Proc. Natl. Acad. Sci. USAVol. 90, pp. 8189-8193, September 1993Physiology

Physicochemical characterization of a ouabain isomer isolated frombovine hypothalamus

(endogenous digitalis/active sodium transport/sodium pump/circular dichroism spectroscopy/mass spectrometry)

ADRIENNE A. TYMIAK*, JON A. NORMAN*t, MARK BOLGAR*, GERALD C. DIDONATO*, HELEN LEE*,WILLIAM L. PARKER*, LEE-CHIANG Lot, NINA BEROVAt, KoJI NAKANISHIt, EDGAR HABER§,AND GARNER T. HAUPERT, JR.II*Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543; tDepartment of Chemistry, Columbia University, New York, NY 10027; andlRenal Unit, Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114

Communicated by Avram Goldstein, May 25, 1993 (received for review February 1, 1993)

ABSTRACT Recent reports have shown the presence of aouabain-like inhibitor of Na+/K+-ATPase in humans. Wehave purified a bovine hypothalamic Na+/K+-ATPase inhib-itory factor (HIF) by using affimity chromatography combinedwith HPLC. This inhibitor has a molecular weight of 584 asdetermined by ion-spray mass spectrometry, making it isobaricwith ouabain. Glycosidase treatment or acid hydrolysis of HIFreleased only L-rhamnose, the hexose isomer found in ouabain,as detected by chiral GC/MS. Additionally, enzymaticallygenerated desrhamnosyl HIF was found to have a molecularweight of 438, as does ouabagenin, the aglycone of ouabain.HIF and its aglycone were indistinguishable from ouabain andouabagenin, respectively, by reversed-phase HPLC retentiontimes. However, derivatization with naphthoylimidazole fol-lowed by HPLC revealed different retention times for naph-thoylation products of HIF and ouabain. Subsequent CDspectroscopy on isolated naphthoylation products of HIF andouabain confirmed that they were different. This study pro-vides chromatographic and spectroscopic evidence that oua-bain and HIF are isomeric cardenolides. The structural dif-ference is presumed to account for the significant differences inbiological properties observed for HIF and ouabain.

The search for an endogenous inhibitor of Na+/K+-ATPasehas been supported by the occurrence of digitalis-like car-diotonic steroids in plants and the presence of a highlyconserved binding site for ouabain in mammalian tissues (1).Obstacles faced in attempts to identify such endogenousinhibitors have included the very low concentrations presentin mammals and the difficulty ofobtaining stringently purifiedsamples for analysis (2, 3). Hamlyn and associates (4, 5)described a ouabain-like inhibitor of Na+/K+-ATPase fromhuman plasma that was indistinguishable from ouabain (Fig.1, structure 1) by several criteria including mass spectrom-etry, immunoreactivity, and biological assays. Their resultsraised interesting speculation about the ability of humans tosynthesize ouabain, previously known only as a plant prod-uct. Both portions of ouabain, specifically the highly func-tionalized steroid ouabagenin (Fig. 1, structure 2) and thesugar L-rhamnose (structure 3), are unprecedented in mam-malian biosynthesis. Outside of the plant kingdom, onlycardiotonic steroidal aglycones have been isolated from toads(6). In the present study, a Na+/K+-ATPase inhibitor frombovine hypothalamus (7) was purified by an enzyme affinitymethod coupled with preparative HPLC steps. Microanalyt-ical methods were developed to directly compare the affinity-purified hypothalamic inhibitory factor (HIF) with authenticouabain. Both HIF and ouabain were shown to have identical

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4HO@IO

I Ouabain 2 Ouabagenin ^ Rhamnose

FIG. 1. Chemical formulae of ouabain, 1; the aglycone of oua-bain, ouabagenin, 2; and rhamnose, 3.

molecular weights and to yield L-rhamnose and isobaricaglycones upon hydrolysis. The stereochemistry of the twoinhibitors was then compared by microscale naphthoylationfollowed by HPLC separation of derivatives for CD spec-troscopy. HIF and ouabain naphthoates proved to be differ-ent, as evidenced by distinct HPLC retention times andcharacteristic CD spectra. These data demonstrate that thebovine hypothalamic Na+/K+-ATPase inhibitor HIF is anisomer of ouabain and that the structural difference betweenHIF and ouabain accounts for observed differences in theirbiological properties (8-11).

METHODSPurification of HIF from Bovine Hypothalamus. HIF was

extracted from bovine hypothalamus and chromatographedas described (9, 12), yielding samples free of protein, lipid,vanadate, and cations known to interfere with Na+/K+-ATPase activity. Dry extracts were dissolved in 10 ml ofwater, applied to CHP20P resin (MCI gel, Mitsubishi Chem-ical, Tokyo), and eluted with a linear gradient of 0-100%methanol (240 ml, 2 hr). Na+/K+-ATPase inhibitory activitywas monitored by the 86Rb+ uptake assay using humanerythrocytes (12). A peak of activity obtained at 84 mincontained 105-750 pmol ofouabain equivalent bioactivity perkilogram of hypothalamus, where 1 unit of ouabain-equivalent bioactivity is defined as 0.75 pmol of HIF (9, 13).HIF was further purified by an affinity step employing

SDS-extracted canine kidney Na+/K+-ATPase coupled toparamagnetic iron particles (Bio Mag 4100, Advanced Mag-

Abbreviations: HIF, hypothalamic inhibitory factor; FAB, fast-atombombardment.tPresent address: Vical Inc., San Diego, CA 92121.§Present address: Division of Biological Sciences, Harvard School ofPublic Health, Boston, MA 02115."To whom reprint requests should be addressed.

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netics, Cambridge, MA) via glutaraldehyde crosslinking ofprimary amino groups. CHP20P-purified HIF was mixedgently with the Na+/K+-ATPase-coupled magnetic beadsovernight at 4°C in 20mM imidazole, pH 7.4/10mM MgCl2/2mM H3PO4/1 mM EDTA/250 mM sucrose. The beads-HIFcomplex was separated from supernatant with a magneticbar, and HIF was selectively eluted with 5 mM EDTA inimidizole (pH 7.4) (37°C, overnight) because HIF inhibition,and consequently ATPase binding, is Mg2+-dependent. Con-centrated eluant was then chromatographed by reconstitu-tion in 250 ,ul of water injection into a Waters Resolvereverse-phase C18 column at a flow rate of 1 ml/min, andelution with water for 3 min followed by a linear gradient to13:87 acetonitrile/water in 4 min and continuing as anisocratic separation for 40 min. HIF (a single peak at 18-21min) was concentrated to dryness, rechromatographed asabove, and stored under argon gas at -80°C. Maximum yieldwas 1 ug of purified HIF from 5 kg of bovine hypothalamus.

Liquid Chromatography/Mass Spectrometry. Sampleswere dissolved in 10 ,ul of a 9:1 mixture of 10 mM pyridineacetate buffer (pH 4.7) and methanol, injected onto a PLRP-Scolumn (4.4 x 150 mm; Polymer Laboratories, Amherst,MA), and eluted at 1 ml/min with a gradient from 9:1 to 8:2of 2 mM ammonium acetate/acetonitrile in a Waters 600MSHPLC. The HPLC effluent was interfaced to a Sciex API IIImass spectrometer (Thornhill, Ontario, Canada) with a 20:1flow split for ion-spray spectrometry (from 350 to 1000 Da in2.8 sec).Gas Chromatography/Mass Spectrometry. Sugars were

detected by chemical ionization GC/MS with a Finnigan TSQ4600 mass spectrometer. For hexose identification, driedsamples were derivatized (14), injected (splitless at 225°C) ona 30-m DB-1 capillary column (J & W Scientific, RanchoCordova, CA; 122-1032), chromatographed with a steppedtemperature program (120°C for 1 min; 16°C/min linear rampfrom 120°C to 180°C; 5°C/min linear ramp from 180°C to250°C), and ionized with ammonia gas. Summed ion chro-matograms (m/z 470 and 380) provided retention times for aand anomers of each sugar. For chirality determinations,dried samples were derivatized (15) and analyzed by GC/MSusing splitless injection and methane ionization under columnconditions described previously (16). Assignments weremade from (M+H)+ ion (m/z 549) traces with referencestandard coinjections (D-rhamnose kindly provided by L. M.Lerner, State University of New York, Brooklyn).

Naringinase Hydrolysis. Naringinase (Sigma N-1385; 50 Alof a 2-mg/ml solution in 10 mM pyridine acetate, pH 4.7/methanol, 9:1) was added to either ouabain (0.55 ug, intriplicate), a bioactive equivalent amount of HIF, or a waterblank, each dried in a microcentrifuge tube. Samples weresealed, incubated with shaking at 37°C for 24 hr, subjected tocentrifugal ultrafiltration at 13,000 x g for 5 min (AmiconMicrocon-10 ultrafiltration device prewashed with the 9:1buffer), and combined with an ultrafiltered wash (50 ul of 10mM pyridine acetate, pH 4.7/methanol, 1:1) of its respectivehydrolysis tube. Ultrafiltrates were dried and stored sealed at-20°C prior to LC/MS analysis.Acid Hydrolysis. Subsamples (10 pmol of each in water) of

ouabain or HIF were dried in 100-,ul microconical glass vials,dissolved in 50 ,ul of2M HCl (Sequenal grade, Pierce), heatedat 110°C for 5 min in a dry block chilled with ice, andconcentrated prior to derivatization.

Naphthoylation and CD Spectroscopy. Each sample of oua-bain or affinity-purified HIF (500 pmol) was dried in a silylatedvial, dissolved in 250 Al of anhydrous acetonitrile, and thenstirred for 3 hr at room temperature with 1.5 mg of naphtho-ylimidazole and 0.4Au1 of 1,8-diazabicyclo[5.4.0]undec-7-ene(17). Reactions were quenched with 1 ml of acetonitrile/water,1:4, and applied to Waters C18 SepPak cartridges. The car-tridges were washed sequentially with the following acetoni-

trile/water mixtures: 2 ml of 1:4, 8 ml of 2:3, and 5 ml of 1:1.The products were eluted with 5 ml of acetonitrile and thensubjected to a Phenomenex C18 column (4.6 x 250 mm, 10 ,um)with isocratic elution at 1 ml/min with methanol/water, 92:8,and fluorescence detection (Shimadzu RF-551 detector;234-nm excitation, 374-nm emission). A Vydac C18 column(4.6 x 250 mm, 10-,um particle size) was employed with 1ml/min isocratic flow of methanol/water, 82:18, for coelutionof ouabain and HIF pentanaphthoates (I and II), as well as forseparation of HIF derivatives IIIa and IIIb. CD spectra (inmethanol) of HPLC-purified samples were obtained with aJasco J-720 spectropolarimeter.Mass Spectrometry of Naphthoates. After CD analysis,

naphthoate samples were subjected to fast-atom bombard-ment (FAB) MS analysis on a JEOL HX110-110 instrument,using a matrix consisting of 3-nitrobenzyl alcohol.

RESULTSIdentification ofL-Rhamnose in HIF. The glycosidic linkage

in HIF was first probed with naringinase, an enzyme prep-aration containing a-L-rhamnosidase activity. Bioassay mon-itoring of naringinase hydrolysates showed that ouabain andHIF were equally susceptible to this enzymatic inactivation,each being cleaved to its respective 100-fold-less-active ag-lycone. Sugar analysis on naringinase hydrolysates was notpossible because the enzymatic blanks contained a variety ofsugars at picomolar concentrations, comparable to the sugarconcentration expected in the HIF hydrolysate. Acid hydro-lysis proved to be a suitable method for generating free sugarsfor analysis, although ouabagenin decomposed during hydro-lysis. Conditions to separate rhamnose and other isobaricsugars by GC/MS detection of persilylated derivatives wereestablished. By this method, acid hydrolysates of HIF andouabain were found to contain only identical mixtures of a-and ,3rhamnose derivatives (Table 1). Subsequently, chiralGC/MS analysis of pertrifluoroacetyl derivatives demon-strated that HIF and ouabain hydrolysates contained onlyL-rhamnose. Since derivatized rhamnose isomers were nar-rowly resolved in these chiral GC/MS analyses of pertriflu-oroacetyl derivatives, HIF-derived rhamnose was unambig-uously identified as the L isomer by coinjection with authen-tic rhamnose standards.

Molecular Weight of HIF. Ion-spray LC/MS proved to bethe most sensitive method for detecting nanogram quantitiesofouabain. To control for any drifts in HPLC retention times,ouabain samples were analyzed before and immediately afteranalysis of the HIF sample. Full-scan mass spectra (Fig. 2)provided the first direct evidence that native HIF and oua-bain have the same molecular weight, 584. Also, by obtainingfull-scan mass spectra to m/z 1000, the possibility that HIFwas multiply glycosylated could be discounted.

Molecular Weight ofHIF Aglycone. Since HIF and ouabainare isobaric and both inhibitors contain rhamnose, the mo-lecular weight ofHIF aglycone can be assigned by difference

Table 1. GC/MS retention times for persilylated sugars isobaricwith rhamnose

Retention time ofderivatized products,

Underivatized sugar min:sec

L-Rhamnose 7:01, 7:52L-Fucose 7:34, 8:042-Deoxy-D-galactose 8:02, 8:252-Deoxy-D-glucose 8:08, 9:04Quinovose 8:27, 9:13Ouabain acid hydrolysate 7:00, 7:53HIF acid hydrolysate 6:59, 7:52

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5

4A

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602

L60750~~00 ----0

585

602

0 500 600nhz

*1

3t

It%100

.ia

at700 800

C a,1c

'IA 50

700 800 at

2A

3

1

1 ~~~Ouabain

HEF Hydrolysate

0 2;0 4;0 60 8.0Minutes

4563 5OO 60M/Z

~~~~~~C. .1.

300 400 600

FIG. 2. LC/MS data for ouabain and HIF determined that bothhave a molecular weight of 584 as indicated by the pseudo-molecularions at m/z 585, 602, and 607, (M+H)+, (M+NH4)+, and (M+Na)+,respectively. (A) Overlaid extracted ion chromatograms (m/z 585)for ouabain and HIF. (B) Full-scan ion-spray mass spectrum summedover the peak for ouabain (retention time, 6.85 min) and background-subtracted. (C) Full-scan ion-spray mass spectrum summed over thepeak for HIF (retention time, 6.78 min) and background-subtracted.

to be identical to that of ouabagenin (see Fig. 1). However,a direct measure ofthe molecular weight ofHIF aglycone wasdesirable. In parallel experiments, HIF and ouabain weretreated with naringinase, lyophilized, and analyzed by LC/MS. The LC/MS proffles obtained for ouabain and HIFhydrolysates showed that both glycosides and aglyconeswere indistinguishable by HPLC retention time and molec-ular weight (Fig. 3). Both hydrolysates contained similaramounts of starting material and product, confirming the Rb+uptake measurements which suggested that ouabain and HIFare equally susceptible to glycolysis by naringinase.

Naphthoylation of Ouabain and HIF. The previous charac-terization experiments did not distinguish HIF from ouabain.To clarify this crucial point, acylation of reactive hydroxylswas used as a means of amplifying any structural differencesbetween these two steroidal glycosides. Naphthoyl deriva-tives were chosen for this purpose because they are obtainedin high yield, give rise to fluorescent products, exhibit intenseUV absorptions, and, most importantly, exhibit characteris-tic exciton-coupled CD spectra (17). Samples (300 ng) ofHIFand ouabain were naphthoylated under conditions specifi-cally optimized for ouabain pentanaphthoate production.Reaction mixtures were analyzed by HPLC with fluores-cence-detection (Fig. 4). As expected, ouabain yielded onlythe 1,19,2',3',4'-pentanaphthoate. Although similar in thecrude reaction HPLC traces, coinjection demonstrated thatpeak II purified from the HIF reaction mixture was differentfrom ouabain pentanaphthoate I (Fig. 5A).Because of the minute amounts of peaks II and III (Fig. 4),

unexpected difficulties were encountered during FAB MS(positive-ion mode). However, the FAB mass spectrum ofpeak II showed two important diagnostic ions at m/z = 609(intense) and 1354 (signal/noise = 2), corresponding, respec-

FIG. 3. LC/MS data for naringinase hydrolysates of ouabain andHIF determined that both have a molecular weight of438 as indicatedby the pseudo-molecular ions at m/z 439 and 456, (M+H)+ and(M+NH4)+, respectively. (A) Overlaid extracted ion chromatograms(sum of m/z 439 and 585) for ouabain (upper trace) and HIF (lowertrace) hydrolysates. Peak 1, solvent front; peak 2, aglycone; peak 3,glycoside. (B) Full-scan ion-spray mass spectrum summed over thepeak for ouabagenin (retention time, 5.53 min) and background-subtracted. (C) Full-scan ion-spray mass spectrum summed over thepeak for HIF aglycone (retention time, 5.51 min) and background-subtracted.

tively, to that of rhamnose trinaphthoate and a pentanaph-thoate isobaric with that of ouabain. In addition to thepentanaphthoate II, a more lipophilic peak, III, was observedin the HIF reaction mixture (Fig. 4B) which, during subse-quent HPLC, was further separated into IIIa and IlIb (Fig. 6).

0.01~

0.0 5.0 10.0 15.0Minutes

20.0 25.0 30.0

FIG. 4. Fluorescence-detected HPLC profiles for crude naph-thoylation reactions. Peaks eluted before 10 min represent by-products ofthe derivatizing reagent. (A) Ouabain reaction (I, ouabainpentanaphthoate). (B) HIF reaction (II, HIF pentanaphthoate; III,HIF hexanaphthoate).

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|0.2 I8 [I10 4 8 12 16Minutes

I+1

i0-1

-2200 250 300 350

Wavelength (nm)

+1

0

C

-l

-2

200 250 300 350Wavelength (tm)

FIG. 5. (A) Fluorescence-detected HPLC trace for coinjectedsamples of ouabain pentanaphthoate (I) and HIF pentanaphthoate(II). (B) CD spectrum (acetonitrile) of ouabain pentanaphthoate (I).(C) CD spectrum (acetonitrile) of HIF pentanaphthoate (II). mdeg,Millidegrees.

Although direct HPLC coinjection comparisons were notmade, IlIa and IlIb were eluted near the retention timeexpected for ouabain hexanaphthoate. Assignment of IlIaand IIIb as hexanaphthoates of HIF followed from their UVabsorption and fluorescence, C18 lipophilicity, and measure-ment of m/z 609 in their individual FAB mass spectra.However, these assignments are tentative because it was notpossible to detect the M+ peak in the FAB mass spectrum;further, the pentanaphthoate peak II has not been furthernaphthoylated to check for its conversion to IIIa and IlIb,due to lack of material.CD Comparison of Ouabain and HIF Naphthoates. An

authentic sample of ouabain 1,19,2',3',4'-pentanaphthoategave a clearly split CD curve with a positive Cotton effect,reflecting exciton coupling (18) between the various naph-thoate groups (Fig. 5B); ouabain pentanaphthoate peak I (Fig.4) also showed the same CD. In sharp contrast, HIF pen-tanaphthoate II exhibited no distinct CD Cotton effect (Fig.5C). This difference could not be explained simply by a lowerconcentration of the HIF derivative, since the UV intensitiesfor solutions of I and II submitted to CD measurements werenearly identical. The CD spectrum of II is very diagnostic anddemonstrates that the exciton coupling among the five naph-thoate groups is internally compensated, a fact that has to beaccounted for in the structure of HIF pentanaphthoate. TheCD spectra of solutions of the "HIF hexanaphthoates" Illaand IlIb showed nearly opposite split CD curves (Fig. 6B andC), but these CD data can not yet be interpreted.

DISCUSSIONDuring the course of isolation, no physical or chemicalevidence was found to differentiate HIF from ouabain. Earlysingle-ion-monitoring ion-spray tandem MS measurements(selecting for m/z 602 and observing daughter ions at m/z

439) suggested that HIF was isobaric with ouabain and that

0.3 A

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-10

10.0 20.0Minutes

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250 300Wavelength (mm)

+5 1

30.0

-5 V

-10200 250 300 350

Wavelength (mm)

FIG. 6. Characterization of lipophilic HIF naphthoylation prod-ucts. (A) Fluorescence-detected HPLC trace showing the separationofHIF hexanaphthoates IlIa and IlIb. (B) CD spectrum (acetonitrile)of IIIa. (C) CD spectrum (acetonitrile) of IlIb. mdeg, Millidegrees.

it was also a glycoside. Each isolation step successful inconcentrating HIF would also have isolated any ouabainpresent. Even by HPLC, native HIF and ouabain werecoeluted. For this reason, a sequential blank procedure wasused to control for the possibility ofcontamination essentiallyas described for the isolation of morphine and codeine frombovine brain (19). All purification columns and apparatus,including HPLC pumps and injectors, were dedicated for thepurification of HIF, and blank runs carried through all stepsat the scale of tissue-purification reactions showed no traceof Na+/K+-ATPase inhibitory activity.Approximately 1 ug of affinity-purified HIF was accumu-

lated for structural analysis. Due to our initial observationsand the small amount of purified product, characterizationstudies were carefully designed to be sample-sparing andmost likely to reveal any subtle differences between HIF andouabain. To achieve this end, we relied heavily on thesensitivity afforded by derivatization for MS, fluorescence-detected HPLC, and CD spectroscopy of naphthoate deriv-atives (17). Microscale derivatization, fluorescence-detectedanalytical chromatography, and CD spectroscopy each inde-pendently provided evidence that ouabain and HIF weredifferent.

Sugar analysis and LC/MS measurements before and afterhydrolysis unambiguously established that HIF was anL-rhamnoside of an aglycone isobaric with ouabagenin. Pre-viously, L-rhamnose was identified in rabbit skin extracts(20), although its biosynthesis was not established. Rham-nose, commonly found as a glycoconjugate in plants andenteric bacteria, is readily available to mammals from dietaryand environmental sources. The ubiquitous presence of thissugar may also explain detection of rhamnose-selective lec-tins in mammalian cells (21). While identification of L-rham-nose does not rule out the possibility that HIF is an endog-enous substance, it also does not distinguish HIF fromouabain.

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With the sugar portion assigned, subsequent characteriza-tion studies were designed to probe the structure and stereo-chemistry of HIF aglycone. The versatile exciton chiralitymethod, based on through-space coupling of chromophores,has been used extensively in assigning absolute configura-tions of various compounds, including steroids and glyco-sides (18, 22). Although the approach ofderivatization for CDspectroscopy was theoretically useful for characterizingHIF, the small sample size necessitated methods develop-ment. Studies with ouabain showed that naphthoylation toouabain pentanaphthoate, a product with a characteristic CDcurve, could be achieved without forming the CD-inactiveouabain hexanaphthoate derivative. In addition, incorpo-ration of multiple naphthoyl groups allowed high-sensitivitydetection of products by their fluorescence and, after puri-fication, adequate CD sensitivity to measure the sign andintensity of exciton coupling for submicrogram samples (17).Derivatization of native HIF was then attempted, since itpermitted comparison of the relative stereochemistry be-tween the steroidal and L-rhamnose subunits of HIF andouabain.

Naphthoylation of HIF provided several clues for under-standing how it differs from ouabain. Pentanaphthoates ofouabain and HIF, although similar chromatographically, hadvery distinct CD spectra. The results demonstrate that thespatial arrangement of chromophores is different for the twoderivatives, as might be expected for regio- or stereochemicalisomers. Also, the formation of HIF hexanaphthoates underconditions where none forms for ouabain suggests that atleast one acylation site is more accessible in the HIF struc-ture. The greater reactivity of HIF could result from adecrease in steric hindrance or an increase in availability ofreactive hydroxyls (as might occur for an isomeric rhamno-side of ouabagenin). Since two hexanaphthoates ofHIF (IIIaand IIIb) were isolated, it is likely that two hydroxyl groupsin HIF are altered relative to ouabain. More precise inter-pretation of these data will be possible after a stereochemicalanalysis of ouabagenin and HIF aglycone.

This subtle structural difference has a significant effect onbiological activity. Thus, the binding and dissociation of HIFin intact renal tubular cells showed positive cooperativity inbinding reactions and a relatively rapid dissociation rateconstant, both characteristics different from those of ouabainand consistent with physiologic regulation in vivo (23). Par-tially purified HIF was further shown to have positiveinotropic effects equipotent with ouabain in cultured ratmyocytes, but at doses nearly 3 orders of magnitude less thanthose required for ouabain (8). These studies also showedthat at the same intracellular Ca2+ concentration in myo-cytes, ouabain was toxic whereas HIF was not. Whileouabain was without effect, isolated vascular rings from ratpulmonary artery and aorta were potently constricted by HIFin vitro (11), consistent with the proposed role of an endog-enous Na+/K+-ATPase inhibitor in the regulation of vaso-constriction and pathogenesis ofhypertension (24, 25). Whilethe abundance and biological properties of HIF suggest itsrole as an endogenous inhibitor of Na+/K+-ATPase, it is notknown whether HIF is truly the product of de novo mam-malian biosynthesis. Although impossible to exclude at thispoint, we feel it extremely unlikely that HIF is ouabain thatis isomerized during the isolation process. Methanolic ex-traction used in the first step of the isolation suppressedenzymatic activity, and Sephadex LH-20 gel filtration re-moved any remaining macromolecules from ouabain or HIF.Isolation conditions were intentionally chosen to be mild,minimizing the likelihood of chemical isomerization. Finally,ouabain isomerization was not observed during the develop-ment of various derivatization reactions.

Regardless of its origin, physical evidence detailed in thisstudy shows that HIF is an isomer of ouabain; this structuraldifference accounts for the biological properties which dis-tinguish the pharmacologic inhibitor ouabain from the puta-tive physiologic mammalian analogue HIF. Determination ofthe exact structure of HIF, while technically challenging, isdesirable since it would probably reveal a general strategy forimproving the therapeutic index of cardiac glycosides andprovide a probe to study the proposed role of endogenousNa+/K+-ATPase inhibition in the pathogenesis of hyperten-sive disease.

We are grateful to Dr. Ron Orlando (Suntory Institute for Bioor-ganic Research, Osaka, Japan) for MS measurements and Ms. S.Taylor (Bristol-Myers Squibb Pharmaceutical Research Institute)for chromatographic studies. This work was supported by a grantfrom the Bristol-Myers Squibb Pharmaceutical Research Institute(G.T.H.), by National Institutes of Health Grant 34509 (K.N.), andby National Science Foundation grant INT90-15531 (K.N. andN.B.).

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