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

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

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

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

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 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

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

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

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-

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

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

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.
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