Am J Physiol Heart Circ Physiol 307: H635–H639, 2014;
doi:10.1152/ajpheart.00404.2014.
Perspectives
Why isn’t endogenous ouabain more widely accepted?
Mordecai P. Blaustein
Departments of Physiology and Medicine and the Center for Heart, Hypertension and Kidney Disease, University of
Maryland School of Medicine, Baltimore, Maryland
Submitted 11 June 2014; accepted in final form 30 June 2014
A new idea is the most quickly acting antigen known to
science.—Wilfred Trotter
Address for reprint requests and other correspondence: M. P. Blaustein,
Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore
St., Baltimore, MD, 21201 (e-mail: mblaustein@som.umaryland.edu).
http://www.ajpheart.org
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During the past few years, several distinguished senior
colleagues who visited our department and inquired about the
status of endogenous ouabain (EO) have asked, “How come I
didn’t know about these [published] data?” And, “Why isn’t
endogenous ouabain more widely accepted?” I usually simply
shrug and express disappointment that EO is not yet a “mainstream hormone,” even though the seminal reports published
more than two decades ago in leading journals (cited below)
have been confirmed and supplemented (6, 7, 22). This has
now happened often enough to give me pause.
Fierce competition for resources undoubtedly slowed the
evolution of the EO story, with its several surprises and
multiple components, making it difficult to sustain attention
outside the immediate field. Research has also been stymied
because the biosynthetic pathway has been only partly elucidated, and there are no commercial EO assays or clinically
approved antagonists.
Upon further reflection, however, I have concluded that
there is at least a partial explanation based on well-known
human behavior to which we, as scientists, are particularly
prone (28). We are all very wary of new, paradigm-shifting
ideas that don’t fit our preconceived notions. By profession, we
are skeptics and we demand proof, but we are also often quick
to express negative opinions without carefully assessing the
data. We tend to ignore ideas and data that don’t fit our
preconceptions; in fact, we often don’t even read (or digest)
articles that fall outside our comfort zone. “Mea culpa!” As
Darwin noted, established investigators are usually set in their
ways; new ideas are for newcomers (9). And we are in good
company (although this isn’t a rationale): recall Einstein’s
famous put down of Heisenberg’s uncertainty principle, “He
[God] does not throw dice” (15).
Below I illustrate the problem with some examples, primarily from my own slow journey of recognition and acceptance of
groundbreaking observations about which I was initially very
skeptical. And, if I was so suspicious, how could I expect
others to rapidly grasp and accept ideas and data that ostensibly
don’t fit existing dogma? Nevertheless, if the facts are inconsistent with the prevailing paradigm, progress requires a new
one. This is “scientific revolution” (28).
In 1991, Hamlyn and colleagues (23) purified an endogenous cardiotonic steroid from human plasma and analytically identified it as ouabain. The following year, Doursout
and coworkers (14) and, a year later, Yuan and colleagues
(60) demonstrated that prolonged ouabain administration
induces hypertension in normal rats. These remarkable ob-
servations have been replicated in a number of highly
respected laboratories [reviewed in Blaustein et al. (7)]. The
early studies were rapidly followed by reports that circulating EO is elevated in many patients with essential hypertension and mineralocorticoid hypertension (52) and in
those with congestive heart failure (21). As in any research
field, however, a few investigators failed to replicate the
original findings, or reported false positives, and some have
continued to question the validity of the original reports
[e.g., Baecher et al. (2), Doris et al. (11), and Nicholls et al.
(40). It is inappropriate to critique those studies here, but
see, for example, Manunta et al. (35).] Thus it might seem
understandable that individuals working outside the immediate field may be left in a quandary and would therefore
simply ignore these data and arguments, but this is not the
whole story.
Leenen’s 1992 discovery of “brain ouabain,” including its
key roles in salt-sensitive hypertension (25) and heart failure
(31), was another seminal contribution. Numerous subsequent
reports from that laboratory confirmed and extended those
results by showing that brain ouabain was a distal component
of a hypothalamic chronic renin-angiotensin II (ANG II)aldosterone-epithelial Na⫹ channel-brain ouabain pathway
(Fig. 1) (7, 18, 30). This slow brain pathway, which is activated
by high salt and/or ANG II modulates central sympathoexcitatory neurons (i.e., the acute mechanisms) that are involved in
both hypertension and heart failure (16, 29, 30, 43, 62). Note
that high dietary salt/salt retention suppresses the peripheral
renin-angiotensin-aldosterone system (RAAS) (1, 36), but,
paradoxically, activates the brain RAAS (18, 43). The more
proximal components of the slow pathway, ANG II and ANG
II type 1 receptors, aldosterone and mineralocorticoid receptors, and epithelial Na⫹ channel (19, 20, 44), are often mentioned in the literature, but brain ouabain has been ignored.
Indeed, even though I was aware of it, I, too, long disregarded
brain ouabain; it didn’t fit my biased, vasculocentric view of
how EO works in hypertension. Finally, in 2010, John Hamlyn
and I sat down with Frans Leenen and listened to each other’s
ideas. We agreed that the brain EO data and plasma EO data
were both valid and needed to be reconciled. The outcome was
the joint proposal of a new, comprehensive view of the pathogenesis of salt- and ANG II-dependent hypertension that involves both brain EO and circulating EO and links the kidneys,
brain, and arteries (7). This led to collaboration and to the
discovery that the chronic brain pathway regulates the plasma
EO level (24).
Following the demonstration that ouabain induces hypertension (14, 60), Hamlyn, Manunta, and associates performed
another key experiment: they infused digoxin into rats; after
all, ouabain and digoxin are both cardiotonic steroids that
indistinguishably inhibit Na⫹ pumps (5, 59). Astonishingly,
digoxin did not induce hypertension; in fact, it normalized
Perspectives
H636
ENDOGENOUS OUABAIN: WHY THE MYSTERY?
High Salt
RAAS
Fast, Acute
Pathway
Heart Failure
ANG II
(Brainstem)
Slow, Chronic
Pathway
(Hypothalamus)
(AT1R)
Aldosterone
Brain
(MR)
Modulation
EO
Adrenals
Sympathetic
Activity
Plasma EO
Arteries and Heart
Fig. 1. Diagram of the proposed fast (acute) and slow (chronic) brain pathways
that regulate the cardiovascular system. They are located in, respectively, the
brainstem (rostroventrolateral medulla) and hypothalamus. Both pathways are
activated by excess dietary salt and/or increased angiotensin II (ANG II). The
peripheral renin-angiotensin-aldosterone system (RAAS) is activated in heart
failure; the increased circulating ANG II stimulates both pathways. High salt
suppresses the peripheral RAAS but paradoxically activates the brain RAAS
(see text for references). Activation of the slow brain pathway by either
circulating or brain ANG II modulates the acute pathway and increases
circulating endogenous ouabain (EO) by as yet unknown mechanisms. The
effects of circulating EO on the arteries and heart are diagrammed in Fig. 2.
AT1R, losartan-blockable ANG II type 1 receptors; MR, eplerenone-blockable
mineralocorticoid receptors; ENaC, benzamil-blockable epithelial Na⫹ channels. “Brain EO” is locally synthesized and secreted; most circulating EO
apparently comes from the adrenals. See text for further details.
blood pressure in ouabain-infused rats. In direct contradiction
to more than 60 years of pharmacological dogma, digoxin
could also behave as an “ouabain antagonist.” These amazing
observations appeared as an abstract in 1993 (38); the full
paper wasn’t published until 2000 (37), largely because of
reviewer skepticism, but the results have been confirmed and
extended to salt-sensitive hypertension (26, 63). These results
were indisputable, but I didn’t understand how digoxin and
ouabain could have different effects, so I long ignored the
digoxin data (55).
The Na⫹ pump catalytic subunit-␣, which contains the
ouabain binding site, has four isoforms. All cells express
pumps (␣␤-dimers) with an ␣1-isoform and pumps with one
other ␣-isoform (5, 33); cardiac and smooth muscles express
␣1 and ␣2 Na⫹ pumps, but the large majority, ⬃75– 80%, are
␣1 (13, 53, 61). Rodent ␣1 pumps are notoriously resistant to
ouabain (EC50 ⬇ 100 ␮M) (5, 41); they cannot be the physiological receptors for EO because plasma EO levels are normally subnanomolar (17, 36, 50). Accordingly, Lingrel and colleagues (12, 13) generated mice with mutant, ouabain-resistant, ␣2
Na⫹ pumps. These ␣R/R
mice do not develop hypertension when
2
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ENaC
injected chronically with ouabain or adrenocorticotropic hormone, whereas wild-type (ouabain sensitive) ␣2 mice do (12,
13, 34). Mice in which ␣2 Na⫹ pumps are selectively knocked
out in the cardiovascular system are also resistant to adrenocorticotropic hormone -induced hypertension (50). Additionally, recent studies indicate that either cardiac-specific overexpression (8) or knockout (51) of ␣2 Na⫹ pumps attenuates the
development of pressure overload-induced cardiac hypertrophy and dysfunction. Clearly, the ␣2 Na⫹ pump and its endogenous ligand play a crucial role in at least some forms of
hypertension and heart failure: additional remarkable, but
largely ignored, data that are complementary to, but independent of, EO measurements.
In 2010, Golovina and colleagues (48) reported that expression of several Ca2⫹ transporter proteins is markedly increased
in arterial smooth muscle (ASM) from rats with ouabaininduced hypertension. The proteins include Na⫹/Ca2⫹ exchanger-1 (NCX1); transient receptor potential cation channel,
subfamily C, member 6 (TRPC6, component of some receptoroperated channels, ROCs); and sarcoplasmic reticulum Ca2⫹
ATPase pump-2 (SERCA2). Furthermore, they and others
found that NCX1 and several other Ca2⫹ transporters are
overexpressed in ASM in many common hypertension models
[reviewed in Blaustein et al. (7) and Pulina et al. (47)]. These
proteins are also upregulated in primary cultured normal rodent
and human ASM cells incubated with nanomolar ouabain for
72–96 h (32, 48). In contrast, digoxin does not upregulate these
proteins either in vivo or in vitro; in fact, digoxin antagonizes
the effects of ouabain (Fig. 2) (63), consistent with the aforementioned blood pressure data (26, 37, 38). Ouabain-digoxin
antagonism could no longer be overlooked simply because it
did not fit the dogma that all cardiotonic steroids act only as
Na⫹ pump inhibitors (5, 56). Also, another key discovery
about ouabain that is inconsistent with this conventional wisdom could no longer be disregarded; namely, reports from
Askari and colleagues (39, 45) that ouabain binding to Na⫹
pumps activates a C-Src-dependent protein kinase signaling
cascade. In ASM, both in vivo and in vitro, this cascade is
activated by ouabain, an effect antagonized by digoxin (Fig. 2)
(63). This verifies ouabain-digoxin antagonism [and see Song
et al. (55)] and implies that activation of the cascade and the
protein upregulation do not depend on Na⫹ pump inhibition
and NCX-mediated enhancement of Ca2⫹ signaling. Thus the
Na⫹ pump is both an ion transporter and a hormone receptor
(Fig. 2). The binding of EO and digoxin have similar shortterm, but different long-term, effects.
In heart failure, the RAAS is activated (16, 29, 62) and ANG
II-stimulated, EO-dependent mechanisms (Figs. 1 and 2) may
also contribute to cardiac remodeling. In the heart, ouabain
stimulates extracellular matrix formation (27, 49) and activates
C-Src (39), and NCX1 overexpression is a common, but
unexplained, feature of heart failure that, paradoxically, may
impair cardiac contractility (42, 58).
The several aforementioned independent and seminal observations, together, reveal a new axis that links all these factors
directly to hypertension (7) and heart failure (see above). The
components include (Figs. 1 and 2) a stimulus (ANG II and/or
high salt), the ANG II-stimulated central control system (the
brain slow neurohumoral regulatory pathway), an hormonal
messenger (EO), “biased” EO receptors (␣2 Na⫹ pumps, which
exhibit ouabain-digoxin antagonism), an EO-activated trans-
Perspectives
ENDOGENOUS OUABAIN: WHY THE MYSTERY?
A
Arteries
Plasma EO
Acute
Chronic
Digoxin
Digoxin
α2 Na+ pump
α2 Na+ pump (non-transport function)
C-Src, ERK1/2, MAPKs
[Na+]CYT
NCX1, TRPC6, SERCA2b expression
NCX1
NCX1
[Ca2+]CYT
SERCA2b
SERCA2b
[Ca2+]SR
[Ca2+]SR
Arterial
Arterial
Constriction
Constriction
Heart
Plasma EO
Acute
Chronic
Digoxin?
Digoxin?
α2 Na+ pump
α2 Na+ pump (non-transport function)
C-Src, ERK1/2, MAPKs
[Na+]CYT
NCX1 expression
NCX1
[Ca2+]CYT
[Ca2+]CYT
SERCA2a
SERCA2a
Cardiac
Contraction
[Ca2+]SR
[Ca2+]SR
Fig. 2. Proposed effects of EO on the arteries (A) and heart
(B). The well-documented acute action of EO (inhibition
of ␣2 Na⫹ pumps), but not its chronic effect (␣2 Na⫹
pump-mediated activation of the C-Src, ERK1/2, MAPK,
and protein kinase cascade), is mimicked by digoxin.
Sustained elevation of plasma EO leads, apparently via the
protein kinase cascade, to increased expression of several
arterial Ca2⫹ transporter proteins. These include Na⫹/
Ca2⫹ exchanger-1 (NCX1), transient receptor potential
cation channel, subfamily C, member 6 (TRPC6; component of receptor-operated channels), and the sarcoplasmic
reticulum (SR) Ca2⫹ ATPase pump-2b (SERCA2b). These
chronic effects of EO are blocked by digoxin (26, 37, 63),
as indicated; digoxin may either mimic (not shown) or
antagonize the acute effect of EO, depending upon the
relative concentrations (55). (Note: ouabain-digoxin antagonism has been demonstrated in arteries and neurons, but
not yet in heart, as indicated by the question marks in B.)
Chronically elevated plasma EO may also increase NCX1
expression in the heart by a C-Src-activated protein kinase
cascade (39, 45). Acute Na⫹ pump inhibition by EO raises
the cytosolic Na⫹ concentration ([Na⫹]CYT). This reduction in the Na⫹ electrochemical gradient, in turn, promotes
net Ca2⫹ gain via NCX1, and a rise in both cytosolic and
SR calcium concentrations, [Ca2⫹]CYT and [Ca2⫹]SR, respectively, and, thus enhances Ca2⫹ signaling and contraction in both the heart and arteries [acute (green) pathways
in A and B]. With increased NCX1 expression due to
chronically elevated plasma EO, however, the heart and
arteries apparently respond differently. The main role of
NCX1 in the heart is to mediate Ca2⫹ extrusion and
promote relaxation during diastole (3), whereas in arteries,
its main role seems to be to mediate Ca2⫹ entry and
maintain tone (61). Thus NCX1 upregulation in the heart
should decrease contractility (B, chronic pathway), but in
arteries it should increase contractility (A, chronic pathway). In other words, the acute and chronic effects of EO
should be synergistic in arteries but antagonistic to one
another in the heart. See text for further details.
Cardiac
Contraction
ducer (protein kinase cascade), protein kinase-modulated peripheral signal mechanisms (Ca2⫹ transporters and channels,
e.g., NCX1, ROCs), a second messenger (Ca2⫹), and Ca2⫹activated effectors (arterial and cardiac contractile apparatus).
Because NCX1 promotes Ca2⫹ exit in heart (3) but Ca2⫹ entry
in arteries (61), chronically elevated EO, which is pivotal, and
upregulated NCX1 should make hearts hypocontractile and
arteries hypercontractile (Fig. 2). Thus EO should promote
both heart failure and hypertension. This is, of course, easy to
see with hindsight but was certainly not anticipated when EO
was discovered.
These considerations should provide novel insight into
therapeutics. For example, plasma EO was not measured in
the most widely cited, long-term trial of the therapeutic
effectiveness of digoxin in heart failure (10). Yet, ouabaindigoxin antagonism implies that patients with low- and
high-ambient EO are likely to have different outcomes when
treated with digoxin. Thus a golden opportunity to predict
who might benefit most from digoxin, the primary goal of
the trial, was lost. Furthermore, it is now difficult to dismiss
the striking correlation between the highest plasma EO
levels and the worst morbidity and mortality statistics in
patients with heart failure and related cardiovascular diseases (4, 21, 46, 54, 57). Surely, it is time to bring EO, a key
neuroendocrine and cardiovascular hormone, in from the
cold.
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[Ca2+]CYT
B
H637
Perspectives
H638
ENDOGENOUS OUABAIN: WHY THE MYSTERY?
ACKNOWLEDGMENTS
I thank J. M. Hamlyn, F. H. H. Leenen, and W. G. Wier for helpful critiques
of a preliminary version of this manuscript and H. Song for help with the
figures.
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grants R01-HL-045215 (to M. P. Blaustein and J. M. Hamlyn) and R01-HL107555 (to M. P. Blaustein).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
M.P.B. conceived and designed research; analyzed data; interpreted results
of experiments; prepared figures; and drafted, edited, revised, and approved
final version of manuscript.
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