At best, however, the cascade is initiated by ouabain concentrations several orders of mag-nitude higher than the measured plasma concentrations of putative endogenous ouabain.. The almo
Trang 1H Y P O T H E S I S
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 Ca2+
con-centration Whether this increase in intracellular Ca2+
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-nitude higher than the measured plasma concentrations of
putative endogenous ouabain The experimentally high
ouabain concentration may be critical for another reason
Most tissues contain various isoforms of the catalytic a-peptide of Na+/K+-ATPase with an individual sublocali-zation and, in rats, with different ouabain-sensitivity The almost ouabain-insensitive a1-isoform of Na+/K+-ATPase
is essentially unaffected by the high ouabain concentration, whereas ouabain-sensitive a-isoforms, possibly confined to membrane structures near cytosolic microdomains and
Na+/Ca2+exchangers, may be totally blocked Classifying endogenous ouabain as a physiological inducer of the sign-aling system on this background seems hazardous Keywords: a isoforms; endogenous ouabain; Na+/K+ -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 crabnerves [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
of endogenous mammalian digitalis-like factors (EDLF,
reviewed in [4]) The occurence of a ouabain isomer (often
called ouabain-like factor, OLF) or even ouabain itself in
normal plasma seemed to support these claims [5]
Goto et al [4] pointed out that a number of criteria should be fulfilled for substances to be considered as candidates for EDLF Inhibition of Na+/K+-ATPase, as exhibited by, e.g., unsaturated fatty acids [6] and lyso-phospholipids, does not suffice Neither does EDLF immunoreactivity, as many steroids, lipids and bile acids cross-react with antibodies to cardiac glycosides [4] Hansen [7] emphasized the vast pool of Na+/K+-ATPase in skeletal muscles, and thus receptors for EDLF/OLF, compared to the measured low secretion rate of OLF from the adreno-cortical gland
A novel role of ouabain in signal-transduction Recently, however, a novel physiological role was attributed
to ouabain and thus to endogenous ouabain in addition to the inhibition of Na+/K+-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 Ca2+ accumulation due to pump inhibition, a reduced Na+ gradient and reduced Na+/Ca2+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 Ca2+ 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,
E-mail: oh@fi.au.dk
Abbreviations: EDLF, endogenous digitalis-like factor; ER,
endo-plasmic reticulum; MAPK, mitogen-activated protein kinase; NF-jB,
nuclear factor kappa B; OLF, ouabain-like factor, a ouabain-like
isomer; ROS, reactive oxygen species; SR, sarcoplasmic reticulum.
(Received 16 January 2003, revised 26 February 2003,
accepted 4 March 2003)
Eur J Biochem 270, 1916–1919 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03554.x
Trang 2hypothetical cascade of events initiated by ouabain
occu-pancy of a small fraction of the sodium pumps One of them
had advocated the role of EDLF for years [15]
Possible pitfalls for a novel role of cardiac glycosides
It is generally accepted that the sodium pump and the
external aspect of the purified, though still
membrane-embedded, Na+/K+-ATPase is the specific receptor for
cardiac glycosides, that the binding can be described as a
simple bimolecular (second order) reaction with a
stoichio-metry of 1 : 1 [12] and that identified amino acid residues
of the external loops of the hydrolytic a-peptide are involved
in ouabain sensitivity [16] Caution is needed, however,
before classifying OLF or even ouabain as normal steroid
hormones functioning in signal-transduction pathways
under physiological conditions The described individual
steps [8–11], that may be part of a cascade of events, are not
questioned under the conditions of the experiments but
serious flaws and failures may invalidate the interpretation
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-sition, has a very high affinity to the specific inhibitor,
ouabain This is true except for a few rodent species (mainly
the rat) in which pumps containing the a1-isoform have an
extremely low affinity for cardiac glycosides Most rat
tissues moreover have a mixture of ouabain-insensitive and
ouabain-sensitive isoforms, of which the latter may have a
sublocalization critical for the observations looking at
ouabain as a signal-transducer
Experiments pointing to a pivotal role of ouabain
in signal-transduction
In the following, more details are discussed concerning the
experiments with ouabain that led to the explicit
formula-tion of a cascade of events Based mainly on observaformula-tions
with rat myocytes, additional roles in signal-transducing
function of ouabain’s interaction with Na+/K+-ATPase
were attributed to tyrosine protein kinase, Src, epidermal
growth factor receptor and the Ras/MAPK
(mitogen-activated protein kinase)-dependent cascade with NF-jB
activation and generation of reactive oxygen species (ROS)
[9–11] In each case, the cascade is hypothesized to be
elicited by the interaction of a minor fraction of
ouabain-altered Na+/K+-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+] and also occasionally independent of the subsequent
increase in [Ca2+]i [10], whereas in other publications, activation of the Ras/MAPK cascade was interpreted as a prerequisite for the increase in [Ca2+]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+-ATPase gave rise to the release of Ca2+from endoplasmic reticulum (ER) and Ca2+influx via voltage-gated and calcium release-activated calcium channels in the plasma membrane The resulting slow-vawe [Ca2+]ioscillations in turn were inter-preted as activating the transcription factor, NF-jB that, after translocation to the nucleus, interacted with target genes initiating growth and differentiation Lowering of the extracellular [K+] did not mimic ouabain in renal epithelial cells as [Ca2+]idid not change with low extracellular [K+], though an increase in [Na+]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-menon into a normal physiological framework, the authors [8–11] (a) more or less ignore the presence of a-peptide isoforms of Na+/K+-ATPase with different ouabain affinities in rats; (b) ignore the extremely high concentra-tions of ouabain applied and (c) assume a similar role by putative endogenous ouabain or OLF
Ouabain-sensitive and -insensitive a-isoforms
of Na+/K+-ATPase in rats All isoforms of the catalytic a-peptide of Na+/K+-ATPase have similar, high affinity for ouabain [17] with Kdin the order of one or a few nanomolar [12,17] An exception from this general rule is the almost ouabain-insensitive a1-isoform seen in rats ([for references, see [18]) In most tissues in the rat, however, this a1-isoform, as well as the highly ouabain-sensitive a2- (skeletal and heart muscle) and a3-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 a1-isoform [19] This explains why large concentrations of ouabain (50–500 lM) are needed to obtain Ca2+-oscillations in a reasonable fraction of the rat kidney cells [8], though oscillations were seen in a few cells exposed to 10–100 nMouabain for 3 h As ouabain binding
to Na+/K+-ATPase is a simple second order reaction [12],
3 h incubation at 37C does not seem reasonable for obtaining equilibrium as demonstrated by the statement that …ouabain binding…is expected to increase as a function of time [8] However, irrespective of the lower or higher concentration range employed, they are both far
FEBS 2003 Endogenous ouabain in normal signal-transduction? (Eur J Biochem 270) 1917
Trang 3beyond the reported plasma concentration of the putative
steroid hormone ouabain or OLF (Fig 1) In this context,
we are even ignoring the fact that Na+/K+-ATPase
containing the ouabain-sensitive a2- and a3-isoforms
else-where in the rat would have been totally blocked at
micromolar or submicromolar concentrations of ouabain
Plasma concentrations of putative endogenous ouabain
In another recent minireview in Eur J Biochem [20]
endogenous ouabain was claimed to be a novel steroid
hormone and the adrenal gland the major place of synthesis
of ouabain However, it is still much debated whether
ouabain or OLF really exist in mammalian blood and
tissues, and if they do, whether low level production from,
e.g the adrenal glands would suffice [7,21] In the original
publication by Hamlyn et al [5] on OLF
…indistinguish-able from the cardenolide ouabain… they stated an
immu-noreactive plasma concentration of 80 ± 18 pmolÆL)1in
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)1in normotensive and
68–76 pmolÆL)1in 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+-ATPase and related topics didn’t lend much support to the existence of endogenous ouabain and OLF Assigning ouabain a novel physiological role given this background seems hazardous
Distribution of a-isoforms of Na+/K+-ATPase
As to the observations at 100 lM ouabain with rat ventricular myocytes (100 nMwith HeLa cells) containing
a1- as well as a2-isoforms of Na+/K+-ATPase the explan-ation for the apparent signal-tranduction may differ [9–11]
In one of the papers [9] it is mentioned that the rat
a1-isoform is less sensitive to ouabain but not the significant content of ouabain-sensitive a2 in rat cardiac muscle [23–25] The ratio between the a1-containing insensitive and
a2/a3-containing sensitive Na+/K+-ATPase in rat cardiac tissue is not well known as only the latter can be determined
in [3H]ouabain binding studies In a membrane fraction from cardiac ventricles of adult rats, high-affinity Na+/K+ -ATPase containing a2 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 applied [26] The a2-population of pumps should be totally blocked by the 100 lMouabain used in experiments with rat ventricular myocytes [9–11] Due to the preferential locali-zation of the ouabain-sensitive pumps to membrane struc-tures overlying junctional SR/ER [27], an alternative explanation to an apparent signal-transduction role could
be a local rise in [Na+]iresulting in reduced Na+/Ca2+ -exchange locally Cellular compartments with larger chan-ges in intracellular Na+and Ca2+may thus be the result of
a ouabain interaction with subpopulations of Na+/K+ -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 a1-isoform
or derived ouabain-resistant a2- or a3-isoforms [28] The endogenous ouabain-sensitive Na+/K+-ATPase is knocked-out by incubation of the cells in micromolar concentrations of ouabain [16] Were signal-transduction
of significance in this situation, the phenomenon should be expected in those cells and the results interpreted accordingly
Fig 1 Ouabain affinities to rat Na+/K+-ATPase isoforms and actual
ouabain or OLF concentrations The figure summarizes the huge
vari-ation in ouabain affinities of Na + /K + -ATPase depending on species
and a-isoforms, actual concentrations of OLF in rat plasma, actual
concentrations of ouabain employed in experiments with renal cells
and ventricular myocytes of rats, and the Na + /K + -pump density in rat
skeletal muscle.
Trang 4Giving ouabain a novel physiological role in
signal-trans-duction appears erroneous unless putative endogenous
ouabain is produced and present in plasma at reasonable
concentrations compared to the affinities with Na+/K+
-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 a1-isoforms in
the rat
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