HYPOTHESIS
No evidence for a role in signal-transduction of Na
+
/K
+
-ATPase
interaction with putative endogenous ouabain
Otto Hansen
Department of Physiology, Aarhus University, DK-8000 A
˚
rhus C, Denmark
A cascade of events (signal-transduction), mainly seen in rat
cardiac myocytes and renal cells, is thought to occur after
ouabain interaction with a minor fraction of Na
+
/K
+
-
ATPase. A higher intracellular Na
+
concentration followed
sodium pump inhibition by ouabain with a subsequent
gradual increase or oscillations in intracellular Ca
2+
con-
centration. Whether this increase in intracellular Ca
2+
concentration is part of the cascade, a result of the cascade or
a totally independent phenomenon are conflicting interpre-
tations that are discussed. At best, however, the cascade is
initiated by ouabain concentrations several orders of mag-

+
-
ATPase; ouabain-like factors; OLF; ouabain-insensitive;
signal-transduction; sodium pumps.
Introduction
Ouabain and other cardiac glycosides have been known as
specific inhibitors of the sodium pump and the purified
Na
+
/K
+
-ATPase ever since the very first observation of
inhibition of the sodium pump of red cells [1] and inhibition
of the isolated Na
+
/K
+
-ATPase from crab nerves [2].
Ouabain inhibition of both phenomena was in fact one of
the important criteria for the identity between the sodium
pump and the purified, though still membrane-embedded
Na
+
/K
+
-ATPase [3]. Before the inhibitory mechanism had
been appreciated, the pharmacological action of cardiac
glycosides in congestive heart failure had been known for
centuries. The amazing interaction of a plant-derived drug
with a membrane receptor of animal cells led to speculations

-ATPase traditionally associated
with the pharmacological action of cardiac glycosides.
Based mainly upon experiments with renal cells [8] and
ventricular myocytes [9–11], in both cases isolated from the
rat, ouabain was suggested to have a hormonal role in signal
transduction in addition to the traditional role of Ca
2+
accumulation due to pump inhibition, a reduced Na
+
gradient and reduced Na
+
/Ca
2+
exchange resulting in a
positive inotropic effect in susceptible tissues (for references
see [12]). Generation of the cascade of secondary events was
associated with transient oscillations [8] or a more gradual,
in some cases independent [10] increase in intracellular Ca
2+
concentration [9–11]. The original observations were
accompanied by an enthusiastic commentary in Nature
Medicine [13] and a minireview in Eur. J. Biochem. [14].
These authors [13,14] added a more comprehensive and
Correspondence to Otto Hansen, Department of Physiology,
Aarhus University, Ole Worms Alle
´
160, DK-8000 A
˚
rhus C,
Denmark. Fax: + 45 86129065, Tel.: + 45 89422806,

and the supposed physiological implication [13,14]. First is
the questionable existence of OLF that at best is at an
insignificantly low plasma concentration compared to the
huge pool of sodium pumps in the mammalian body. Of
equal importance is the often ignored fact, that the rat,
contrary to most other mammalian species, contains an
almost ouabain-insensitive isoform of Na
+
/K
+
-ATPase.
In most tissues, the Na
+
/K
+
-ATPase is a heterodimer
consisting of a 112 kDa hydrolytic a-peptide, through
which the vectorial transport of Na
+
and K
+
takes place,
and the small a-glycoprotein (35 kDa for the protein) with
chaperone-like function. Four isoforms of the a-peptide
have been described (one in testes). In most mammalian
species, Na
+
/K
+
-ATPase, irrespective of isoform compo-

+
-ATPase with neighbouring proteins (Src,
growth factor receptors and Ras), i.e. by protein–protein
interaction. In myocytes, the events from signal-transduc-
tion were interpreted as independent of a small increase in
[Na
+
]
i
and also occasionally independent of the subsequent
increase in [Ca
2+
]
i
[10], whereas in other publications,
activation of the Ras/MAPK cascade was interpreted as a
prerequisite for the increase in [Ca
2+
]
i
[11]. Nevertheless,
simple inhibition of pump function, by lowering the
extracellular K
+
concentration, was able to mimic ouabain
with respect to the cascade in rat cardiac myocytes [9].
In rat renal cells [8] on the other hand, ouabain
interaction with a minor fraction of the Na
+
/K

i
was seen [8]. A possible
explanation in the latter case could be that pump inhibition
is counteracted by hyperpolarization rather than membrane
depolarization due to the increased K
+
gradient according
to the Goldman constant-field equation. The Na
+
/K
+
-
ATPase is indeed electrogenic, contributing in the order of
10 mV to the membrane potential due to the stoichiometry
of (3 : 2) Na
+
/K
+
-exchange by the pump. On the other
hand, according to the pump-leak model, the membrane
potential is not established exclusively by the pump.
The mentioned, often contradictory observations and
interpretations do not add to the credibility of the
hypothesis. The main objections to a normal signal-
transducing function of ouabain interaction with Na
+
/K
+
-
ATPase, however, are the following. To place this pheno-

1
-isoform, as well as the highly ouabain-
sensitive a
2
- (skeletal and heart muscle) and a
3
-isoform
(nerve tissue and brain) are present. The exception is the
kidney where more than 99.9% of the enzyme consists of
the ouabain-insensitive a
1
-isoform [19]. This explains why
large concentrations of ouabain (50–500 l
M
) are needed to
obtain Ca
2+
-oscillations in a reasonable fraction of the rat
kidney cells [8], though oscillations were seen in a few cells
exposed to 10–100 n
M
ouabain for 3 h. As ouabain binding
to Na
+
/K
+
-ATPase is a simple second order reaction [12],
3 h incubation at 37 °C does not seem reasonable for
obtaining equilibrium as demonstrated by the statement
that Ô…ouabain binding…is expected to increase as a

normal rats as measured by ELISA. OLF were quantified in
plasma from two strains of rat by another group in
collaboration with Hamlyn [21] by means of two independ-
ent assays, a radioimmunoassay using an anti-ouabain Ig
and an enzymatic assay using ouabain-sensitive Na
+
/K
+
-
ATPase from dog kidney. The concentration range of OLF
found in plasma was 25–27 pmolÆL
)1
in normotensive and
68–76 pmolÆL
)1
in Milan hypertensive rats, i.e., concentra-
tions almost 3–6 orders of magnitude lower than employed
in experiments focusing on signal-transduction [8–11], see
Fig. 1. Higher values based solely on immunoassays have
been reported [15] (e.g., during volume expansion in dogs or
strenous exercise in man, though still much lower than the
concentrations used for signal-transduction) while others
have been unable to detect immunoreactive OLF in plasma,
adrenal glands and tissues of man at all [22]. Endogenous
ouabain was a theme at the 10th International Conference
on Na,K-ATPase and Related Cation Pumps in Elsinore,
Denmark, in August 2002. The meeting of 250 specialists on
Na
+
/K

content of ouabain-sensitive a
2
in rat cardiac muscle
[23–25]. The ratio between the a
1
-containing insensitive and
a
2
/a
3
-containing sensitive Na
+
/K
+
-ATPase in rat cardiac
tissue is not well known as only the latter can be determined
in [
3
H]ouabain binding studies. In a membrane fraction
from cardiac ventricles of adult rats, high-affinity Na
+
/K
+
-
ATPase containing a
2
seemed to represent 26% of total
activity [25]. A similar distribution could be estimated by
using isoform-specific antibodies and Western blots to
which homogenates of rat cardiac ventricles had been

ATPase [27]. An overall increase in intracellular Na
+
was
seen in the experiments with low concentration of extracel-
lular [K
+
] [8,9], that also mimicked ouabain with respect to
the cascade in cardiac myocytes [9].
Other implications of signal-transduction by ouabain
Finally, I would like to discuss that a great many studies on
rat wild-type, as well as mutant Na
+
/K
+
-ATPase, have been
carried out with mammalian cell lines transfected with
cDNA constructs encoding a ouabain-resistant a
1
-isoform
or derived ouabain-resistant a
2
-ora
3
-isoforms [28]. The
endogenous ouabain-sensitive Na
+
/K
+
-ATPase is
knocked-out by incubation of the cells in micromolar

+
-
ATPase. In both respects, convincing data fulfilling a
number of criteria for EDLF/OLF are unavailable. More-
over, experiments on rat tissue are unfeasible for the
demonstration of signal-transduction as ouabain-sensitive
a-isoforms of Na
+
/K
+
-ATPase, possibly confined to
membrane structures near cytosolic microdomains, would
be totally blocked at ouabain concentrations necessary for
limited binding to almost ouabain-insensitive a
1
-isoforms in
the rat.
References
1. Schatzmann, H J. (1953) Herzglycoside als Hemmstoffe fu
¨
rden
aktiven Kalium- und Natriumtransport durch die Erythrocyten-
membran. Helv. Physiol. Acta 11, 346–354.
2. Skou, J.C. (1960) Further investigations on a Mg
2+
+Na
+
-acti-
vated adenosinetriphosphatase, possibly related to the active,
linkedtransportofNa

Ouabain, a steroid hormone that signals with slow calcium oscil-
lations. Proc. Natl. Acad. Sci. USA 98, 13420–13424.
9. Haas, M., Askari, A. & Xie, Z. (2000) Involvement of Src and
epidermal growth factor receptor in the signal-transducing func-
tion of Na
+
/K
+
-ATPase. J. Biol. Chem. 275, 27832–27837.
10. Liu, J., Tian, J., Haas, M., Shapiro, J.I., Askari, A. & Xie, Z.
(2000) Ouabain interaction with cardiac Na
+
/K
+
-ATPase
initiates signal cascades independent of changes in intracellular
Na
+
and Ca
2+
concentrations. J. Biol. Chem. 275, 27838–
27844.
11. Tian, J., Gong, X. & Xie, Z. (2001) Signal-transducing function of
Na
+
-K
+
-ATPase is essential for ouabain’s effect on [Ca
2+
]

18. Hansen, O. (2001) The a
1
isoform of Na
+
,K
+
-ATPase in rat
soleus and extensor digitorum longus. Acta Physiol. Scand. 173,
335–341.
19. Lu
¨
cking, K., Nielsen, J.M., Pedersen, P.A. & Jørgensen, P.L.
(1996) Na-K-ATPase isoform (a
3
, a
2
, a
1
) abundance in rat kidney
estimated by competitive RT-PCR and ouabain binding. Am. J.
Physiol. 271, F253–F260.
20. Schoner, W. (2002) Endogenous cardiac glycosides, a new class of
steroid hormones. Eur. J. Biochem. 269, 2440–2448.
21. Ferrandi, M., Manunta, P., Balzan, S., Hamlyn, J.M., Bianchi, G.
& Ferrari, P. (1997) Ouabain-like factor quantification in mam-
malian tissues and plasma. Comparison of two independent
assays. Hypertension 30, 886–896.
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authentic endogenous mammalian substance derived from the
adrenal? Hypertension 23, 632–638.