1674 CLINICALCHEMISTRY, Vol. 40, No. 9, 1994

CUN. CHEM. 40/9, 1674-1685 (1994)

Understanding the Sodium Pump and Its Relevance to DiseaseAndrea M. Rose’ and Roland Valdes, Jr.”

I

Na,K-ATPase (sodium pump; EC 3.6.1.37) is present inthe membrane of most eukaryotic cells and controlsdirectly or indirectly many essential cellular functions.Regulation of this enzyme (ion transporter) and its indi-vidual isoforms is believed to play a key role in theetiology of some pathological processes. The sodiumpump is the only known receptor for the cardiac glyco-sides. However, endogenous ligands structurally similarto digoxin or ouabain may control the activity of thisimportant molecular complex. Here we review the struc-ture and function of Na,K-ATPase, its expression anddistribution in tissues, and its interaction with knownligands such as the cardiac glycosides and other sus-pected endogenous regulators. Also reviewed are vari-ous disorders, including cardiovascular, neurological,renal, and metabolic diseases, purported to involve dys-function of Na,K-ATPase activity. The escalation inknowledge at the molecular level concerning sodiumpump function foreshadows application of this knowl-edge in the clinical laboratory to identify individuals atrisk for Na,K-ATPase-associated diseases.

IndexIng Terms: Na,K-ATPase/isoforms/cardiac glycosides/ age-related effects/digoxin/ouabain/hypertenslon/diabetes/Alzheimerdisease/neurological disorders

The sodium-potassium-activated adenosine triphos-phatase (Na,K-ATPase; sodium pump; EC 3.6.1.37) is aplasma membrane-associated protein complex that is ex-pressed in most eukaryotic cells.4’5 The “pump” couplesthe energy released in the intracellular hydrolysis ofadenosine tnphosphate (ATP) to the transport of cellularions, a major pathway for the controlled translocation ofsodium and potassium ions across the cell membrane.Na,K-ATPase therefore controls directly or indirectlymany essential cellular functions, e.g., cell volume, freecalcium concentration, and membrane potential. Regula-

Departments of ‘ Pathology and 2Biochemistry, University ofLouisville School of Medicine, Louisville, KY 40292.

‘Mdress correspondence to this author at: Department of Pa-thology, University of Louisville, Louisville, KY 40292. Fax 502-852-1771; E-mail ROVALDO1@ULKYVM.LOUISVILLE.EDU.

4Nonstandard abbreviations: Na,K-ATPase, Na,K-activatedadenosine triphosphatase; DLIF, digoxin-like immunoreactive fac-tors; EDLF, endogenous digoxin-like factors; OLF, ouabain-likefactors; and AD, Alzheimer disease.

5Although technically an enzyme, Na,K-ATPase functions pri-marily as an ion transporter. Therefore, most investigators refer tothe alpha and beta gene products as isoforms instead of isoen-zymes.

Received December 27, 1993; accepted June 16, 1994.

tion of this enzyme (transporter) and its individual iso-forms is thought to play a key role in the etiology of somepathological processes.

The sodium pump is the only known receptor for thecardiac glycosidesused to treat congestive heart failureand cardiac arrhythmias. This suggests that endoge-nous ligands structurally similar to cardiac glycosidesmay act as natural regulators of the sodium pump inheart and other tissues. Identification of naturally cc-

curring regulators of Na,K-ATPase could initiate thediscovery of new hormone-like control systems involvedin the etiology of selected disease processes, hence theimportance of understanding the relation of the sodiumpump and its ligands to disease. In this article, we re-view recent information related to structure and func-tion, genetic expression and distribution in tissues, andinteraction of the sodium pump with ligands such as thecardiac glycosides and other suspected endogenouscounterparts. Several diverse disease processes-includ-ing cardiovascular, renal, neurological, and metabolic

disorders, having in common a dysfunction in salt and

water homeostasis-are emphasized, as is the clinicalneed for understanding the function and control of thisubiquitous ion transporter system.

Structure and FunctIon of Na,K-ATPaseNa,K-ATPase couples the energy released in the in-

tracellular hydrolysis of ATP to the export of three in-tracellular Na ions and the import of two extracellularK ions. The continuous operation of this macromolec-War machine ensures the generation and maintenanceof concentration gradients of Na and K across the cellmembrane. This electrochemical gradient provides en-ergy for the membrane transport of metabolites andnutrients, e.g., glucose and amino acids, and such ionsas protons, calcium, chloride, and phosphate. The elec-trochemical gradient is essential also for regulation ofcell volume and for the action potential of muscle andnerve. The relative intra- and extracellular concentra-tions of Na and K ions maintained primarily by thesodium pump and the cofactors required for activity areshown in Fig. 1 (1, 2).

The functional macromolecule is a membrane-span-

ning 270-kDa tetramer consisting of two dimers, each

composedof noncovalently interacting alpha (112 kDa)and beta (55 kDa) subunits. A model of the transportercomplex relative to its membrane location is shown inFig. 2. The presence of a smaller gamma subunit (10

(150 mmol/L)

Digitalis

(5 mmol/L)

OUT

Fig. 1. SchematIc diagram of the Na,K-ATPase-associated cofactorsand Ions.Modifiedfrom Akera (2).

Extracellular

IN

Intracellular

a a

IN

Fig.2. Schematic diagram of the plasma membrane-spanning Na,K-ATPase transporter complex, indicating the positions of the alpha,beta, and putative gamma subunits.

I AlP

E1 . E2-K2

2K

A

CLINICALCHEMISTRY, Vol. 40, No. 9, 1994 1675

kDa) has been suggested; however, its role, if any, hasnot been defined (3).

The alpha (catalytic) subunit, is proposed to have 7(4)or 8(5) transmembrane domains; however, the numbercan vary from 6 to 10, based on interpretation of hydrop-athy profile data (6) and identification of specific li-gand-receptor interactions that predict alpha chain to-pology (7-9). The alpha subunit contains all the bindingsites for ligands known to stimulate or inhibit the en-zyme (10-13). Tentative models representing mem-brane-spanning segments of the alpha subunit are de-tailed in Fig. 3.

The beta subunit has a single hydrophobic transmem-brane domain and is highly glycosylated on its noncy-tosolic surface (14). Hiatt et al. postulate that the betasubunit may serve to orient and stabilize the alphasubunit in the membrane (15). Cellular expression ofthe beta subunits and assembly with the alpha subunits

are necessary for correct conformation and activity ofthe Na,K-ATPase holoenzyme (16). Although the role ofthe beta subunit in ion translocation is uncertain, itspresence appears essential for function of the sodiumpump (16).

Phosphorylation is an important step in the function

of Na,K-ATPase. The molecule undergoes an alpha-he-lix to beta-sheet transition between two principal reac-tive states, E1 and E2, in the multistep reaction bywhich Na ions and K ions traverse the membrane(17, 18). The conformational transition results via a

‘)63K327 Y1016

1263 0737 Y1018R166

Fig. 3. Foldingmodels for the Na,K-ATPase alpha transmembranesegments: (A) 10 membrane-spanning segments; (B) 8 membrane-spanning segments.Letters refer to the one-letter amino acid code. Numbers represent the topo-logical location of particular amino adds. From Kailish et at. (7); used withpermission.

cyclic reaction in which the enzyme is phosphorylatedby ATP in the presence of Mg2 ions and Na ions andthen dephosphorylated in the presence of K ions. Amodel depicting this thermodynamic cycle is shown inFig. 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 independentgenes (20-22). The sequence conservation among differ-

3Na2l(

E2-P.Na3 .p r E2P.K2

Nat. Mi2 ______________________Ei-P.Naa1,...

ATP

Fig.4. Principal reactive states (E1 and E) involved in the transportof sodium (Na) and potassium (K) ions across the cytoplasmicmembrane.The enzyme (E) Isphosphorylated(P), with ATP as thephosphatedonor andMg2 as a cofactor In the reaction. From MacGregor and Walker (19), asmodified from Sen et at. (18); used with permission.

EyeHeart (ventricle)

KidneyLungSkeletal muscle

ThyroidUterus

HumanRatRatRatHumanHumanHuman

al, a2, a3a

al, a3 neonate#{176}

al, a2 adult#{176}al, a3 aged#{176}alaalaal,

Rat a1,a2,l,i32aHuman al, a2Human ala

Superscripts referto all thepreceding isoforms onthe same line: a isoform-specific expression of mRNA identified byNorthernanalysis;b isoform-specificexpressionof proteinidentifiedbyWesternanalysis.

1676 CLINICAL CHEMISTRY, Vol. 40, No. 9, 1994

ent species suggests that individual roles for the iso-forms, though not yet determined, arose early and weremaintained throughout evolution.

The three alpha isoforms (alpha 1, 2, and 3) are ex-pressed in a developmental and tissue-specific manner.Utilizing monoclonal antibodies that recognize the inch-vidual alpha isoforms, Lucchesi and Sweadner haveshown (23) that rat ventricular muscle-membrane prep-arations express alpha 1 in all stages of development;alpha 3 is present at birth through days 14 to 21, and isthen replaced by alpha 2 in the adult rat; alpha 3 againpredominates in aged rats. The physiological signifi-cance of this shift in subunit isoform expression is un-known. Tissue specificity for the different isoforms hasbeen identified both at the mRNA and protein level forvarious species (22, 24), and is summarized for isoformsfrom rat and human tissues in Table 1. Table 1, al-though not comprehensive, serves to illustrate the di-versity of isoform distribution in tissues. The vast ma-jority of studies defining isoform distribution have beendone with animals, less data being available for humantissues. Nevertheless, studies in both humans and ratssuggest that alpha 1 is the only isoform expressed ap-preciably in the kidney (33), whereas alpha 3 is associ-ated primarily with the nervous system (22, 28, 29).Isoform specificity even extends to cell type within aparticular tissue, as evidenced by studies with tissuefrom brain (34), heart (35), and eye (30), supporting thehypothesis that the isoforms have different physiologi-cal functions (36, 37).

Three Na,K-ATPase beta isoforms and one relatedH,K-ATPase (another member of the cation-transport-ing ATPases) beta isoform have been identified. Beta 1has been isolated from several vertebrate species in awide range of tissues. Beta 2 is expressed largely inbrain (26,27) and ocular ciliary epithelia (38); however,recent studies suggest that beta 2 is also expressed inglycolytic fast-twitch muscles of the rat hindlimb (32).

Table 1. Tissue dIstribution of Na,K-ATPase Isoforms.Tissu. source Species Subunit

Adipose Rat al, a2’

Brain Rat f32#{176}Human al a2, a

Human a3b

Human al, a2, a3ab

Human al, a2, a3, a

Interestingly, the beta 2 isoform appears to serve a dualfunction, both as a subunit of the Na,K-ATPase and asa mediator of neuron-astrocyte adhesion (39). This sug-gests the possibility that these proteins, as a family,may play other roles besides their traditionally definedtransport function. Finally, a protein referred to as beta3 has been isolated from Xenopus (40), and the beta-isoform of the H,K-ATPase has been characterized fromseveral vertebrate species (41-43). The beta isoformsare less similar to each other in amino acid sequencethan are their alpha isoform counterparts to each other;the beta isoforms also vary in their number of aspar-agine-linked glycosylation sites (27).

The expression of all the alpha isoforms and beta 1 aredifferentially regulated by hormones (44). Horowitz etal. (45) determined that thyroid status affected the al-pha 1, alpha 2, and beta isoform-specific expression ofmRNA and protein in rat heart, skeletal muscle, andkidney; and Gick and Ismail-Beigi (46) found that incu-bation of a rat liver cell line with thyroid hormoneresulted in an increase in alpha 1 and beta mRNAexpression and Na,K-ATPase activity. Increased so-dium concentration in response to corticosteroids is re-ported to recruit an intracellular latent pool of Na,K-ATPase 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 sequencesof the human alpha isoform genes, found that each con-tains a number of potential transacting and hormone-binding sites that do not appear to be conserved amongthe three alpha isoform genes, thus allowing for differ-ential regulation.

The ability to detect nucleotide changes that result inrestriction fragment length polymorphisms has led tothe discovery of sequence variation in some families inthe human alpha (50) and beta (51) subunit genes.Whether these genotypic differences, or others not yetidentified, correlate with a particular pathology is atpresent undetermined.

Regulators of Sodium Pump ActivityReferences Cardiac Glycosides

25 The Na,K-ATPase alpha subunit is the only known26, 27 receptor for the cardiac glycosides, which underscores

29 the clinical significance of the pump in treatment of30 congestive heart failure and arrhythmias. MacGregor

31 and Walker have written a short review of the cardiac28 glycosides (19). These inhibitors of the sodium pump are23 derived from extracts of the plant genera Digitalis, Stro-

23 phanthus, and Acocanthera. Digoxin and digitoxin are23 products of species of the foxglove plant, Digitalis, and28 ouabain is obtained from the East African Ouabaio tree28 or seeds of the plant Strophanthus gratus (52,53). These28 compounds are the most potent inotropic agents known,32 and their cardiac effects are believed to be mediated28 through their ability to inhibit the sodium pump. His-28 torically, preparations of these substances have been

used therapeutically for perhaps 3000 years, includinguse of plant extracts containing cardiac glycosides bythe ancient Egyptians (54).

Digoxin, which can be administered orally and isreadily absorbed by the gastrointestinal tract, is themost widely used cardiac glycoside clinically; ouabain isthe most widely used experimentally. These compounds,all cardenolides, have a sterol skeleton. Cardenolidesare C steroids having one or more sugar residues atC-3 and a flve-membered lactone ring at C-17 (see Fig.5). As previously described, cardiac glycoside binding toNa,K-ATPase occurs on the extracellular face of theintegral membrane protein. However, recent data sug-gest that a hydrophobic binding pocket that containsmembrane-spanning amino acids may be involved aswell (55). Alpha subunit amino acids important for car-diac glycoside binding have been determined (6, 56),and recent studies with site-directed mutagenesis tomake amino acid substitutions at proposed binding siteshave helped determine which regions recognize cardiacglycoside sugars (57). A first-order-approximation bind-ing model of the interaction of digoxin with Na,K-

ATPase has been proposed by Thomas (58) and involvesa folding of the receptor-binding epitopes around theligand. Fig. 6 depicts the intermolecular forces thatmight play a role in the interaction between the lactonering, sterol section, and sugar residues of digoxin. Re-gardless 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 ac-tivity of Na,K-ATPase.

Seminal work demonstrating the inhibitory effect ofcardiac glycosides on Na.,K-ATPase has been performedby Akera and Brody (60). Akera et al. (61) were the firstto compare the in vivo sensitivity of the sodium pump toouabain in dog, sheep, guinea pig, and rat with the invitro sensitivity of Na,K-ATPase in cardiac microsomalfractions from these same species. Inhibition of the so-dium pump by cardiac glycosides increases the strength

OH

OH RHAMNOSE:

RHAMNOSE

OH OH

H

DIGrTOXOSE

DIGITOXOSE: HDIGITOXOSE

DIGITOXOSE NO OH

OH

Fig. 5. Structures of the cardiacglycosides,digoxinand ouabain.Three digitoxosesugars (Indigoxin)and one rhamnose sugar (inouabain) areattached at the C-3 position of the steroid backbone.

AECP7(:

SITED

H-BINDING SITES

11

Fig. 6. A model for drug (ouabain, digoxin) interactionwith thereceptor (Na,K-ATPase).FromThomas (58); used with permission.

of contraction (inotropic effect) and slows the beating(chronotropic effect) of the heart. Membrane excitationof cardiac myocytes is characterized by opening of theNa channel and depolarization of the sarcolemmalmembrane in response to increased intracellular so-dium. Consequently, Ca2 channels open and the Ca2ion influx triggers the release of Ca2 stores from thesarcoplasmic reticulum into the cytosol. The increase inintracellular free Ca2 activates contractile proteins,resulting in myocardial contraction. Cardiac glycosidesinhibit the exchange of Na and K via Na,K-ATPase;the result is a relative transient increase in intracellu-lar sodium. The Na/Ca-ion exchanger, present in thesarcolemmal membrane, mediates the exchange of Naions for Ca2 ions. This exchange mechanism probablyresults in a relative increase in the concentration ofintracellular Ca2. The increased intracellular Ca2 istaken up into the sarcoplasmic reticulum via a Ca2pump. After depolarization, the extra Ca2 released re-sults in enhanced contractile force (1). Somberg et al.(62) determined that a 25% reduction in pump activitywas associated with a 20% increase in contractilestrength.

The cardenolides that specifically interact with thesodium pump also have well-documented effects onother cardiovascular organs such as the peripheral vas-cular tissue (see below). Even though digoxin is widelyused in the treatment of heart disease, the therapeuticindex is low, and Smith et al. (63) cite digoxin intoxica-tion as the most widely encountered adverse drug reac-tion in clinical practice. Symptoms of digoxin toxicitycommonly involve the gastrointestinal tract and thecentral nervous system and include: anorexia, nausea,vomiting, diarrhea, headache, delirium, cardiac rhythmdisturbances, manic syndrome, and depressive syn-drome (64, 65). One explanation is due to what Langerterms the “sodium pump lag” effect (66) explained above

CUNICAL CHEMISTRY, Vol. 40, No. 9, 1994 1677

1678 CLINICALCHEMISTRY, Vol. 40, No. 9, 1994

and described in the flow diagram in Fig. 7. The desir-able outcome to the proposed sequence of events de-scribed in Fig. 7 is positive inotropy, but some individ-uals may experience symptoms of cardiac glycosidetoxicity as a result of abnormally high intracellularconcentrations of calcium.

Low tolerance to cardiac glycosides has been associ-ated with old age, acute myocardial infarction/ischemia,hypoxemia, magnesium depletion, renal insufficiency,hypercalcemia, carotid sinus massage, electrical cardi-oconversion, hypothyroidism, and hypokalemia (67). In-teraction with coadministered drugs such as quinidine,verapamil, and cyclosporine is a frequent cause of toxicaccumulation of diguxin. Recent evidence suggests thatthese drugs inhibit renal excretion of digoxin by inhib-iting the MDR1 gene product, P-glycoprotein, shown tobe present on the apical membrane of mammalian kid-ney (68). P-glycoprotein is overexpressed in multidrug-resistant cells and functions as a drug-efflux pump, rec-ognizing a variety of therapeutic agents, includingvinblastine, a known P-glycoprotein substrate. Signifi-cant accumulation of digoxin or vinblastine has beenreported in both a multidrug-resistant chinese hamsterovary cell line and the drug-sensitive parent cell linewhen cyclosporine, verapamil, or quinidine was addedto the culture medium (69). This suggests digoxin ex-cretion is also mediated by P-glycoprotein.

Understanding the pharmacokinetics of the cardiacglycosides in relation to renal excretion, age-related tol-erance, and interaction with coadministered drugs hasled to better patient management and a decrease intoxicity and mortality. However, therapeutic drug mon-itoring practices for this drug (e.g., reference ranges,toxic concentrations, dosing regimens) stifi vary consid-erably throughout clinical laboratories. Examining 666institutions participating in Q-Probes (a subscriptionquality-improvement program of the College of ClinicalPathologists), Howanitz and Steindel (70) found thatparticipants 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 ION

CONCENTRATION

1INCREASE IN INTRACELLULAR Ca2 ION CONCENTRATION

POSITIVE INOTROPY Ca2 OVERLOAD LEADINGTO CARDIAC GLYCOSIDE TOXICITY

Fig. 7. Schematic representation of the extra- and intracellularevents that lead to increasedcontractileforce (positiveinotropy)orpossible cardiac glycoside toxicity.From MacGregor and Walker (19); used with permission.

g/L) and 16 different upper limits (1.1 to >2.7 pg/L) fortheir therapeutic digoxin ranges. Some blood sampleswere drawn <6 h after dosing. Blood sampling at inap-propriate times may result in digoxin serum concentra-tions in excess of the therapeutic range, and therebyincrease the likelihood of an erroneous decision to with-hold digoxin or even to administer immunoglobulinfragments of digoxin-specific antibodies (used in thetreatment of digoxin toxicity) (70).

Additionally, in some individuals, digoxin is biotrans-formed into metabolites with variable cross-reactivityin digoxin immunoassays (71, 72). Even if these metab-olites are not biologically active, their cross-reactivity indigoxin immunoassays could result in digoxin underdos-ing (72, 73). However, we (72) and others (74) havefound that some but not all of these metabolites havesignificant Na,K-ATPase inhibitory activity, the clini-cal importance of which has not been fully addressed.

Recent important findings suggest that the alpha sub-units of the Na,K-ATPase exhibit species and isoformvariation in their affinity for binding of cardenolides(75). Differences in binding affinities, previously as-cribed to variables such as assay conditions and ionconcentrations, now include differences attributable tothe presence of high- and low-affinity alpha subunitmolecular forms. Originally termed “a” and “a+ ,“ themolecular cloning of the Na,K-ATPase from variousspecies demonstrated that a+ is actually represented bythe two distinct isoforms now referred to as alpha 2 andalpha 3. The alpha 2 and alpha 3 subunits are theisoforms with greatest sensitivity to ouabain in the rat(76-78). Charlemagne et al. (79) describe high- andlow-affinity ouabain-binding sites in rat heart, with re-spective apparent dissociation constants in the 10-8 to10_6 mol/L range. Age, ion concentration, hormone con-centrations, and pathological conditions have all beendemonstrated to correlate with changes in isoform ex-pression (37). Isoform variation in affinity for digoxinand other cardiac glycosides should be considered incases where the response to cardiac glycoside therapy isinappropriate. However, work in this area is still pre-liminary. For example, Schmidt et al. (80) quantifiedthe digitalis receptor concentration in the left ventricleat autopsy, comparing patients without heart diseasewith those with end-stage heart failure who had re-ceived digitalis therapy. Previous work, based on theuse of in vitro systems and tissue culture, had shown anincrease in expression of Na,K-ATPase in response toincubation with digitalis, leading to speculation thatpatients might develop tolerance to digitalis therapy(81). However, rather than increased expression,Schmidt et al. (80) showed a lower concentration ofdigitalis receptors in failing hearts than in the controlsubjects. These investigators did not address the pres-ence or absence of specific high- or low-affinity alphaisoforms, because their quantification of digitalis recep-tors was based on [3Hlouabain binding. Affinity for oua-ham is affected by the presence or absence of certainamino acid residues at the amino terminus of the alphasubunit that correspond to isoform type (82); therefore,

CLINICALCHEMISTRY, Vol. 40, No. 9, 1994 1679

the down-regulation of high-affinity alpha isoformswithout an overall loss in holoenzyme concentrationcould give the same experimental results as a decreasein overall Na,K-ATPase expression. Phenomena such aschanges in subunit expression, distribution in tissues,ligand binding affinity, and endogenous ligand concen-trations hold promise for establishing a new under-standing of the basic mechanisms underlying patho-physiology. These findings maybe of central importancein establishing hypotheses regarding the clinical role ofendogenous ouabain- or digitalis-like factors.

EndogenousLigands:Implicationsin PathologyThe ubiquitous nature of the sodium pump and its

involvement in diverse physiological processes suggeststhat alteration of pump activity by endogenous or xeno-biotic factors may play a key role in many fundamental

physiological processes (e.g., modulation of cardiac con-tractility, control of sodium in the kidney, vascular con-tractility, neurotransmitter release and processing)(19). The presence of a highly conserved Na,K-ATPasebinding domain for cardiac glycoside drugs implies theexistence of natural ligands that act as endogenousmodulator(s) of this transporter.

Substantial evidence suggests that endogenous digi-talis-like and ouabain-like factors exist. In the process ofmonitoring therapeutic digoxin concentrations by vari-ous immunoassay procedures, several investigatorsnoted increased digoxin values in subjects not treatedwith cardiac glycosides (83, 84). Also noted were in-creases in serum digoxin measurements in subjectswhose digitalis therapy had been discontinued (85).Factors giving rise to these apparent digoxin valueswere termed digoxin-like immunoreactive factors(DLIF) (84) or endogenous digitalis-like factors (EDLF)(86). Detectable concentrations of these factors havebeen observed in serum and plasma from healthy adults(87), plasma from volume-expanded dogs (86), new-bOrns (88, 89), pregnant women (90), patients with re-nal impairment (91), and patients with liver dysfunc-tion (92, 93). Aside from DLIF interference with theaccurate measurement of digoxin in human serum,these molecules, because of their structural similarity to

digoxin itself (94), may interact with the Na,K-ATPaseat the digitalis-binding site on the alpha subunit. Pre-sent evidence suggests that the likely tissue source ofthis factor is the adrenal cortex (94, 95).

In addition to the discovery of DLIF, there is compel-ling evidence that ouabain-like factors (OLF) are pre-sent in mammals (96). Hamlyn and Manunta (97) andother investigators (98) have isolated a factor from bothserum and adrenals with ouabain-like properties thatinclude structural similarity to ouabain, Na,K-ATPase

inhibitory activity, and increased concentration inpathophysiological conditions. A review of this workand the potential role of this ouabain-like factor in dis-ease are detailed by Blaustein (53), who summarizes theproposedphysiological effects of endogenous ouabain incontrol of intracellular calcium stores and cell respon-

siveness. Controversy remains, however, about thesource of this endogenous ouabain-like molecule (99).

Substantial arguments still prevail as to what ismeant by digitalis-like activity (100). It is important tounderstand that immunoreactivity does not imply func-tional activity or vice versa. Thus, DLIF should not bemistaken or confused with EDLF or OLF (84), even if,as is suspected, the identity of these factors converges asmore is learned about them. For example, these mole-cules may be related precursors, metabolic products ofeach other, or the same molecule.

Substantial evidence links endogenous digitalis-likefactors with vasoreactivity. Data supporting the hypoth-esis that endogenous digitalis-like factors interact withNa,K-ATPase to induce peripheral vasoconstriction in-clude the following

1) Subjects with some forms of essential hypertensionhave increased sodium-potassium pump inhibitory ac-tivity (101, 102), natriuretic activity (103), and digoxinimmunoactivity in their plasma (104).

2) Spontaneously hypertensive rhesus monkeys havehigh serum concentrations of digoxin-like activity(105).

3) Crude preparations of the natriuretic activity con-strict third-order arterioles, making them more respon-sive to other vasoconstrictive agents such as norepi-nephrine (106).

4) Crude preparations of the natriuretic activity fromurine cause dose-dependent contractions of isolated ano-coccygeus muscle of the rat (which resembles thesmooth muscle of blood vessels) (107).

5) Infusion of ouabain (108, 109) or digoxin (110) intohumans specifically induces peripheral vasoconstric-tion.

6) Injection of antibodies to digoxin lowers the bloodpressure of deoxycorticosterone-salt-retamning hyper-tensive rats (111).

7) Preparations containing digoxin-like immunore-activity from human urine raise blood pressure andprotect rats from acute digitalis toxicity (112).

8) Bolus infusion of digoxin induces vasoconstrictionof epicardial coronary arteries in humans (113).

9) Ouabain-like compounds isolated from human se-rum demonstrate vasoreactivity comparable with thatof ouabain (114).

Cumulatively, the digitalis-like activity of these fac-tors strongly implicate endogenous regulators of theNa,K-ATPase as vasoconstrictive agents involved in theetiology of some hypertensive states.

Clinical Conditions Linked to Dysfunction orModification of Na,K-ATPase ActivityCardiovascularDisease and Hypertension

Pathological conditions in animals and humans in-volving salt and water homeostasis have been associ-ated with alterations in Na,K-ATPase activity and (or)the presence of circulating endogenous digoxin- or oua-bain-like factors (germane articles and reviews are pro-vided in Table 2). Two of the most notable disordersinvolving salt and water homeostasis-heart disease

Table 2. Clinical conditions correlating with alteredNa,K-ATPase activity or presence of modifiers.

Condition Reference.

Cardiovascular disease and hypertensionHeart disease 80 115 116

Hypertension (HTN) 52. 97, 101, 117, 118, 119

HTN, pregnancy-induced 120, 121, 122

HTN, 123hypothyroidism-related

Early experiments by Pamnani et al. aimed at under-standing the effects of hypertension on the myocardiumand vasculature at the molecular level by using rat

.

one-kidney, one-clip, and reduced renal mass-saline.

models of hypertension (102, 139). They demonstrateddecreased myocardial and vascular Na,K-ATPase activ-ity, suggesting that reduced pump activity might becommon in low-renin and other models of essential hy-pertension. In a more recent study, uninephrectomized

Impaired renal function 91 animals treated with deoxycorticosterone or angioten-Renal disease 84, 85, 124 sin II provided insight into the molecular mechanisms

Diabetes and other metabolic disorders that may be involved in some forms of hypertensionAcromegaly (140). Separation of mRNA by Northern analysis of ratAldosteronism cardiac left ventricle, aorta, and skeletal muscle RNA,Diabetes mellltus 128 by use of Na,K-ATPase alpha isoform-specific cDNAObesity

Digoxin toxicityAge-relatedDisease-related .

MultipledruginteractionFetal abnormalities

Growth retardation 130Renal abnormality 130H droce halus 130

aneuploidy ‘

Nonimmune hydrops 130

probes, showed tissue-specific changes in isoform ex-pression of mRNA transcripts in response to increasedintravascular pressure.

Greater concentrations of DLIF and OLF have beennoted in women with pregnancy-induced hypertension

.

(preeclampsia) than in normal pregnancy (84, 141).Pregnancy is a volume-expanded condition, and in bothnormal and hypertensive pregnancy the anomalousDLIF values resolve rapidly upon delivery (90, 120-122). Increased concentrations of DLIF are associated

Low birth weight 131 with acute and chronic renal disease (91, 124) and withPreterm infants 132 fluid retention due to hepatic failure (92, 93). Endoge-

Neurological disorders nous OLF is reported markedly increased in hyperten-Alzheimer disease 133, 134, 135, 136 sion that is due to hypothyroidism (123) and congestiveBipolar disorder 64 heart failure (116). DLIF is also significantly increased

Pulmonary conditions in the plasma of human subjects with electrocardio-Pulmonary disease 137 graphic evidence of reversible cardiac dysfunction in-Chronic obstructive 138

pulmonary diseaseduced by physical exhaustion (142). Weinberg et al.(143) used peritoneal dialysis fluid from patients withchronic renal failure to chromatographically isolate

three molecular species having DLIF activity. Of theand hypertension-are causally related. Hypertension three, one had a retention time identical to ouabain, andincreases the risk of myocardial infarction, congestive one had a retention time identical to digoxin. And Yuanheart failure, renal failure, and cerebral stroke (52). et al. (144) showed that administration of chronic lowThe initial reduction in myocardial contractility that doses of ouabain was associated with the development ofoccurs in some forms of heart failure results in vasocon- hypertension in normotensive rats as well as in ratsstriction and peripheral resistance. The constriction of having various degrees of reduced renal mass. Mean

the vascular beds in the kidneys causes salt and water blood pressure increased with the degree of mass reduc-retention. Alterations in Na,K-ATPase activity or ex- tion but was significantly greater than in controls evenpression can alter vascular or cardiac contractility by for rats with no renal mass reduction.affecting sodium homeostasis (37). Thus, the sequence In an effort to determine whether ouabain itself actsof events previously described strongly correlates with as a hypertensive agent or simply exacerbates the hy-the involvement of a circulating inhibitor of sodium pertensive action of mineralocorticoids, Sekihara et al.pump activity in the pathogenesis of both cardiovascu-lar disease and hypertension.

Hypertension, as evidenced by persistently high arte-

(145) treated mononephrectomized rats with ouabain (1mg), deoxycorticosterone acetate (5 mg), or a combina-tion of both, weekly for 6 weeks. Both ouabain and

rial blood pressure, can be idiopathic or secondary to deoxycorticosterone acetate lacked hypertensive actionunderlying conditions. Essential (idiopathic hyperten- individually at the dosage given but, in combination,sion) is a poorly understood though relatively common they produced a significant increase in blood pressure asdisease. An inherited predisposition has been suggested, well as cardionephromegaly and histopathologicaland such individuals may be especially sensitive to di- changes consistent with the effects of an elevation inetary salt (117). Hypertension secondary to primary blood pressure. The authors concluded that, in thosealdosteronism (126), other endocrine disorders, and hypertensive individuals secreting greater concentra-pregnancy often tends to resolve once the underlying tions of mineralocorticoids, ouabain might amplify theproblem is alleviated, hypertensive effect.

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119, 125126118, 119, 127,119

64,65,6769

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DLIF, isolated from urine by use of cross-reactivity todigoxin antibody as evidence of activity, was admiriis-tered to normotensive rats to determine its cardiovas-cular effects (112). After an initial 60-mm stabilizationperiod, infusion of DLIF in 10 animals caused the meanarterial pressure to rise from 124.3 (±1.9) to 140.4(±5.2) mmHg at 7.5 mm, induced diuresis, and slowedthe heart rate. These results, coupled with data docu-menting the presence of endogenous digitalis-like com-pounds 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 hyper-tension (127), found that not only do type I (insulin-depen-dent) diabetic individuals have a familial predispositionfor essential hypertension, but also normotensive offspringof parents who are nondiabetic but have essential hyper-tension show increased concentrations of plasma insulinand reduced insulin sensitivity; moreover, Na retentionis characteristic of both type I and type II (non-insulin-dependent) diabetics. These authors also report that intra-cellular calcium is increased in adipocytes, in part viainsulin’s inhibition of Ca2,Mg-ATPase, and that insu-lin may increase renal sodium retention and influence theactivity of transmembrane electrolyte pumps. Insulin reg-ulation of vascular Na,K-ATPase gene expression is citedby Tirupattur et al. (148) as an important factor in thedevelopment of hypertension in diabetes. mRNA encoding

both the alpha 1 and alpha 2 isoforms was identified invascular smooth muscle cells derived from embryonic ratthoracic aorta. Although the predominant isoform wasalpha 1, only the concentrations of the alpha 2 isoformincreased in response to insulin treatment. The overallincrease in ouabain-inhibitable Na,K-ATPase activity invascular smooth muscle cells in response to insulin treat-ment suggests that, in the absence of insulin or in insulin-resistant states, Na,K-ATPase activity could decrease, re-sulting in increased vascular contractility and bloodpressure.

In their review, Clerico and Giampietro (119) citereports of decreased sodium pump activity in the nerves,heart, and aorta of diabetic humans, and in rats withstreptozotocin- or alloxan-induced diabetes; however,there was a paradoxical increase in Na,K-ATPase activ-ity in the kidneys of streptozotocin-induced diabeticrats. The authors speculate that these tissue differencescould be due to tissue-specific regulation of Na,K-ATPase activity or metabolism. They further suggestthat in metabolic diseases such as diabetes meffitus,obesity, and acromegaly (which have in common in-creased sodium retention and volume expansion) in-creased sodium intake, hyperinsulinemia, or increasedconcentrations of growth hormone could trigger the re-lease of an endogenous digitalis-like factor that couldmodulate the pump and increase blood pressure. A morerecent study by Chen et al. (128), using a similar dia-betic rat model system, confirmed the previous findings.In addition, Chen et al. found greater amounts of adigitalis-like factor in the plasma and urine of their

hypertensive diabetic rats than in controls. A review bySewers and Khoury (118) highlights the additional riskof cardiovascular disease when hypertension accompa-nies diabetes mellitus and discusses additional risk fac-tors such as obesity, genetics, and ion transport control.

Digoxin Toxicity

Although advanced age is not a pathological condi-tion, age-related changes in renal clearance and volumeof distribution can increase the likelihood of digoxintoxicity (65). In addition, evidence based on basal met-abolic rate measurements indicates that whole-bodyNa,K-ATPase activity decreases with age (64, 129).This suggests that, even though their serum cligoxinconcentrations may be within the therapeutic range,digitalis toxicity in the elderly might result, in part,from inhibition of an already less-active sodium/potas-sium transport system. Low tolerance to digoxin is alsoassociated with certain disease states, particularly thoseinvolving hypokalemia, and with multiple drug interac-tion, as discussed previously.

Fetal Abnormalities

The neonatal period is associated with volume expan-sion and sodium imbalance. As stated previously, age-related changes in Na,K-ATPase alpha isoform expres-sion in the rat have been observed (23). DLIF issignificantly increased in the plasma of newborn infantsbut usually returns to normal within several days (88,132). Increased concentrations of digoxin-like immuno-reactivity have been observed in fetuses having growthretardation, renal abnormality, hydrocephalus, aneu-ploidy, and nonimmune hydrops (reviewed in ref. 130),again suggesting a role for endogenous pump modifiersin the regulation and genetic expression of Na,K-ATPase and in the etiology of some pathological pro-cesses.

Neurological Disorders

Bipolar illness (manic depression) is characterized bysevere mood swings that alternate between episodes ofirritability, excessive energy, and distractibility (ma-nia), and mental and motor slowdown to the point ofstupor (depression). Patients with bipolar illness exhibitaltered 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-ATPaseactivity could lead to mania by increasing membraneexcitability and neurotransmitter release. A greater de-gree of pump inhibition, and consequently depolariza-tion block, could result in depression by decreasing neu-rotransmitter release (149). Patients in manic statesshow increased sodium retention and intracellular cal-cium concentrations, and therapeutic modalities such aslithium or calcium-channel blockers are theorized toaffect sodium-calcium exchange (64). Interestingly,symptoms mimicking those observed in bipolar patientsoccur with digitalis toxicity. These can include confu-sion, disorientation, drowsiness, lethargy, agitation,and hallucinations (137), further implicating Na,K-

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ATPase as an etiological agent in bipolar illness. Recentevidence in our laboratory suggests that DLIF (mea-sured with a digoxin-specific immunoassay) is increasedin acutely psychiatrically ill bipolar patients relative torecovered bipolar patients (unpublished). Increased en-dogenous ouabain-like compounds that excessively sup-press Na,K-ATPase have been proposed as a mechanismin this disorder (133).

Alzheimer disease (Al)) is an age-related neurodegen-erative disorder of unknown etiology. In addition towell-documented neuropathological changes such asneurofibrillary tangles and neuritic plaque formationwith accumulation of beta-amyloid (136), AD is associ-ated with lower cerebral blood flow and decreased use ofoxygen and glucose, especially in areas exhibiting neu-ropathological changes (134). About 50% of the energyexpenditure of resting brain is believed to supportNa,K-ATPase activity. Hank et al. (134) found a de-crease in ouabain binding of brain tissue from AD pa-tients in comparison with age-matched controls. Thereduction in ouabain binding was especially evident inthe neocortex, an area predominantly affected in Al)patients. A decrease in ouabain-inhibitable Na,K-ATPase activity in the brain subcortical but not corticalareas of patients with Al) was also noted by Liguni et al.(135). Although sodium pump dysfunction may not becausal in this disorder, the progressive dementia asso-ciated with AD may in part be due to alterations inNa,K-ATPase activity. To date, the presence of possibleendogenous pump modifiers in relation to AD has notbeen described.

Pulmonary Conditions

Little has been published in this area. Varsano et al.(138) used a digoxin radioimmunoassay to show thatpatients with advanced chronic respiratory failure hadgreater concentrations of DLIF than did controls.Chronic obstructive pulmonary disease and other formsof advanced respiratory disease are frequently associ-ated with water and sodium retention, and the authorssuggested that increased DLIF might be an attempt tocontrol water and sodium metabolism.

In summary, conditions associated with volume ex-pansion or alterations in sodium homeostasis (see Table2), many of which have been described here, also showchanges in Na,K-ATPase activity, increases in endoge-nous digitalis-like substances, or both. Modification ofpump activity can occur in response to changes in iso-form expression or modulation by endogenous pumpinhibitors or activators. As to whether altered regula-tion of Na,K-ATPase activity is directly linked to any ofthese conditions remains to be elucidated. Preliminarydata identifying polymorphisms in the human Na,K-ATPase alpha and beta subunit genes offer the potentialof linking these and other genetic differences to specificclinical disorders. Locating genetic mutations of Na,K-ATPase or of the endogenous modifiers may soon beadded to the repertoire of test capabifities in the clinicalchemistry laboratory, providing early identification of

individuals at risk for a variety of Na,K-ATPase-asso-ciated diseases.

This work was supported in part by Grant RO1-HL36172 fromthe National Institutes of Health.

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