], but not in dog and man [42,43]. Ó FEBS 2002 Endogenous cardiac glycosides (Eur. J. Biochem. 269) 2443 antagonists may lower blood pressure. In fact, an new compound resembling cardiotonic steroids, PST 2238, shows such an antihypertensive action [75,76] (Fig. 1). Micromolar concentrations of PST 2238 lower the ouabain- induced hypertension in rats when given orally. This new prototype of an antihypertensive drug acts also in Milan hypertensive rats, where a genetic alteration of adducin genes is associated with hypertension and up-regulation of renal Na + /K + -ATPase. Hence, PST 2238 might be used for the treatment of human essential hypertension caused by an alteration of the cytoskeletal protein adducin [77]. Identification of other cardenolides as endogenous inhibitors of the sodium pump A number of observations indicate that additional cardio- tonic steroids of the cardenolide or bufadienolide group may play a role in the circulation (Fig. 1) [19,21,78,79]. The existence of an endogenous digoxin can not excluded so far. Approximately 7.9 lg of a substance indistinguishable from digoxin was isolated from 100 tons of human urine [80]. Its properties in FAB-MS, proton NMR, several different HPLC systems and in its reactivity with digoxin antibodies were identical with digoxin [81], but a digoxin-like immuno- reactive factor from bovine adrenals appears to be slightly different. This latter factor exists in a deglycosylated and a reduced form [82,83]. It was found in blood plasma, urine, adrenal glands, and breast cyst fluid [17]. There is, however, no evidence so far that digoxin is synthesized in mammalian cells. It may be taken up from the gut with the diet and stored in the adrenals [26]. Endogenous digoxin immuno- reactivity is increased in renal failure, in newborn infants, and under conditions of hypertensive pregnancy as well as during prolonged, strenuous exercise [15,17,84]. Plasma digoxin immunoreactivity is increased about 2.5-fold in patients with acute myocardial infarction [85]. Most inter- esting is that digoxin counteracts the hypertensinogenic effect of ouabain in rats [48,59] (Fig. 1). This may be due to a decrease in plasma renin activity and angiotensin II, aldosterone, and epinephrine levels, and a significant increase in the levels of atrial and brain natriuretic peptides [86]. Additionally, the digoxin-induced arterial baroreflex opposes the sympathetic excitatory pressor responses to ouabain in the periphery and in the brain [48,59,87] and digoxin no longer activates the chemoreflex in patients with chronic heart failure [88] (Fig. 2). It is thus unclear how two substances that are both specific inhibitors of the sodium pump can produce opposing physiological effects. One reason may lie in the higher hydrophobicity of digoxin as compared with ouabain, which would lead to a different tissue distribution. However, other reasons may also exist. Identification of bufadienolides as endogenous inhibitors of the sodium pump In addition to the endogenous cardenolides ouabain and digoxin, endogenous bufadienolides have also been identi- fied (Fig. 1) [19,78,89–91]. Material cross-reacting with antibodies against the bufadienolides bufalin and proscil- laridin A has been found to increase in concentration in blood and to correlate with systolic blood pressure [79,92]. Bufalin-like immunoreactivity rose in the serum of Dahl-S rats under the conditions of a high-salt diet [92]. Proscillar- idin A-immunoreactive material was purified from bovine adrenals. The compound, with a molecular mass of 600 Da, had a UV maximum at 250 nm, which indicates that this hydrophilic compound differs from a classical bufadienolide with a UV maximum at 300 nm [21]. Another compound, 3b-hydroxy 14a 20:21-bufenolide, has been purified and identified from human placenta [78]. Is marinobufagenin, an endogenous a 1 sodium pump inhibitor, a natriuretic hormone? Marinobufagenin (3b,5b-dihydroxy-14,14-epoxybufadieno- lide) (Fig. 1) was originally discovered in amphibians and more recently isolated from the urine of patients with myocardial infarction [90]. In contrast to ouabain, marino- bufagenin exhibits a greater affinity for the ouabain- resistant a1 subunit of Na + /K + -ATPase [93,94]. As one of the factors associated with blunted natriuresis, salt- sensitive Dahl rats have a mutation in the a1 subunit of Na + /K + -ATPase [95]. The mutated renal sodium pump exhibits an abnormal Na + /K + -ATPase pumping ratio, which upon a high salt intake results in an inability of the kidneys to fully excrete sodium [96,97]. When salt-sensitive Dahl rats were exposed to an acute NaCl load, a transient increase in the plasma endogenous ouabain concentration was observed that was accompanied by a sustained increase in the level of endogenous marinobufagenin [94]. It was suggested that the increased blood plasma marinobufagenin concentration is due to secretion that promotes natriuresis and compensates for the genetically impaired pressure natriuretic mechanism [98]. The bufadienolide is vasocon- strictive [90]; it is elevated in volume expansion and pre- eclampsia and, like ouabain, is increased upon voluntary hypoventilation of human volunteers [99]. 19-Norbufalin, a cardiotonic steroid causing cataract formation In normal lenses, immunoreactivity against bufalin and ouabain-like factor is sevenfold to 30-fold higher in the capsular epithelial layer than in the lens fiber region [100]. In human cataractous lenses, the concentration of the sodium pump inhibitor was much higher than in normal lenses. Hence, it was isolated from cataractous lenses and identified as 19-norbufalin (Fig. 1) and its Thr-Gly-Ala tripeptide derivative [91]. Cardiac glycosides alter the osmotic balance of lenses and induce cataract formation by crystalline degradation and protein leakage that initiate opacity. Inhibition of the sodium pump in rat lenses down- regulates the expression of the intracellular signaling protein 14-3-3 without a significant change in c-crystalline gene expression. Because 14-3-3 proteins are multifunctional regulatory proteins, a reduction in the abundance of various isoforms would have profound effects on cell function [101]. BINDING OF CARDIOTONIC STEROIDS TO SERUM PROTEINS As hydrophobic substances, steroid hormones are trans- ported in blood as complexes bound to specific binding globulins [102]. Serum albumin is presumed to fulfill this role for cardiac glycosides but at concentrations of the 2444 W. Schoner (Eur. J. Biochem. 269) Ó FEBS 2002 compounds much above the pharmacologically effective concentrations [103]. If one assumes that cardiotonic steroids are in fact vertebrate hormones, one would expect that a specific cardiac glycoside binding protein might exist. Search for such a protein led to the isolation of 53- and 26-kDa proteins from bovine serum [104], and a 14.4-kDa protein from human plasma [105]. The bovine protein exhibited K d values of 1.5 and 75 n M for ouabain [104]. Unexpectedly, the 14.4-kDa protein from human plasma turned out to be identical with the plasmin-generated Fc fragment of IgG. The plasmin-fragment pFc (but not native IgG or the papain-generated Fc-fragments) protected THP-1 cells against the ouabain-induced arrest of cell division [105]. Hence, it was speculated that pFc might be generated on tissue surfaces during fibrinolysis and is involved in tissue remodeling during inflammation. CONCLUSIONS Much information has now accumulated indicating that the plant toxin ouabain is a mammalian steroid hormone [18,20–22] that is under the rapid control of catecholamines and/or angiotensin [28,34,36–38,42]. The picture is emer- ging that increased plasma Na + concentrations [47], hypoxemia [39,40], and physical exercise [42] stimulate the release of endogenous ouabain and possibly other cardio- tonic steroids from hypothalamic and adrenocortical cells. The increase in plasma ouabain leads in turn to an increase in blood pressure (Fig. 2). This rise is not associated with plasma renin activity [106]. The release of ouabain from zona fasciculata cells of the adrenal gland is stimulated via the angiotensin and adrenergic systems, which are inti- mately interconnected [107]. Increased endogenous ouabain concentrations are associated with about 50% of hyperten- sion cases diagnosed in Caucasians with low renin hyper- tension [50]. According to Hamlyn et al., elevated levels of ouabain primarily affect the central nervous system and activate angiotensin II-dependent pathways in the brain that mediate sympathetic nerve activity [16]. Circulating ouabain and its interplay with the tissue renin-angiotensin and sympathetic nervous systems might also induce func- tional and structural cardiovascular changes by peripheral mechanisms [16]. There are other endogenous cardiotonic steroids, inclu- ding endogenous digoxin, marinobufagenin, and proscil- laridin A, that are released under the more restricted conditions mentioned above. It is presently unclear how these other cardiotonic steroids might interfere with each other and modulate the ouabain system. Marinobufagenin shows a preference for the a1 isoform of the sodium pump, which is the major isoform of the kidney. It may therefore have natriuretic properties that would lower plasma volume. Digoxin evidently counteracts the hypertensive effect of ouabain. Hence, the result of the actions of the different endogenous cardiotonic steroids seems to be a cooperative effect in handling salt and water homeostasis. Because ouabain and digoxin are both inhibitors of the sodium pump, the hitherto used rationale of digoxin therapy becomes muddled. How can this paradoxical physiological action of ouabain and digoxin be explained on a molecular level? Is there a different tissue distribution of the differ- ent cardiac glycosides, a difference in their affinities at the various pump isoforms, differences in the signal transduction pathway [9] or are there other receptors for cardiac glycosides besides the sodium pump [104,105]? ACKNOWLEDGEMENTS The author’s own research was supported by the Deutsche Fors- chungsgemeinschaft, Bonn-Bad Godesberg, the Fonds der Chemischen Industrie, Frankfurt/Main, and the Akademie fu ¨ r Tiergesundheit, Bonn. REFERENCES 1. Withering, W. (1785) An account on the foxglove, and some of its medical uses with practical remark on dropsy and other diseases. G. G. J. & J. Robinson, London. 2. Szent-Gyo ¨ rgyi, A. (1953) Chemical Physiology of Contraction. in Body and Heart Muscle pp. 86–91. Academic Press, New York. 3. Ringer, S. (1885) Regarding the influence of the organic constit- uents of the blood on the contractility of the ventricle. J. Physiol. 6, 361–381. 4. Schatzmann, H.J. (1953) Herzglykoside als hemmstoffe fu ¨ rden aktiven kalium- und natriumtransport durch die erythrocyten- membran. Helv. Physiol. Pharmacol. Acta 11, 346–354. 5. Skou, J.C. (1957) The influence of some cations on an adeno- sinetriphosphatase from peripheral nerves. Biochim. Biophys. Acta 23, 394–401. 6. Eisner,D.A.&Smith,T.W.(1992)TheNa-Kpumpandits effectors in cardiac muscle. In The Heart and Cardiovascular System (Fozzard,H.A.,Haber,E.,Jennings,R.B.,Katz,A.M.& Morgan, H.E., eds), pp. 863–902. Raven Press, New York. 7. Blaustein, M.P., Juhaszowa, M. & Golovina, V.A. (1998) The cellular mechanism of action of cardiotonic steroids: a new hypothesis. Clin. Exp. Hypertens. 20, 691–703. 8. Juhaszova, M. & Blaustein, M.P. (1997) Na + pump low and high ouabain affinity a subunit isoforms are differently distributed in cells. Proc. Natl Acad. Sci. USA 94, 1800–1805. 9. Scheiner-Bobis, G. & Schoner, W. (2001) A fresh facet for oua- bain action. Nat. Med. 7, 1288–1289. 10. Dahl,L.K.,Knudsen,K.D.,Heine,M.&Leitl,G.(1967)Effects of chronic excess salt ingestion. Genetic influence on the devel- opment of salt hypertension in parabiotic rats. Evidence for a circulating factor. J. Exp. Med. 126, 687–699. 11. deWardener, H.E. & Clarkson, E.M. (1985) Concept of natri- uretic hormone. Physiol. Rev. 65, 658–759. 12. Blaustein, M.P. (1977) Sodium ions, calcium ions, blood pressure regulation and hypertension: a reassessment and a hypothesis. Am.J.Physiol.232, C167–C173. 13. Hamlyn, J.M., Ringel, R., Schaeffer, J., Levinson, P.D., Hamil- ton, B.P., Kowarski, A.A. & Blaustein, M.P. (1982) A circulating inhibitor of (Na+K)-ATPase associated with essential hyper- tension. Nature 300, 650–652. 14. Blaustein, M.P. (1993) Physiological effects of endogenous oua- bain: control of intracellular Ca 2+ stores and cell responsiveness. Am.J.Physiol.264 (Cell Physiol. 33), C1367–C1387. 15. Goto, A. & Yamada, K. (1998) Ouabain-like factor. Curr. Opin. Nephrol. Hypertens. 7, 189–196. 16. Hamlyn,J.M.,Lu,Z.,Manunta,P.,Ludens,J.H.,Kimura,K., Shah,J.R.,Laredo,J.,Hamilton,J.P.,Hamilton,M.J.& Hamilton, B.P. (1998) Observations on the nature, biosynthesis, secretion and significance of endogenous ouabain. Clin. Exp. Hypertens. 20, 523–533. 17. Schoner, W. (2001) Endogenous cardiotonic steroids. Cell Mol. Biol. 47, 273–280. 18.Hamlyn,J.M.,Blaustein,M.P.,Bova,S.,DuCharme,D.W., Mandel, F., Mathews, W.R. & Ludens, J.H. (1991) Identification and characterization of a ouabain-like compound from human plasma. Proc.NatlAcad.Sci.USA88, 6259–6263. Ó FEBS 2002 Endogenous cardiac glycosides (Eur. J. Biochem. 269) 2445 19. Li, S Q., Eim, C., Kirch, U., Lang, R.E. & Schoner, W. (1998) Bovine adrenals and hypothalamus are a major source of pros- cillaridin A- and ouabain-immuno-reactivities. Life Sci. 62, 1023– 1033. 20. Kawamura,A.,Guo,J.,Itagaki,Y.,Bell,C.,Wang,Y.,Haupert, J.,Garner,T.,Magil,S.,Gallagher,R.T.,Berova,N.&Naka- nishi, K. (1999) On the structure of endogenous ouabain. Proc. Natl Acad. Sci. USA 96, 6654–6659. 21. Schneider,R.,Wray,V.,Nimtz,M.,Lehmann,W D.,Kirch,U., Antolovic, R. & Schoner, W. (1998) Bovine adrenals contain, in addition to ouabain, a second inhibitor of the sodium pump. J. Biol. Chem. 273, 784–792. 22. Komiyama,Y.,Nishimura,N.,Munakata,M.,Mori,T.,Okuda, K.,Nishino,N.,Hirose,S.,Kosaka,C.,Masuda,M.& Takahashi, H. (2001) Identification of endogenous ouabain in culture supernatant of PC12 cells. J. Hypertens. 19, 229–236. 23. Goto, A., Yamada, K., Ishii, M., Yoshioka, M., Ishiguro, T., Eguchi, C. & Sugimoto, T. (1988) Purification and characteriza- tion of human urine-derived digitalis-like factor. Biochem. Biophys. Res. Commun. 154, 847–853. 24. Inagami, T. & Tamura, M. (1988) Purification and character- ization of specific endogenous ouabain-like substance from bovine adrenal. Am.J.Med.Sci.295, 400–405. 25. Mathews, W.R., DuCharme, D.W., Hamlyn, J.M., Harris, D.W., Mandel, F., Clark, M.A. & Ludens, J.A. (1991) Mass spectral characterization of an endogenous digitalis-like factor from human plasma. Hypertension 17, 930–935. 26. Kitano, S., Morimoto, S., Nishibe, A., Fukuo, K., Hirotani, A., Nakahashi, T., Ysuda, O. & Ogihara, T. (1998) Exogenous ouabain is accumulated in the adrenals and mimics the kinetics of endogenous digitalis-like factor in rats. Hypertens. Res. 21, 47–56. 27. Greef, K. & Wirth, K.E. (1981) Pharmakokinetics of stro- phanthin glycosides. Handbook Exp. Pharmacol. 56/II, 57–85. 28. Doris, P.A., Hayward-Lester, A., Bourne, D. & Stocco, D.M. (1996) Ouabain production by cultured adrenal cells. Endocrinology 137, 533–539. 29. Masugi, F., Ogihara, T., Hasegawa, T., Sagakuchi, K. & Kuma- hara, Y. (1988) Normalization of high plasma level of ouabain- like immunoreactivity in primary aldosteronism after removal of adenoma. J. Hum. Hypertens. 2, 17–20. 30. Boulanger, B.R., Lilly, M.P., Hamlyn, J.M., Laredo, J., Shurtleff, D. & Gann, D.S. (1993) Ouabain is secreted by the adrenal gland of the awake dogs. Am. J. Physiol. 264, E413–E419. 31. Manunta,P.,Evans,G.,Hamilton,B.P.,Gann,D.,Resau,J.& Hamlyn, J.M. (1992) A new syndrome with elevated plasma ouabain and hypertension secondary to an adrenocortical tumor. J. Hypertens. 10, S27. 32.Komiyama,Y.,Nishimura,N.,Munakata,M.,Okuda,K., Nishino,N.,Kosaka,C.,Masuda,M.,Mori,T.,Matsuda,T.& Takahashi, H. (1999) Increases in plasma ouabainlike immunoreactivity during surgical extirpation of pheochromo- cytoma. Hypertens. Res. 22, 135–139. 33. Qazzaz, H.M., El-Masri, M.A. & Valdes, R.J. (2000) Secretion of a lactone-hydrogenated ouabain-like effector of sodium, potassium-adenosine triphosphatase activity by adrenal cells. Endocrinology 141, 3200–3209. 34. Laredo, J., Hamilton, B.P. & Hamlyn, J.M. (1994) Ouabain is secreted by bovine adrenocortical cells. Endocrinology 135, 794–797. 35. Perrin,A.,Brasmes,B.,Chambaz,E.M.&Defaye,G.(1997) Bovine adrenocortical cells in culture synthesize an ouabain-like compound. Mol. Cell. Endocrinol. 126, 7–15. 36. Laredo, J., Hamilton, J.P. & Hamlyn, J.M. (1995) Secretion of endogenous ouabain from bovine adrenal cells. Role of zona glomerulosa and zona fasciculata. Biochem. Biophys. Res. Com- mun. 212, 487–493. 37. Laredo, J., Shah, J.R., Lu, Z., Hamilton, B.P. & Hamlyn, J.M. (1997) Angiotensin II stimulates secretion of endogenous ouabain from bovine adrenal cortical cells via angiotensin II receptors. Hypertension 29, 401–107. 38. Laredo, J., Shah, J.R., Hamilton, B.P. & Hamlyn, J.M. (2000) Alpha-1 adrenergic receptors stimulate secretion of endogenous ouabain from human and bovine adrenocortical cells. In Na/K-ATPase and Related ATPases (Taniguchi, K. & Kayas, S., eds), pp. 671–679. Elsevier Science, Amsterdam. 39. De Angelis, C. & Haupert, G.T. Jr (1998) Hypoxia triggers release of an endogenous inhibitor of Na + -K + -ATPase from midbrain and adrenal. Am. J. Physiol. 274, F182–F188. 40.Paci,A.,Marrone,O.,Lenzi,S.,Prontera,C.,Nicolini,G., Ciabatti, G., Ghione, S. & Bonsignore, G. (2000) Endogenous digitalis-like factors in obstructive sleep apnea. Hypertens. Res. 23S, S87–S91. 41. Lichtstein,D.,Steinitz,M.,Gati,I.,Samuelov,S.,Deutsch,J.& Orly, J. (1998) Biosynthesis of digitalis-compound in rat adrenal cells: hydroxycholesterol as a precursor. Life Sci. 62, 2109–2126. 42. Bauer,N.,Neu,H.,Kirch,U.,Gru ¨ nbaum, E G. & Schoner, W. (2001) Relelase of endogenous ouabain to blood upon exercise of circulation is under the control of epinephrinde and angiotensin II. Biol. Chem. 382, S109. 43. Butt,A.N.,Semra,Y.K.,Lane,S.J.,Lee,T.&Swaminathan,R. (1998) Endogenous ouabain secretion in man is not regulated by ACTH. J. Steroid Biochem. Mol. Biol. 66, 151–157. 44. Yamada, K., Goto, A. & Omata, M. (1997) Adrenocortico- tropin-induced hypertension in rats: role of ouabain-like com- pound. Am. J. Hypertens. 10, 403–408. 45. Antolovic,R.,Bauer,N.,Mohadjerani,M.,Kost,H.,Neu,H., Kirch, U., Gru ¨ nbaum, E G. & Schoner, W. (2000) Endogenous ouabain and its binding globulin: effect of physical exercise and study on its tissue distribution. Hypertens. Res. 593–598. 46. Gottlieb,S.S.,Rogowski,A.C.,Weinberg,M.,Krichten,C.M., Hamilton, B.C. & Hamlyn, J.M. (1992) Elevated concentrations of endogenous ouabain in patients with congestive heart failure. Circulation 86, 420–425. 47. Hasegawa, T., Masugi, F., Ogihara, T. & Humahara, Y. (1987) Increase in plasma ouabainlike inhibitor of Na+,K+-ATPase with high sodium intake in patients with essential hypertension. J. Clin. Hypertens. 3, 419–429. 48. Manunta, P., Hamilton, J., Rogowski, A.C., Hamilton, B.P. & Hamlyn, J.M. (2000) Chronic hypertension induced by ouabain but not digoxin in the rat: antihypertensive effect of digoxin and digitoxin. Hypertens. Res. 23, S77–S85. 49. Pierdomenico, S.D., Bucci, A., Manunta, P., Rivera, R., Ferrandi,M.,Hamlyn,J.M.,Lapenna,D.,Cuccurullo,F.& Mezzetti, A. (2001) Endogenous ouabain and hemodynamic and left ventricular geometric patterns in essential hypertension. Am. J. Hypertens. 14, 44–50. 50. Manunta, P., Stella, P., Rivera, R., Ciurlino, D., Cusi, D., Ferrandi, M., Hamlyn, J.M. & Bianchi, G. (1999) Left ventricular mass, stroke volume and ouabain-like factor in essential hyper- tension. Hypertension. 34, 450–456. 51. Adair, C.D., Buckalew, V., Taylor, K., Ernest, J.M., Frye, A.H. & Veille, J.C. (1996) Elevated endoxin-like factor complicating a multifetal second trimester pregnancy: treatment with digoxin- binding immunoglobulin. Am. J. Nephrol. 16, 529–531. 52. Goodlin, R.C. (1988) Antidigoxin antibodies in eclampsia. New Engl. J. Med. 318, 518–519. 53. Manunta, P., Rogowski, A.C., Hamilton, B.P. & Hamlyn, J.M. (1994) Ouabain-induced hypertension in the rat: relation- ships among plasma and tissue ouabain and blood pressure. J. Hypertens. 12, 549–560. 54. Pamnani, M.B., Chen, S., Yuan, C.M. & Haddy, F.J. (1994) Chronic blood pressure effects of bufalin, a sodium-potassium ATPase inhibitor in rats. Hypertension 23, I106–I109. 2446 W. Schoner (Eur. J. Biochem. 269) Ó FEBS 2002 55. Veerasingham, S.J. & Leenen, F.H. (1999) Ouabain- and central sodium-induced hypertension depend on the ventral ante- roventral third ventricle region. Am. J. Physiol. 276, H63–H70. 56. Yuan,C.M.,Manunta,P.,Hamlyn,J.M.,Chen,S.,Bohen,E., Yeun,J.,Haddy,F.J.&Pamnani,M.B.(1993)Long-termoua- bain administration produces hypertension in rats. Hypertension 22, 178–187. 57. Manunta, P., Hamilton, B.P. & Hamlyn, J.M. (2001) Structure- Activity Relationship for hypertensinogeniv activity of ouabain. Role of the sugar and lactone ring. Hypertension 37, 472–477. 58. Wang, H., Yuan, W Q. & Lu, Z.R. (2000) Differential regulation of the sodium pump alpha-subunit isoform gene by ouabain and digoxin in tissues of rats. Hypertens. Res. 23, S55–S60. 59. Huang, B.S., Kudlac, M., Kumarathasan, R. & Leenen, F.H. (1999) Digoxin prevents ouabain and high salt intake-induced hypertension in rats with sinoaortic denervation. Hypertension 34, 733–738. 60. Xie, Z. & Askari, A. (2002) Na + /K + -ATPase as a signal trans- ducer. Eur. J. Biochem. 269, 2434–2439. 61. Aizman, O., Uhle ´ n, P., Lal, M., Brismar, H. & Aperia, A. (2001) Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc. Natl Acad. Sci. USA 98, 13420–13424. 62. Nakagawa, Y., Rivera, V. & Larner, A.C. (1992) A role for the Na/K-ATPase in the control of human c-fos and c-jun tran- scription. J. Biol. Chem. 267, 8785–8788. 63. Akashi,M.,Loussararian,A.H.,Adelman,D.C.,Saito,M.& Koeffler, H.P. (1990) Role of lymphotoxin in expression of interleukin 6 in human fibroblasts. Stimulation regulation. J. Clin. Invest. 85, 121–129. 64. Yamato, K., El-Hajjaoui, Z., Kuo, J.F. & Koeffler, H.P. (1989) Granulocyte-macrophage colony-stimulating factor: signals for its mRNA accumulation. Blood 74, 1314–1320. 65. Numazawa, S., Inoue, N., Nakura, H., Sugiyama, T., Fujino, E., Shinoki, M., Yoshida, T. & Kuroiwa, Y. (1996) A cardiotonic steroid bufalin-induced differentiation of THP-1 cells. Involve- ment of Na + ,K + -ATPase inhibition in the early changes in proto-oncogene expression. Biochem. Pharmacol. 52, 321–329. 66. Szabo, E., Francis, J. & Birrer, M.J. (1998) Alterations in dif- ferentiation and apoptosis induced by bufalin in c-jun over- expressing U-937 cells. Int. J. Oncol. 12, 403–409. 67. Zhang, L., Nakaya, K., Yoshida, T. & Kuroiwa, Y. (1992) Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers. Cancer Res. 52, 4634–4641. 68. Rap, Z.M., Schoner, W., Czerniki, Z., Hildebrandt, G., Mueller, W. & Hoffmann, O. (1994) Changes in the endogenous ouabain- like substance in the brain and cerebrospinal fluid after cold- induced vasogenic brain edema in the sodium pump. In The Sodium Pump – Structure Mechanism, Hormonal Control and its Role in Disease (Bamberg, E. & Schoner, W., eds), pp. 779–782, Steinkopff Springer, New York. 69. Huang, B.S. & Harmsen, E., Yu, H. & Leenen, F.H.H. (1992) Brain ouabain-like activity and the sympathexcitatory and pressor effects of central sodium in rats. Circ. Res. 71, 1059–1066. 70. Takahashi, H., Matsuzawa, M., Okabayashi, H., Suga, K., Ikeg- aki,I.,Yoshimura,M.,Bichi,H.,Okamura,H.,Murakami,S. & Ibata, Y. (1987) Evidence for digitalis-like substance in the hypothalamopituitary axis in rats: implications in the central cardiovascular regulation associated with excess intake of sodium. Jap. Circ. J. 51, 1199–1207. 71. Huang, B.S., Ganten, D. & Leenen, F.H.H. (2001) Responses to central Na + and ouabain are attenuated in transgenic rats defi- cient in brain angiotensogen. Hypertension 37, 683–686. 72. Zhang, L., Nakaya, K., Yoshida, T. & Kuroiwa, Y. (1991) Bufalin as a potent inducer of differentiation of human myeloid leukemia cells. Biochem. Biophys. Res. Commun. 178, 686–693. 73. Zhao,X.,White,R.,Huang,B.S.,VanHuysse,J.&Leenen, F.H.H. (2001) High salt intake and the brain renin-angiotensin system in Dahl-sensitive rats. J. Hypertens. 19, 89–98. 74. Kawaguchi, H., Sawa, H. & Yasuda, H. (1991) Effect of endothelin on angiotensin converting enzyme activity in cultured pulmonary artery endothelial cells. J. Hypertens. 9, 171–174. 75. Ferrari, P., Torielli, L., Ferrandi, M., Padoani, G., Duzzi, L., Florio,M.,Conti,F.,Melloni,L.,Vesci.,L.,Corsico,N.& Bianchi, G. (1998) PST 2238: a new antihypertensive compound that antagonizes the long term pressor effect of ouabain. J. Pharmacol. Exp. Therapeut. 285, 83–94. 76.Quadri,L.,Bianchi,G.,Cerri,A.,Fedrizzi,G.,Ferrari,P., Gobbini, M., Melloni, P., Sputore, S. & Torri, M. (1997) 17b-(3Furyl)-5b-androstane-3b-14b-17a-triol (PST 2238). A very potent antihypertensive agent with a novel mechanism of action. J. Med. Chem. 40, 1561–1564. 77. Ferrari, P., Ferrandi, M., Tripoldi, G., Torielli, L., Padoani, G., Minotti, E., Melloni, E. & Bianchi, G. (1999) PST 2238: a new antihypertensive compound that modulates Na,K-ATPase in genetic hypertension. J. Pharmacol. Exp. Therapeut. 288, 1074–1083. 78. Hilton, P.J., White, R.W., Lord, G.A., Garner, G.V., Gordon, D.B., Hilton, M.J., Forni, L.G., McKinnon, W., Ismail, F.M., Keenan, M., Jones, K. & Morden, W.E. (1996) An inhibitor of the sodium pump obtained from human placenta. Lancet 348, 303–305. 79. Sich, B., Kirch, U., Tepel, M., Ziedek, W. & Schoner, W. (1996) Pulse pressure correlates with a proscillaridin A immunoreactive compound. Hypertension 27, 1073–1078. 80. Goto, A., Ishiguro, T., Yamada, K., Ishii, M., Yoshioka, M., Eguchi, C., Shimura, M. & Sugimoto, T. (1990) Isolation of an urinary digitalis-like factor indistinguishable from digoxin. Biochem. Biophys. Res. Commun. 173, 1093–1101. 81. Goto, A. & Yamada, K. (1998) Purifcation of endogenous digi- talis-like factors from normal human urine. Clin. Exp. Hypertens. 20, 551–556. 82. Qazzaz, H.M.A.M., Goudy, S.L. & Valdes, R.J. (1996) Degly- cosylated products of endogenous digoxin-like immunoreactive factor in mammalian tissue. J. Biol. Chem. 271, 8731–8737. 83. Shaikh, I.M., Lau, B.W.C., Siegfried, B.A. & Valdes, R. (1991) Isolation of digoxin-like immunoreactive factors from mamma- lian adrenal cortex. J. Biol. Chem. 266, 13672–13678. 84. Valdes, R. Jr (1985) Endogenous digoxin-immunoactive factor in human subjects. Fed. Proc. 44, 2800–2805. 85. Bagrov, A.Y., Fedorova, O.V., Maslova, M.N., Roukoyatkina, N.I., Ukhanova, M.A. & Zhabko, E.P. (1991) Endogenous plasma Na,K-ATPase inhibitory activity and digoxin-like immunoreactivity after acute myocardial infarction. Cardiovasc. Res. 25, 371–377. 86. Tsutamoto, T., Wada, A., Maeda, K., Hisanaga, T., Fukai, D., Maeda, Y., Ohnishi, M., Mabuchi, N. & Kinoshita, M. (1997) Digitalis increases brain natriouretic peptide in patients with severe heart failure. Am. Heart. J. 134, 910–916. 87. Gheorghiade, M. & Ferguson, D. (1991) Digoxin: a neuro- hormone modulator in heart failure? Circulation 84, 2181–2186. 88. Paganelli, F., Maixent, J M., Ge ´ lisse, R., Barnay, P., Dodero, F., Francheschi, F., Le ´ vy, S. & Saadjian, A. (2001) Effect of digoxin on chemoreflex in patients with chronic heart failure. Cell. Mol. Biol. 47, 335–340. 89. Bagrov, A.Y. & Fedorova, O.V. (1998) Effects of two putative endogenous digitalis-like factors, marinobufagenin and ouabain, on the Na + ,K + -pump in human mesenteric arteries. J. Hyper- tens. 16, 1953–1958. Ó FEBS 2002 Endogenous cardiac glycosides (Eur. J. Biochem. 269) 2447 90. Bagrov, A.Y., Fedorova, O.V., Dmitrieva, R.I., Howald, W.N., Hunter, A.P., Kuznetsova, E.A. & Shpen, V.M. (1998) Char- acterization of a urinary bufodienolide Na + ,K + -ATPase inhib- itor in patients after acute myocardial infarction. Hypertension 31, 1097–1103. 91. Lichtstein,D.,Gati,I.,Samuelov,S.,Berson,D.,Rozeman,Y., Landau, L. & Deutsch, J. (1993) Identification of digitalis-like compounds in human cataractous lenses. Eur. J. Biochem. 216, 261–268. 92. Oda, M., Kurosawa, M., Numazawa, S., Tanaka, S., Akizawa, T., Ito, K., Maeda, M. & Yoshida, T. (2001) Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal anti- body. Life Sci. 68, 1107–1117. 93. Fedorova, O.V. & Bagrov, A.Y. (1997) Inhibition of Na/K- ATPase from rat aorta by two endogenous Na/K pump inhibitors, ouabain and marinobufagenin. Evidence of interaction with different a-subunit isoforms. Am. J. Hypertens. 10, 929–935. 94. Fedorova, O.V., Lakatta, E.G. & Bagrov, A.Y. (2000) Endogenous Na,K pump ligands are differentially regulated during acute NaCl loading in DAHL rats. Circulation 102, 3009– 3014. 95. Herrera,V.L.,Xie,H.X.,Lopez,L.V.,Schork,N.J.&Ruiz- Opazo, N. (1998) The a-1 Na,K-ATPase gene is susceptibility hypertension gene in Dahl salt-sensitive HSD rat. J. Clin. Invest. 102, 1102–1111. 96. Canessa, M., Romero, J.R., Ruiz-Opazo, N. & Herrera, V.L.M. (1993) The a-1 Na,K-pump of the Dahl saltsensitive rat exhibits altered Na modulation of K transport in red blood cells. J. Membr. Biol. 134, 107–122. 97. Oroz, D.E. & Hopfer, U. (1996) Pathophysiological con- sequences of changes in the coupling ratio of Na,K-ATPase for renal sodium reabsorption and its implications for hypertension. Hypertension 27, 219–227. 98. Fedorova, O.V., Kolodkin, N.I., Agalakova, N.I., Lakatta, E.G. & Bagrov, A.Y. (2001) Marinobufagenin, an endogenous a-1 sodium pump ligand, in hypertensive Dahl salt-sensitive rats. Hypertension 37, 462–466. 99. Bagrov, A.Y., Roukayatkina, N.I., Fedorova, O.V., Pinaev, A.G. & Ukhanova, M.V. (1993) Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad. Eur. J. Pharmacol. 234, 165–172. 100. Lichtstein, D., Levy, T., Deutsch, J., Steinitz, M., Samuel Zigler, J.,J.&Russell,P.(2000)InvolvementofNa + ,K + -ATPase inhibitors in cataract formation In Na/K-ATPase and Related ATPases (Taniguchi, K. & Haya, S., eds), Elsevier Science, Amsterdam. 101. Lichtstein, D., McGowan, M.H., Russel, P. & Carber, D.A. (2000) Digitalis and digitalislike compounds down-regulate gene expression of intracellular signaling protein 14-3-3 in rat lenses. Hypertens. Res. 23, S51–S53. 102. Rosner, W., Hryb, D.J., Khan, M.S., Nakhla, A.M. & Romas, N.A. (1999) Sex hormone-binding globulin mediates steroid hormone signal transduction at the plasma membrane. J. Steroid Biochem. Mol. Biol. 69, 481–485. 103. Krieglstein, J. (1981) Plasma protein binding of cardiac glyco- sides. Handbook Exp. Pharmacol. 56, 95–104. 104. Antolovic, R., Kost, H., Mohadjerani, M., Linder, D., Linder, M. & Schoner, W. (1998) A specific binding protein for cardiac glycosides exists in bovine serum. J. Biol. Chem. 273, 16259– 16264. 105. Komiyama, Y., Nishimura, N., Nishino, N., Okuda, K., Munakata, M., Kosaka, C., Masuda, M. & Takahashi, H. (1998) Purification and characterization of ouabain-binding protein in human plasma. Clin.Exp.Hypertens.20, 683–690. 106. Rossi, G.P., Manunta, P., Hamlyn, J.M., Pavan, E., De Toni, R., Semplicini, A. & Pessina, A.C. (1995) Immunoreactive endo- genous ouabain in primary hyperaldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels. J. Hypertens. 13, 1181–1191. 107. Dluhy, R.G. & Williams, G.H. (1998) Endocrine hypertension In Williams Textbook of Endocrinology, 9th edn. (Wilson, J.D., Foster, D.W., Kronenberg, H.M. & Larsen, P.R., eds), pp. 729– 749. W.B. Saunders, City, Philadelphia, PA. 2448 W. Schoner (Eur. J. Biochem. 269) Ó FEBS 2002 . bovine hypothalamus is also ouabain [20]. In summary, ouabain, an arrow poison of the African Ouabaio tree and of Strophanthus gratus plants and a long- known. the adrenal gland being a major place of synthesis and/or storage of ouabain, adrenalectomy leads to a lowering of ouabain plasma levels [18,29]. The adrenal