I
CUN. CHEM. 40/9, 1674-1685
(1994)
Understanding the Sodium Pump and Its Relevance to Disease
Andrea
M. Rose’
and Roland Valdes, Jr.”
Na,K-ATPase (sodium pump; EC 3.6.1.37) is present in
the membrane of most eukaryotic cells and controls
directly or indirectly many essential cellular functions.
Regulation of this enzyme (ion transporter) and its individual isoforms is believed to play a key role in the
etiology of some pathological processes. The sodium
pump is the only known receptor for the cardiac glycosides. However, endogenous ligands structurally similar
to digoxin or ouabain may control the activity of this
important molecular complex. Here we review the structure and function of Na,K-ATPase,
its expression and
distribution in tissues, and its interaction with known
ligands such as the cardiac glycosides and other suspected endogenous
regulators. Also reviewed are various disorders, including cardiovascular, neurological,
renal, and metabolic diseases, purported to involve dysfunction of Na,K-ATPase activity. The escalation in
knowledge at the molecular level concerning sodium
pump function foreshadows application of this knowledge in the clinical laboratory to identify individuals at
risk for Na,K-ATPase-associated
diseases.
IndexIng Terms: Na,K-ATPase/isoforms/cardiac
glycosides/ age-
related effects/digoxin/ouabain/hypertenslon/diabetes/Alzheimer
disease/neurological disorders
The sodium-potassium-activated
adenosine triphosphatase (Na,K-ATPase;
sodium
pump; EC 3.6.1.37) is a
plasma membrane-associated
protein complex that is expressed in most eukaryotic
cells.4’5 The “pump” couples
the energy
released
in the intracellular
hydrolysis
of
adenosine
tnphosphate
(ATP) to the transport
of cellular
ions, a major pathway for the controlled translocation
of
sodium and potassium
ions across the cell membrane.
Na,K-ATPase
therefore controls directly or indirectly
many essential cellular functions, e.g., cell volume, free
calcium concentration, and membrane potential. RegulaDepartments
of ‘ Pathology and 2Biochemistry, University of
Louisville School of Medicine, Louisville, KY 40292.
‘Mdress
correspondence to this author at: Department of Pathology, University of Louisville, Louisville, KY 40292. Fax 502852-1771; E-mail [email protected].
4Nonstandard
abbreviations: Na,K-ATPase, Na,K-activated
adenosine triphosphatase;
DLIF, digoxin-like immunoreactive factors; EDLF, endogenous digoxin-like factors; OLF, ouabain-like
factors; and AD, Alzheimer disease.
5Although technically an enzyme, Na,K-ATPase
functions primarily as an ion transporter. Therefore, most investigators
refer to
the alpha and beta gene products as isoforms instead of isoenzymes.
Received December 27, 1993; accepted June 16, 1994.
1674 CLINICAL CHEMISTRY, Vol. 40, No. 9,
1994
tion of this enzyme (transporter) and its individual isoforms is thought to play a key role in the etiology of some
pathological processes.
The sodium pump is the only known receptor
for the
cardiac glycosides used to treat congestive heart failure
and cardiac arrhythmias.
This suggests
that endogenous ligands structurally
similar to cardiac glycosides
may act as natural regulators
of the sodium pump in
heart and other tissues.
Identification
of naturally
cccurring regulators
of Na,K-ATPase
could initiate
the
discovery of new hormone-like
control systems involved
in the etiology of selected disease processes, hence the
importance
of understanding
the relation of the sodium
pump and its ligands to disease. In this article, we review recent information
related
to structure
and function, genetic expression
and distribution
in tissues, and
interaction
of the sodium pump with ligands such as the
cardiac
glycosides
and other suspected
endogenous
counterparts.
Several diverse disease processes-including cardiovascular,
renal, neurological,
and metabolic
disorders, having in common a dysfunction
in salt and
water homeostasis-are
emphasized, as is the clinical
need for understanding
the function
and control of this
ubiquitous
ion transporter
system.
Structure and FunctIon of Na,K-ATPase
Na,K-ATPase
couples the energy released
in the intracellular
hydrolysis
of ATP to the export of three intracellular Na ions and the import of two extracellular
K ions. The continuous operation of this macromolecWar machine ensures the generation and maintenance
of concentration
gradients
of Na and K across the cell
membrane.
This electrochemical
gradient
provides energy for the membrane
transport
of metabolites
and
nutrients, e.g., glucose and amino acids, and such ions
as protons, calcium, chloride,
and phosphate. The electrochemical
gradient
is essential also for regulation of
cell volume and for the action potential of muscle and
nerve. The relative
intra- and extracellular
concentrations of Na and K ions maintained
primarily by the
sodium pump and the cofactors required for activity are
shown in Fig. 1 (1, 2).
The functional macromolecule is a membrane-spanning 270-kDa tetramer
consisting of two dimers, each
composedof noncovalently
interacting alpha (112 kDa)
and beta (55 kDa) subunits. A model of the transporter
complex relative to its membrane location is shown in
Fig. 2. The presence of a smaller gamma subunit (10
A
OUT
(150 mmol/L)
Digitalis
(5 mmol/L)
Y1016
‘)63K327
IN
Fig. 1. SchematIc diagram of the Na,K-ATPase-associated cofactors
and Ions.
Modifiedfrom Akera (2).
Extracellular
1263
R166
0737
Y1018
IN
Intracellular
Fig. 3. Foldingmodels for the Na,K-ATPase alpha transmembrane
segments: (A) 10 membrane-spanning segments; (B) 8 membranespanning segments.
a
a
Fig.2. Schematic diagram of the plasma membrane-spanning Na,KATPase transporter complex, indicating the positions of the alpha,
beta, and putative gamma subunits.
kDa) has been suggested;
however, its role, if any, has
not been defined (3).
The alpha (catalytic) subunit, is proposed to have 7(4)
or 8(5) transmembrane
domains; however, the number
can vary from 6 to 10, based on interpretation
of hydropathy profile data (6) and identification
of specific ligand-receptor
interactions
that predict alpha chain topology (7-9). The alpha subunit contains all the binding
sites for ligands
known to stimulate or inhibit the enzyme (10-13).
Tentative
models representing
membrane-spanning
segments
of the alpha subunit are de-
Letters refer to the one-letter amino acid code. Numbers represent the topological location of particular amino adds. From Kailish et at. (7); used with
permission.
cyclic reaction in which the enzyme is phosphorylated
by ATP in the presence of Mg2 ions and Na ions and
then dephosphorylated
in the presence of K ions. A
model depicting this thermodynamic
cycle is shown in
Fig. 4.
Isoform Regulation and Genetic Expression
With the advent of molecular
biological techniques,
three alpha and four beta isoforms of the Na,K-ATPase
have been identified, which are encoded by independent
genes (20-22). The sequence conservation
among differ3Na
2l(
tailed in Fig. 3.
The beta subunit
has a single hydrophobic
transmem-
brane domain and is highly glycosylated
on its noncytosolic surface (14). Hiatt et al. postulate that the beta
may serve to orient and stabilize
the alpha
subunit in the membrane
(15). Cellular
expression
of
the beta subunits and assembly with the alpha subunits
are necessary for correct conformation
and activity of
the Na,K-ATPase
holoenzyme
(16). Although the role of
the beta subunit
in ion translocation
is uncertain,
its
presence
appears
essential
for function
of the sodium
pump (16).
Phosphorylation
is an important step in the function
of Na,K-ATPase.
The molecule
undergoes an alpha-helix to beta-sheet transition between two principal reactive states, E1 and E2, in the multistep
reaction by
which Na ions and K ions traverse
the membrane
(17, 18). The conformational
transition
results via a
subunit
.p
E2-P.Na3
I
Ei-P.Naa1,...
r
E2P.K2
AlP
Nat. Mi2
E1
ATP
______________________
E2-K2
.
2K
Fig. 4. Principal reactive states (E1 and E) involved in the transport
of sodium (Na) and potassium (K) ions across the cytoplasmic
membrane.
The enzyme (E) Is phosphorylated(P), with ATP as thephosphatedonor and
Mg2 as a cofactor In the reaction. From MacGregor and Walker (19), as
modified from Sen et at. (18); used with permission.
CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994
1675
ent species suggests that individual
roles for the isoforms, though not yet determined,
arose early and were
maintained
throughout evolution.
The three alpha isoforms (alpha 1, 2, and 3) are expressed in a developmental
and tissue-specific
manner.
Utilizing monoclonal antibodies that recognize the inchvidual alpha isoforms, Lucchesi and Sweadner
have
shown (23) that rat ventricular muscle-membrane
preparations express alpha 1 in all stages of development;
alpha 3 is present at birth through days 14 to 21, and is
then replaced by alpha 2 in the adult rat; alpha 3 again
predominates
in aged rats. The physiological
significance of this shift in subunit isoform expression is unknown. Tissue specificity for the different isoforms has
been identified both at the mRNA and protein level for
various species (22, 24), and is summarized
for isoforms
from rat and human tissues in Table 1. Table 1, although not comprehensive,
serves to illustrate the diversity of isoform distribution
in tissues. The vast majority of studies defining isoform distribution have been
done with animals, less data being available for human
tissues. Nevertheless,
studies in both humans and rats
suggest that alpha 1 is the only isoform expressed appreciably in the kidney (33), whereas
alpha 3 is associated primarily
with the nervous system (22, 28, 29).
Isoform specificity even extends to cell type within a
particular tissue, as evidenced by studies with tissue
from brain (34), heart (35), and eye (30), supporting the
hypothesis that the isoforms have different physiological functions (36, 37).
Three Na,K-ATPase
beta isoforms and one related
H,K-ATPase
(another member of the cation-transporting ATPases) beta isoform have been identified. Beta 1
has been isolated from several vertebrate species in a
wide range of tissues. Beta 2 is expressed largely in
brain (26,27) and ocular ciliary epithelia (38); however,
recent studies suggest that beta 2 is also expressed in
glycolytic fast-twitch muscles of the rat hindlimb
(32).
Table 1. Tissue dIstribution of Na,K-ATPase Isoforms.
Tissu. source
Species
Adipose
Rat
Brain
Rat
Human
Human
Human
Human
Human
Eye
Heart (ventricle)
Rat
Rat
Rat
Kidney
Lung
Skeletal muscle
Human
Human
Human
Rat
Thyroid
Uterus
Human
Human
References
Subunit
Regulators of Sodium Pump Activity
Cardiac Glycosides
al, a2’
f32#{176}
al a2, a
25
26, 27
The Na,K-ATPase
alpha subunit
receptor for the cardiac glycosides,
a3b
29
30
the clinical significance
congestive
heart failure
31
and Walker have written a short review of the cardiac
glycosides (19). These inhibitors of the sodium pump are
derived from extracts of the plant genera Digitalis, Strophanthus,
and Acocanthera.
Digoxin and digitoxin
are
products of species of the foxglove plant, Digitalis,
and
ouabain is obtained from the East African Ouabaio tree
or seeds of the plant Strophanthus
gratus (52,53). These
compounds are the most potent inotropic agents known,
and their cardiac effects are believed to be mediated
through their ability to inhibit the sodium pump. Historically,
preparations
of these substances
have been
used therapeutically
for perhaps 3000 years, including
use of plant extracts containing
cardiac glycosides by
the ancient Egyptians
(54).
al, a2, a3ab
al, a2, a3,
a
al, a2, a3a
al, a3 neonate#{176}
al, a2 adult#{176}
al, a3 aged#{176}
ala
ala
al,
a1,a2,l,i32a
al, a2
ala
28
23
23
23
28
28
28
32
28
28
Superscripts referto all the preceding isoforms onthe same line: a isoformspecific expression of mRNA identified byNorthernanalysis;b isoform-specific
expressionof proteinidentifiedby Westernanalysis.
1676
Interestingly,
the beta 2 isoform appears to serve a dual
function, both as a subunit of the Na,K-ATPase
and as
a mediator of neuron-astrocyte
adhesion (39). This suggests the possibility
that these proteins, as a family,
may play other roles besides their traditionally
defined
transport
function. Finally, a protein referred to as beta
3 has been isolated from Xenopus (40), and the betaisoform of the H,K-ATPase
has been characterized
from
several vertebrate
species (41-43).
The beta isoforms
are less similar to each other in amino acid sequence
than are their alpha isoform counterparts to each other;
the beta isoforms also vary in their number of asparagine-linked glycosylation sites (27).
The expression of all the alpha isoforms and beta 1 are
differentially
regulated by hormones (44). Horowitz et
al. (45) determined that thyroid status affected the alpha 1, alpha 2, and beta isoform-specific
expression of
mRNA and protein in rat heart, skeletal muscle, and
kidney; and Gick and Ismail-Beigi (46) found that incubation of a rat liver cell line with thyroid
hormone
resulted in an increase in alpha 1 and beta mRNA
expression
and Na,K-ATPase
activity.
Increased
sodium concentration
in response to corticosteroids
is reported to recruit an intracellular
latent pool of Na,KATPase complexes to the cell membrane in the cortical
collecting tubules of rat (47) and rabbit (48) kidney.
Lingrel
et al. (49), examining
the 5’-flanking sequences
of the human alpha isoform genes, found that each contains a number of potential transacting
and hormonebinding sites that do not appear to be conserved among
the three alpha isoform genes, thus allowing for differential regulation.
The ability to detect nucleotide changes that result in
restriction
fragment
length
polymorphisms
has led to
the discovery of sequence variation in some families in
the human alpha (50) and beta (51) subunit genes.
Whether these genotypic differences, or others not yet
identified, correlate
with a particular pathology is at
present undetermined.
CLINICAL CHEMISTRY, Vol. 40, No. 9,
1994
is the only known
which underscores
of the pump
in treatment
of
MacGregor
and arrhythmias.
Digoxin,
which can be administered
orally
and is
readily absorbed
by the gastrointestinal
tract, is the
most widely used cardiac glycoside clinically; ouabain is
the most widely used experimentally.
These compounds,
all cardenolides,
have a sterol skeleton.
Cardenolides
are C steroids having one or more sugar residues at
C-3 and a flve-membered
lactone ring at C-17 (see Fig.
5). As previously described, cardiac glycoside binding to
Na,K-ATPase
occurs on the extracellular
face of the
integral
membrane protein. However, recent data suggest that a hydrophobic
binding pocket that contains
membrane-spanning
amino acids may be involved as
well (55). Alpha subunit amino acids important
for cardiac glycoside binding
have been determined
(6, 56),
and recent studies
with site-directed
mutagenesis
to
make amino acid substitutions
at proposed binding sites
have helped determine
which regions recognize cardiac
glycoside sugars (57). A first-order-approximation
binding model of the interaction
of digoxin with Na,KATPase has been proposed by Thomas (58) and involves
a folding of the receptor-binding
epitopes around the
ligand. Fig. 6 depicts the intermolecular
forces that
might play a role in the interaction between the lactone
ring, sterol section, and sugar residues of digoxin. Regardless of the details, the interaction is very specific,
with dissociation
constants in the iO
mol/L range
(59), and clearly leads to selective inhibition
of the activity of Na,K-ATPase.
Seminal work demonstrating
the inhibitory effect of
cardiac glycosides on Na.,K-ATPase
has been performed
by Akera and Brody (60). Akera et al. (61) were the first
to compare the in vivo sensitivity of the sodium pump to
ouabain
in dog, sheep, guinea pig, and rat with the in
vitro sensitivity
of Na,K-ATPase
in cardiac microsomal
fractions from these same species. Inhibition
of the sodium pump by cardiac glycosides increases the strength
OH
OH
RHAMNOSE:
RHAMNOSE
OH
OH
H
DIGrTOXOSE
DIGITOXOSE
DIGITOXOSE
DIGITOXOSE:
H
NO
OH
OH
Fig. 5. Structures of the cardiacglycosides,digoxinand ouabain.
Three digitoxose sugars (Indigoxin)and one rhamnose sugar (in ouabain) are
attached at the C-3 position of the steroid backbone.
AECP7(:
SITED
H-BINDING SITES
11
Fig. 6. A model for drug (ouabain, digoxin) interactionwith the
receptor (Na,K-ATPase).
FromThomas (58); used with permission.
of contraction
(inotropic effect) and slows the beating
(chronotropic effect) of the heart. Membrane excitation
of cardiac myocytes is characterized
by opening of the
Na
channel and depolarization
of the sarcolemmal
membrane
in response
to increased
intracellular
sodium. Consequently,
Ca2 channels
open and the Ca2
ion influx triggers
the release of Ca2 stores from the
sarcoplasmic reticulum into the cytosol. The increase in
intracellular
free Ca2 activates
contractile
proteins,
resulting in myocardial
contraction. Cardiac glycosides
inhibit the exchange of Na and K via Na,K-ATPase;
the result is a relative transient increase in intracellular sodium. The Na/Ca-ion exchanger,
present in the
sarcolemmal
membrane, mediates the exchange of Na
ions for Ca2 ions. This exchange mechanism
probably
results in a relative increase in the concentration
of
intracellular
Ca2. The increased
intracellular
Ca2 is
taken up into the sarcoplasmic
reticulum
via a Ca2
pump. After depolarization,
the extra Ca2 released results in enhanced contractile force (1). Somberg et al.
(62) determined that a 25% reduction in pump activity
was associated
with a 20% increase
in contractile
strength.
The cardenolides
that specifically
interact with the
sodium pump also have well-documented
effects on
other cardiovascular
organs such as the peripheral vascular tissue (see below). Even though digoxin is widely
used in the treatment of heart disease, the therapeutic
index is low, and Smith et al. (63) cite digoxin intoxication as the most widely encountered
adverse drug reaction in clinical practice. Symptoms
of digoxin toxicity
commonly
involve the gastrointestinal
tract and the
central nervous system and include: anorexia, nausea,
vomiting, diarrhea,
headache, delirium, cardiac rhythm
disturbances,
manic syndrome,
and depressive
syndrome (64, 65). One explanation
is due to what Langer
terms the “sodium pump lag” effect (66) explained above
CUNICAL CHEMISTRY, Vol. 40, No. 9, 1994
1677
and described in the flow diagram in Fig. 7. The desirable outcome to the proposed sequence of events described in Fig. 7 is positive inotropy, but some individuals may experience
symptoms
of cardiac
glycoside
toxicity as a result of abnormally
high intracellular
concentrations
of calcium.
Low tolerance to cardiac glycosides has been associated with old age, acute myocardial infarction/ischemia,
hypoxemia,
magnesium
depletion, renal insufficiency,
hypercalcemia,
carotid sinus massage,
electrical
cardioconversion, hypothyroidism,
and hypokalemia
(67). Interaction with coadministered
drugs such as quinidine,
verapamil, and cyclosporine is a frequent cause of toxic
accumulation
of diguxin. Recent evidence suggests that
these drugs inhibit renal excretion of digoxin by inhibiting the MDR1 gene product, P-glycoprotein,
shown to
be present on the apical membrane of mammalian
kidney (68). P-glycoprotein
is overexpressed
in multidrugresistant cells and functions as a drug-efflux pump, recognizing
a variety
of therapeutic
agents,
including
vinblastine,
a known P-glycoprotein
substrate. Significant accumulation
of digoxin or vinblastine
has been
reported in both a multidrug-resistant
chinese hamster
ovary cell line and the drug-sensitive
parent cell line
when cyclosporine,
verapamil,
or quinidine
was added
to the culture medium (69). This suggests digoxin excretion is also mediated by P-glycoprotein.
Understanding
the pharmacokinetics
of the cardiac
glycosides in relation to renal excretion, age-related tolerance, and interaction with coadministered
drugs has
led to better patient management
and a decrease
in
toxicity and mortality. However, therapeutic drug monitoring practices for this drug (e.g., reference ranges,
toxic concentrations,
dosing regimens) stifi vary considerably throughout clinical laboratories.
Examining
666
institutions
participating
in Q-Probes (a subscription
quality-improvement
program of the College of Clinical
Pathologists),
Howanitz and Steindel (70) found that
participants
used 13 different lower limits (0 to >1.0
CARDIAC GLYCOSIDES
4,
INHIBITION OF Na ,
- ATPase
BY BINDING AT EXTRACELLULAR ENZYME SURFACE
4,
INCREASE IN INTRACELLULARNa
CONCENTRATION
ION
1
INCREASE IN INTRACELLULAR Ca2
POSITIVE INOTROPY
Fig. 7. Schematic
representation
ION CONCENTRATION
Ca2 OVERLOAD LEADING
TO CARDIAC GLYCOSIDE TOXICITY
of the extra-
and intracellular
events that lead to increasedcontractileforce (positiveinotropy)or
possible cardiac glycoside toxicity.
From MacGregor and Walker (19); used with permission.
1678 CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994
g/L) and 16 different upper limits (1.1 to >2.7 pg/L) for
their therapeutic
digoxin ranges. Some blood samples
were drawn <6 h after dosing. Blood sampling at inappropriate times may result in digoxin serum concentrations in excess of the therapeutic
range, and thereby
increase the likelihood of an erroneous decision to withhold digoxin or even to administer
immunoglobulin
fragments
of digoxin-specific
antibodies
(used in the
treatment of digoxin toxicity) (70).
Additionally, in some individuals,
digoxin is biotransformed into metabolites with variable cross-reactivity
in digoxin immunoassays
(71, 72). Even if these metabolites are not biologically active, their cross-reactivity
in
digoxin immunoassays
could result in digoxin underdosing (72, 73). However, we (72) and others (74) have
found that some but not all of these metabolites
have
significant
Na,K-ATPase
inhibitory activity, the clinical importance of which has not been fully addressed.
Recent important findings suggest that the alpha subunits of the Na,K-ATPase
exhibit species and isoform
variation
in their affinity for binding of cardenolides
(75). Differences
in binding
affinities, previously
ascribed to variables
such as assay conditions
and ion
concentrations,
now include differences
attributable
to
the presence of high- and low-affinity
alpha subunit
molecular forms. Originally
termed “a” and “a+ ,“ the
molecular
cloning of the Na,K-ATPase
from various
species demonstrated
that a+ is actually represented by
the two distinct isoforms now referred to as alpha 2 and
alpha 3. The alpha 2 and alpha 3 subunits
are the
isoforms with greatest sensitivity
to ouabain in the rat
(76-78).
Charlemagne
et al. (79) describe high- and
low-affinity ouabain-binding
sites in rat heart, with respective apparent dissociation
constants in the 10-8 to
10_6 mol/L range. Age, ion concentration,
hormone concentrations,
and pathological
conditions have all been
demonstrated
to correlate with changes in isoform expression (37). Isoform variation in affinity for digoxin
and other cardiac glycosides
should be considered in
cases where the response to cardiac glycoside therapy is
inappropriate.
However, work in this area is still preliminary.
For example, Schmidt et al. (80) quantified
the digitalis receptor concentration
in the left ventricle
at autopsy, comparing patients without heart disease
with those with end-stage
heart failure who had received digitalis therapy. Previous work, based on the
use of in vitro systems and tissue culture, had shown an
increase in expression
of Na,K-ATPase
in response to
incubation
with digitalis,
leading to speculation
that
patients might develop tolerance to digitalis therapy
(81). However,
rather
than increased
expression,
Schmidt et al. (80) showed a lower concentration
of
digitalis receptors in failing hearts than in the control
subjects. These investigators
did not address the presence or absence of specific high- or low-affinity
alpha
isoforms, because their quantification
of digitalis receptors was based on [3Hlouabain binding. Affinity for ouaham is affected by the presence or absence of certain
amino acid residues at the amino terminus
of the alpha
subunit that correspond to isoform type (82); therefore,
the down-regulation
of high-affinity
alpha isoforms
without an overall
loss in holoenzyme
concentration
could give the same experimental
results as a decrease
in overall Na,K-ATPase
expression. Phenomena such as
changes in subunit expression,
distribution
in tissues,
ligand binding affinity, and endogenous ligand concentrations hold promise for establishing
a new understanding
of the basic mechanisms
underlying
pathophysiology. These findings maybe of central importance
in establishing
hypotheses regarding the clinical role of
endogenous
ouabain- or digitalis-like
factors.
EndogenousLigands:Implicationsin Pathology
The ubiquitous
nature of the sodium pump and its
involvement
in diverse physiological
processes suggests
that alteration of pump activity by endogenous or xenobiotic factors may play a key role in many fundamental
physiological
processes (e.g., modulation of cardiac contractility, control of sodium in the kidney, vascular contractility,
neurotransmitter
release
and processing)
(19). The presence of a highly conserved
Na,K-ATPase
binding
domain
for cardiac glycoside drugs implies the
existence
of natural ligands
that act as endogenous
modulator(s)
of this transporter.
Substantial
evidence suggests that endogenous
digitalis-like and ouabain-like
factors exist. In the process of
monitoring therapeutic
digoxin concentrations
by various immunoassay
procedures,
several
investigators
noted increased digoxin values in subjects not treated
with cardiac glycosides
(83, 84). Also noted were increases
in serum digoxin
measurements
in subjects
whose digitalis
therapy had been discontinued
(85).
Factors
giving rise to these apparent digoxin values
were
termed
digoxin-like
immunoreactive
factors
(DLIF) (84) or endogenous digitalis-like
factors (EDLF)
(86). Detectable
concentrations
of these factors have
been observed in serum and plasma from healthy adults
(87), plasma from volume-expanded
dogs (86), newbOrns (88, 89), pregnant
women (90), patients with renal impairment
(91), and patients with liver dysfunction (92, 93). Aside from DLIF interference
with the
accurate measurement
of digoxin in human serum,
these molecules,
because
of their structural similarity
to
digoxin itself (94), may interact with the Na,K-ATPase
at the digitalis-binding
site on the alpha subunit. Present evidence suggests that the likely tissue source of
this factor is the adrenal cortex (94, 95).
In addition to the discovery of DLIF, there is compelling evidence that ouabain-like
factors (OLF) are present in mammals (96). Hamlyn and Manunta
(97) and
other investigators
(98) have isolated a factor from both
serum and adrenals with ouabain-like
properties that
include structural similarity
to ouabain, Na,K-ATPase
inhibitory
activity,
and increased
concentration
in
pathophysiological
conditions.
A review of this work
and the potential role of this ouabain-like
factor in disease are detailed by Blaustein (53), who summarizes the
proposed physiological
effects of endogenous ouabain in
control of intracellular
calcium stores and cell respon-
siveness.
Controversy
remains,
however,
about the
source of this endogenous ouabain-like
molecule (99).
Substantial
arguments
still prevail
as to what is
meant by digitalis-like
activity (100). It is important
to
understand that immunoreactivity
does not imply functional activity or vice versa. Thus, DLIF should not be
mistaken
or confused with EDLF or OLF (84), even if,
as is suspected, the identity of these factors converges as
more is learned about them. For example, these molecules may be related precursors, metabolic products of
each other, or the same molecule.
Substantial
evidence links endogenous
digitalis-like
factors with vasoreactivity.
Data supporting the hypothesis that endogenous digitalis-like
factors interact with
Na,K-ATPase
to induce peripheral vasoconstriction
include the following
1) Subjects with some forms of essential hypertension
have increased sodium-potassium
pump inhibitory activity (101, 102), natriuretic activity (103), and digoxin
immunoactivity
in their plasma (104).
2) Spontaneously
hypertensive
rhesus monkeys have
high serum concentrations
of digoxin-like
activity
(105).
3) Crude preparations
of the natriuretic activity constrict third-order arterioles, making them more responsive to other vasoconstrictive
agents such as norepinephrine
(106).
4) Crude preparations of the natriuretic activity from
urine cause dose-dependent
contractions of isolated anococcygeus
muscle of the rat (which resembles
the
smooth muscle of blood vessels) (107).
5) Infusion of ouabain (108, 109) or digoxin (110) into
humans
specifically
induces peripheral
vasoconstriction.
6) Injection of antibodies to digoxin lowers the blood
pressure
of deoxycorticosterone-salt-retamning
hypertensive rats (111).
7) Preparations
containing
digoxin-like
immunoreactivity from human urine raise blood pressure and
protect rats from acute digitalis toxicity (112).
8) Bolus infusion of digoxin induces vasoconstriction
of epicardial coronary
arteries
in humans (113).
9) Ouabain-like
compounds
isolated from human serum demonstrate
vasoreactivity
comparable with that
of ouabain
(114).
Cumulatively,
the digitalis-like
activity of these factors strongly implicate
endogenous
regulators
of the
Na,K-ATPase
as vasoconstrictive
agents involved in the
etiology of some hypertensive
states.
Clinical Conditions Linked to Dysfunction or
Modification of Na,K-ATPase Activity
CardiovascularDisease and Hypertension
Pathological
conditions in animals and humans involving salt and water homeostasis
have been associated with alterations
in Na,K-ATPase
activity and (or)
the presence of circulating endogenous digoxin- or ouabain-like factors (germane articles and reviews are provided in Table 2). Two of the most notable disorders
involving
salt and water homeostasis-heart
disease
CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994 1679
Table 2. Clinical conditions correlating with altered
Na,K-ATPase activity or presence of modifiers.
Condition
.
Cardiovascular disease and hypertension
Heart disease
Hypertension
Reference
52. 97, 101, 117, 118, 119
HTN, pregnancy-induced
120, 121, 122
HTN,
123
hypothyroidism-related
Impaired renal function
91
Renal disease
84, 85, 124
Diabetes and other metabolic disorders
Acromegaly
Aldosteronism
Diabetes
119, 125
126
118, 119, 127, 128
119
mellltus
Obesity
Digoxin toxicity
Age-related
Disease-related
Multipledrug interaction
.
Fetal abnormalities
Growth retardation
Renal abnormality
H droce halus
64,65,
67
69
.
130
130
130
aneuploidy
Nonimmune hydrops
Low birth weight
131
Preterm infants
132
‘
Neurological disorders
Alzheimer disease
Bipolar disorder
Pulmonary conditions
Pulmonary disease
Chronic obstructive
pulmonary disease
.
one-kidney,
one-clip, and reduced renal mass-saline
models of hypertension
(102, 139). They demonstrated
decreased myocardial
and vascular Na,K-ATPase
activity, suggesting that reduced pump activity might be
common in low-renin
and other models of essential hypertension. In a more recent study, uninephrectomized
animals treated with deoxycorticosterone
or angiotensin II provided
insight
into the molecular mechanisms
that may be involved in some forms of hypertension
(140). Separation
of mRNA by Northern
analysis of rat
cardiac left ventricle, aorta, and skeletal muscle RNA,
by use of Na,K-ATPase
alpha isoform-specific
cDNA
probes, showed tissue-specific
changes in isoform expression of mRNA transcripts
in response to increased
intravascular
pressure.
Greater concentrations of DLIF and OLF have been
noted in women with pregnancy-induced hypertension
(preeclampsia)
than in normal pregnancy
(84, 141).
Pregnancy
is a volume-expanded
condition, and in both
normal
and hypertensive
pregnancy
the anomalous
DLIF values resolve rapidly upon delivery (90, 120122). Increased
concentrations
of DLIF are associated
with acute and chronic renal disease (91, 124) and with
fluid retention
due to hepatic failure (92, 93). Endogenous OLF is reported markedly increased in hypertension that is due to hypothyroidism
(123) and congestive
heart failure (116). DLIF is also significantly
increased
in the plasma of human subjects
with electrocardiographic evidence of reversible
cardiac dysfunction
induced by physical
exhaustion
(142). Weinberg
et al.
(143) used peritoneal
dialysis fluid from patients with
.
80 115 116
(HTN)
Early experiments by Pamnani et al. aimed at underthe effects of hypertension on the myocardium
and vasculature
at the molecular level by using rat
standing
130
133, 134, 135, 136
64
137
138
chronic
and hypertension-are
causally
related.
Hypertension
increases the risk of myocardial infarction, congestive
heart failure, renal failure, and cerebral stroke (52).
The initial reduction in myocardial
contractility
that
occurs in some forms of heart failure results in vasoconstriction and peripheral
resistance.
The constriction
of
the vascular beds in the kidneys causes salt and water
retention. Alterations
in Na,K-ATPase
activity or expression can alter vascular
or cardiac contractility
by
affecting sodium homeostasis
(37). Thus, the sequence
of events previously described strongly correlates with
the involvement
of a circulating
inhibitor of sodium
pump activity in the pathogenesis
of both cardiovascular disease and hypertension.
Hypertension,
as evidenced by persistently
high arterial blood pressure, can be idiopathic or secondary
to
underlying
conditions.
Essential
(idiopathic hypertension) is a poorly understood though relatively common
disease. An inherited predisposition
has been suggested,
and such individuals
may be especially sensitive to dietary salt (117). Hypertension
secondary
to primary
aldosteronism
(126), other endocrine
disorders,
and
pregnancy
often tends to resolve once the underlying
problem is alleviated,
1680
CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994
renal
failure
to
chromatographically
isolate
three molecular
species having DLIF activity.
Of the
three, one had a retention time identical to ouabain, and
one had a retention time identical to digoxin. And Yuan
et al. (144) showed that administration
of chronic low
doses of ouabain was associated with the development
of
hypertension
in normotensive
rats as well as in rats
having
various degrees of reduced renal mass. Mean
blood pressure increased with the degree of mass reduction but was significantly
greater than in controls even
for rats with no renal mass reduction.
In an effort to determine whether ouabain itself acts
as a hypertensive
agent or simply exacerbates
the hypertensive action of mineralocorticoids,
Sekihara et al.
(145) treated mononephrectomized
rats with ouabain (1
mg), deoxycorticosterone
acetate (5 mg), or a combination of both, weekly for 6 weeks. Both ouabain
and
deoxycorticosterone
acetate lacked hypertensive
action
individually
at the dosage given but, in combination,
they produced a significant
increase in blood pressure as
well as cardionephromegaly
and histopathological
changes consistent
with the effects of an elevation in
blood pressure. The authors concluded that, in those
hypertensive
individuals
secreting greater concentrations of mineralocorticoids,
ouabain might amplify the
hypertensive
effect.
DLIF, isolated from urine by use of cross-reactivity
to
digoxin antibody
as evidence of activity, was admiriistered to normotensive
rats to determine its cardiovascular effects (112). After an initial 60-mm stabilization
period, infusion of DLIF in 10 animals caused the mean
arterial
pressure to rise from 124.3 (±1.9) to 140.4
(±5.2) mmHg at 7.5 mm, induced diuresis, and slowed
the heart rate. These results, coupled with data documenting the presence of endogenous
digitalis-like
compounds in the serum of hypertensive
animals
(102, 146,
147), suggest
a role for DLIF in hypertension.
Diabetes and Other Metabolic Diseases
Weidmann
and Ferrari, studying diabetes and hypertension (127), found that not only do type I (insulin-dependent) diabetic individuals
have a familial predisposition
for essential hypertension,
but also normotensive
offspring
of parents who are nondiabetic
but have essential hypertension show increased concentrations
of plasma insulin
and reduced insulin sensitivity;
moreover, Na retention
is characteristic
of both type I and type II (non-insulindependent) diabetics. These authors also report that intracellular calcium is increased
in adipocytes, in part via
insulin’s inhibition of Ca2,Mg-ATPase,
and that insulin may increase renal sodium retention and influence the
activity of transmembrane
electrolyte pumps. Insulin regulation of vascular Na,K-ATPase
gene expression is cited
by Tirupattur et al. (148) as an important
factor in the
development
of hypertension
in diabetes. mRNA encoding
both the alpha 1 and alpha 2 isoforms was identified in
vascular smooth muscle cells derived from embryonic
rat
thoracic aorta. Although the predominant
isoform was
alpha 1, only the concentrations
of the alpha 2 isoform
increased in response to insulin treatment.
The overall
increase in ouabain-inhibitable
Na,K-ATPase
activity in
vascular smooth muscle cells in response to insulin treatment suggests that, in the absence of insulin or in insulinresistant states, Na,K-ATPase
activity could decrease, resulting in increased
vascular
contractility
and blood
pressure.
In their review, Clerico and Giampietro
(119) cite
reports of decreased sodium pump activity in the nerves,
heart, and aorta of diabetic humans, and in rats with
streptozotocinor alloxan-induced
diabetes; however,
there was a paradoxical
increase in Na,K-ATPase
activity in the kidneys of streptozotocin-induced
diabetic
rats. The authors speculate that these tissue differences
could be due to tissue-specific
regulation
of Na,KATPase activity or metabolism.
They further suggest
that in metabolic diseases
such as diabetes meffitus,
obesity, and acromegaly
(which have in common increased sodium retention
and volume expansion)
increased sodium intake, hyperinsulinemia,
or increased
concentrations
of growth hormone could trigger the release of an endogenous
digitalis-like
factor that could
modulate the pump and increase blood pressure. A more
recent study by Chen et al. (128), using a similar diabetic rat model system, confirmed the previous findings.
In addition, Chen et al. found greater amounts
of a
digitalis-like
factor in the plasma and urine of their
hypertensive
diabetic rats than in controls. A review by
Sewers and Khoury (118) highlights the additional risk
of cardiovascular
disease when hypertension
accompanies diabetes mellitus and discusses additional risk factors such as obesity, genetics, and ion transport control.
Digoxin Toxicity
Although
advanced age is not a pathological
condition, age-related changes in renal clearance and volume
of distribution
can increase the likelihood
of digoxin
toxicity (65). In addition, evidence based on basal metabolic rate measurements
indicates
that whole-body
Na,K-ATPase
activity decreases
with age (64, 129).
This suggests that, even though their serum cligoxin
concentrations
may be within
the therapeutic
range,
digitalis
toxicity in the elderly might result, in part,
from inhibition of an already less-active
sodium/potassium transport
system.
Low tolerance
to digoxin is also
associated with certain disease states, particularly those
involving hypokalemia,
and with multiple drug interaction, as discussed previously.
Fetal Abnormalities
The neonatal period is associated with volume expansion and sodium imbalance. As stated previously, agerelated changes in Na,K-ATPase
alpha isoform expression in the rat have been observed
(23). DLIF is
significantly
increased in the plasma of newborn infants
but usually returns to normal within several days (88,
132). Increased concentrations
of digoxin-like
immunoreactivity
have been observed in fetuses having growth
retardation,
renal abnormality,
hydrocephalus,
aneuploidy, and nonimmune
hydrops (reviewed in ref. 130),
again suggesting
a role for endogenous pump modifiers
in the regulation
and genetic
expression
of Na,KATPase and in the etiology of some pathological
processes.
Neurological
Disorders
Bipolar illness (manic depression) is characterized
by
severe mood swings that alternate
between episodes of
irritability,
excessive
energy, and distractibility
(mania), and mental and motor slowdown to the point of
stupor (depression). Patients with bipolar illness exhibit
altered ion distribution
and transport.
In one model,
El-Mallakh
et al. (64) propose a biphasic phenomenon,
in which mild or moderate reduction in Na,K-ATPase
activity could lead to mania by increasing
membrane
excitability
and neurotransmitter
release. A greater degree of pump inhibition,
and consequently
depolarization block, could result in depression by decreasing neurotransmitter
release (149). Patients
in manic states
show increased sodium retention and intracellular
calcium concentrations,
and therapeutic modalities such as
lithium
or calcium-channel
blockers are theorized to
affect sodium-calcium
exchange
(64). Interestingly,
symptoms mimicking those observed in bipolar patients
occur with digitalis toxicity. These can include confusion, disorientation,
drowsiness,
lethargy,
agitation,
and hallucinations
(137), further implicating
Na,KCLINICAL CHEMISTRY, Vol. 40, No. 9, 1994
1681
ATPase as an etiological agent in bipolar illness. Recent
evidence in our laboratory suggests that DLIF (measured with a digoxin-specific
immunoassay)
is increased
in acutely psychiatrically
ill bipolar patients relative to
recovered bipolar patients (unpublished).
Increased endogenous ouabain-like
compounds
that excessively
suppress Na,K-ATPase
have been proposed as a mechanism
in this disorder (133).
Alzheimer disease (Al)) is an age-related neurodegenerative disorder of unknown
etiology. In addition to
well-documented
neuropathological
changes
such as
neurofibrillary
tangles and neuritic plaque formation
with accumulation
of beta-amyloid
(136), AD is associated with lower cerebral blood flow and decreased use of
oxygen and glucose, especially in areas exhibiting neuropathological
changes (134). About 50% of the energy
expenditure
of resting brain is believed
to support
Na,K-ATPase
activity. Hank et al. (134) found a decrease in ouabain binding of brain tissue from AD patients in comparison
with age-matched
controls. The
reduction in ouabain binding was especially
evident in
the neocortex, an area predominantly
affected in Al)
patients.
A decrease
in ouabain-inhibitable
Na,KATPase activity in the brain subcortical but not cortical
areas of patients with Al) was also noted by Liguni et al.
(135). Although sodium pump dysfunction may not be
causal in this disorder, the progressive
dementia associated with AD may in part be due to alterations
in
Na,K-ATPase
activity. To date, the presence of possible
endogenous
pump modifiers in relation to AD has not
been described.
Pulmonary Conditions
Little has been published in this area. Varsano et al.
(138) used a digoxin radioimmunoassay
to show that
patients with advanced chronic respiratory failure had
greater
concentrations
of DLIF than did controls.
Chronic obstructive pulmonary disease and other forms
of advanced
respiratory disease are frequently
associated with water and sodium retention, and the authors
suggested that increased DLIF might be an attempt to
control water and sodium metabolism.
In summary, conditions associated with volume expansion or alterations in sodium homeostasis
(see Table
2), many of which have been described here, also show
changes in Na,K-ATPase
activity, increases in endogenous digitalis-like
substances,
or both. Modification
of
pump activity can occur in response to changes in isoform expression
or modulation
by endogenous
pump
inhibitors or activators. As to whether altered regulation of Na,K-ATPase
activity is directly linked to any of
these conditions remains to be elucidated. Preliminary
data identifying
polymorphisms
in the human Na,KATPase alpha and beta subunit genes offer the potential
of linking these and other genetic differences to specific
clinical disorders. Locating genetic mutations of Na,KATPase or of the endogenous
modifiers may soon be
added to the repertoire of test capabifities in the clinical
chemistry laboratory, providing early identification
of
1682 CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994
individuals
at risk for a variety
ciated diseases.
This work was supported
of Na,K-ATPase-asso-
in part by Grant RO1-HL36172
from
the National Institutes of Health.
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