Proc. Nati. Acad. Sci. USAVol. 86, pp. 10080-10084, December 1989Medical Sciences

Positive inotropic effects of the endogenous Na+/K+-transportingATPase inhibitor from the hypothalamus

(endogenous digitalis/contractility/actlve sodium transport/cardiac myocytes/ouabain)

HAIFA A. HALLAQ AND GARNER T. HAUPERT, JR.*Renal and Cardiac Units, Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114

Communicated by Seymour S. Kety, August 11, 1989 (received for review April 4, 1989)

ABSTRACT Bovine hypothalamus contains a nonpeptidicsubstance that inhibits purified Na+/K+-transporting ATPase[ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37]reversibly with high affinity by a mechanism similar to, but notidentical to, that of the cardiac glycosides. It possesses some ofthe characteristics ascribed to a putative endogenous "digitalis-like" compound that has been implicated in the control of renalsodium excretion and the pathogenesis of essential hypertensionin man. To determine whether this hypothalamic Na+/K+-transporting ATPase inhibitor might have physiologic proper-ties in cardiac tissues, its effects on Na+ pump inhibition,accumulation of cytosolic free calcium, and contractile responsewere studied in cultured, spontaneously contracting neonatal ratcardiocytes. The hypothalamic factor potently inhibited the Na'pump in these cells, increased myoplasmic free calcium in adose-dependent manner, and reversibly enhanced myocyte con-tractility by up to 40%, comparable in degree to maximalpositive inotropic effects caused by the cardiac glycoside oua-bain. Comparative studies further indicate that cardiotoxiceffects of ouabain in the myocytes may be more complex thansimple progressive elevation of intracellular free calcium con-centration because at a free calcium concentration in excess ofthat produced by a toxic dose of ouabain, no toxicity with thehypothalamic Na+/K+-transporting ATPase inhibitor oc-curred.

By regulating the electrolyte balance of cells and organs, theplasma membrane Na+/K+-transporting ATPase [ATP phos-phohydrolase (Na+/K+-transporting), EC 3.6.1.37; Na+/K+-ATPase] is responsible for many fundamental cellular andphysiologic processes such as cell volume regulation, thesetting of membrane potential, regulation of cardiac contrac-tility, and renal Na' reabsorption. The enzyme also serves asthe cellular receptor for the digitalis glycosides (1). Relativelylittle is known about the endogenous regulation of the enzyme.Catecholamines (2-4), thyroid hormone (5), aldosterone (6),and vanadium (7) have all been linked to either direct orindirect effects on enzyme activity. However, the fact that theonly known specific regulatory ligand, digitalis, is a substancefrom the plant kingdom has given rise to the notion of theexistence of a specific endogenous physiologic regulator ofmammalian origin, which might functionally resemble thecardiac glycosides. The concept of an "endogenous digitalis"has been made more provocative by a growing body ofexperimental evidence for a circulating Na+/K+-ATPase in-hibitor that would play a physiologic role in the normalnatriuretic response to extracellular fluid volume expansion (8)and a pathogenetic role in a prevalent human disease, hyper-tension (9). The postulated action of this substance is themodulation of renal tubular Na' reabsorption and vascularsmooth muscle tone by regulating Na+/K+-ATPase activity

(10). Indeed, the exogenous analogue of this putative regula-tor, digitalis, a potent inhibitor of the Na+/K+-ATPase, hasbeen shown to cause both natriuresis (11) and an increase invascular resistance (12), although these are not its majorpharmacological effects.Much effort has been expended in many laboratories to

identify and characterize a plasma, urinary, or tissue inhibitorof Na+/K+-ATPase; still a dearth of functional studies exist,and the definitive structure of such an inhibitor is not known(13). Furthermore, the degree to which various candidatecompounds are truly "digitalis-like" in either structure orfunction remains controversial because even those sub-stances characterized in the greatest biochemical detail (14-16) manifest some differences from the cardiac glycosides.One cardinal criterion for biological activity functionally

analogous to the cardiac glycosides would be the demonstra-tion of positive inotropic effects in cardiac muscle. We haveisolated and partially purified from bovine hypothalamus alow-molecular-mass, nonpeptidic substance that satisfies anumber of essential criteria for a physiologic regulator ofNa'pump activity in vivo (17). It inhibits the purified Na+/K+-ATPase reversibly, with high affinity (K, = 1.4 nM) (18);it's effects are specific for the Na+/K+-ATPase in the plasmamembrane (14), and it acts only from the extracellular surfaceof the cell (14, 17), consistent with the concept ofa circulatinginhibitor of the Na+/K+-ATPase. Thus, while certain of thebiologic effects of this hypothalamic substance are the sameas those of the cardiac glycosides, important differences in itsmechanism of inhibition of the Na+/K+-ATPase have beendiscovered (14, 18). Its potential role as regulator of renalNa' excretion was strengthened by the demonstration ofbinding and dissociation reactions in intact renal tubular cellsconsistent with physiologic regulation in vivo (19). But phys-iologic effects in cardiac cells remained unknown, althoughpreliminary experiments documented inhibition of purifiedcardiac Na+/K+-ATPase (20). To further explore the possi-bility that this hypothalamic inhibitory factor (HIF) couldhave physiologic actions in cardiac tissues, its effects on Na'pump activity, cytosolic free Ca2l ([Ca2+]i) accumulation,and contractility were studied in spontaneously contracting,neonatal rat myocytes in culture.We report here that HIF is a potent inhibitor of the Na'

pump in these cells, with effects on [Ca2+]i changes andcontractile response parallel to those of the cardiac glycosideouabain. Our results also show that at the same [Ca2"] in themyocytes, ouabain is toxic, whereas the endogenous factor isnot.

MATERIALS AND METHODSPreparation of HIF. The Na+/K+-ATPase inhibitor was

prepared from bovine hypothalamus as described (14).

Abbreviations: Na+/K+-ATPase, Na+/K+-transporting ATPase;[Ca2+]j, cytosolic free calcium concentration; HIF, factor isolatedfrom hypothalamus; ASM, amplitude of systolic cell motion.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Briefly, hypothalami collected fresh and frozen immediatelyon dry ice were thawed, homogenized, and extracted inmethanol/water (4:1, vol/vol). Methanol was removed byflash evaporation, and lipids were removed by extraction ofthe remaining aqueous phase with petroleum ether and chlo-roform. Initial separation of HIF was done by using lipophilicgel chromatography (14). Further purification was accom-plished by using successive cation- and anion-exchange chro-matographies (21). Purified HIF is free of vanadate (emissionspectroscopy), NH4 ion (reductive amination, Sigma kit170-A), and free fatty acids and lysophospholipids (GC/MS),which have been shown to interfere in pertinent bioassays.One unit of HIF in the myocyte studies is defined as thatamount of inhibitor that inhibits ouabain-sensitive K+ trans-port by 50%, as determined by 86Rb+ uptake into humanerythrocytes (14). One unit of HIF in this assay also inhibitsthe Na+/K+-ATPase purified from canine renal medulla by50%, as measured spectrophotometrically in a kinetic cou-pled-enzyme assay (18). With the latter assay, it could beestimated that one unit per 50 ,ul equals :15 nM HIF (18).

Tissue Culture. Myocardial cells were isolated from ven-trical fragments of the hearts of 1-day-old Sprague-Dawleyrats by serial trypsinizations in a Ca2+- and Mg2+-free Hanks'solution as described (22). For measurements of [Ca2+]f, thecells were plated on rectangular (13 x 30 mm) glass coverslipsand for measurements of cell contractility, cells were platedon circular (12 mm) glass coverslips; both types of coverslipswere placed inside Petri dishes. For measurements of 'Rb+uptake, cells were plated in Petri dishes at 1-1.5 x 106 cellsper 35-mm dish. All cultures were incubated in humidified 5%C02/95% air at 37TC. Confluent monolayers in which anestimated 80% of the cells exhibited spontaneous synchro-nous contractions developed by 3 days, at which time ex-periments were done.'Rb+ Influx Measurements. Na' pump activity was esti-

mated in the cultured cardiac cells as the difference in 86Rb+uptake seen with and without 5 mM ouabain, following themethod of Panet et al. (23). Incubations were continued forup to 10 min (period of linear uptake of the isotope) (24).Measurement of [Ca2+]i. Changes in [Ca2+], were detected

using the fluorescent probe fura-2 (25). Rectangular glasscoverslips with attached myocytes were placed in bufferedsalt solution (BSS; 140 mM NaCI/5 mM KCI/1 mM CaCl2/1mM MgCI2/10 mM glucose/i mM Na2HPO4/10 mMHepes-Tris, pH 7.4) to which was added 5,M fura-2/AM andincubated for 1 hr in humidified 5% C02/95% air at 37°C.Additional loading medium was added, and incubation wascontinued for 15 min to complete the hydrolysis of fura-2/AM. The cells were washed and incubated an additional 30min in BSS (26). Coverslips with loaded myocytes wereinserted into a thermostated (37°C) cuvette containing 2 ml ofBSS and various additions of HIF or ouabain as indicated.The fluorescence was continuously recorded using a PhotonTechnology International (South Brunswick, NJ) DeltaScan1 spectrofluorometer. Values of [Ca2+]i were calculated fromthe ratio R = F34O/F38o by using the formula: [Ca2+]j = KdB(R - Rmin)/(Rmax - R), where Kd is 225 nM, F is fluores-cence, and B is the ratio of fluorescence of free dye to that ofCa2+-bound dye measured at 380 nm. Rmax and Rmin weredetermined in separate experiments by using digitonin toequilibrate [Ca2+]j with ambient [Ca2+] (Rma,), and additionof MnCl2 (0.1 mM) and EGTA (1 mM) (Rmin). Backgroundautofluorescence was measured in unloaded cells and sub-tracted from all measurements.Measurements of Myocyte Contractility. Contractility, de-

termined as amplitude of systolic cell motion (ASM) andbeating frequency were measured in individual cells by usinga phase-contrast microscope video motion-detector systemaccording to the method of Barry et al. (27). A glass coverslipwith attached cultured myocytes was placed in a chamber

provided with inlet and exit ports for medium perfusion. Thechamber was enclosed in a Lucite box at 370C and placed onthe stage of an inverted phase-contrast microscope. The cellswere covered with 1 ml of medium containing HIF orouabain. Motion was monitored by a low-light-level TVcamera attached to the microscope and calibrated with 262raster lines. The motion-detector monitors a selected rasterline segment and provides new position data every 16 msecfor an image border of a microsphere within the cell layermoving along the raster line. The analog voltage output fromthe motion detector is filtered at low-band pass filter andcalibrated to indicate actual Aum of motion, and the derivativeis obtained electronically and recorded as velocity of motionin um/sec. Rate, amplitude, and velocity of contractionremained stable for several hours during control perfusions.The changes in the contractility induced by ouabain or HIFwere calculated in comparison with the contractility of thesame cells before addition of ouabain or HIF. Position ofmaximal relaxation and shifts of this baseline (AMR) whichindicate toxicity can be reliably measured by the videomethod used (26, 28).

RESULTSHIF Effects on Myocyte Na+ Pump Activity. Fig. 1 shows

the effects of HIF and ouabain on total 'Rb+ uptake into thecultured cardiac cells. In this experiment, to ensure satura-tion binding, myocytes were preincubated for 20 min withHIF (2 units/ml) or ouabain (5 mM), or both before adding86Rb+ to run the 10-min flux. Ouabain decreased uptake fromcontrol levels of 810 nmol/mg of protein to 210 nmol/mg(74%), whereas HIF at 2 units/ml decreased the uptake fromcontrol levels to 377 nmol/mg (54%). The combination ofouabain plus HIF was not additive, indicating that HIFinhibitory effects are specific for K+ transport through theNa+/K+-ATPase. In this experiment, HIF at 2 units/ml thusinhibited ouabain-sensitive K+ transport by 74%. In subse-quent experiments, Na' pump activity is defined as thedifference in 86Rb+ uptake seen with and without 5 mMouabain.

Fig. 2 shows the effects of increasing doses of HIF on Na+pump activity in the myocytes. The largest dose ofHIF tested(4 units/ml) inhibited ouabain-sensitive K+ uptake by 90%,whereas the ID50 for pump inhibition occurred at an HIFconcentration of 0.55 unit/ml. Inhibition of transport by HIFprogressed as a function of exposure time of cells to inhibitor

a

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FIG. 1. Inhibition of uptake of Rb+ by neonatal rat cardiacmyocytes exposed to HIF (2 units/ml), ouabain (5 mM), or both. HIFinhibited 54% of total Rb+ uptake and 74% of the ouabain-sensitiveuptake. Incubation of the cells with both ouabain and HIF producedno additive inhibition. Values are means ± SEM for n = 6 determi-nations for each condition. Concentration of HIF is in arbitrary unitsas described in the text.

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FIG. 2. Concentration dependence of HIF inhibition of ouabain-sensitive Rb+ uptake in neonatal rat cardiac myocytes. Ninetypercent of ouabain-sensitive K' (Rb+) uptake was inhibited by themaximal dose of HIF tested. Values are means + SEM for n = 4determinations at each concentration. (Inset) Inhibition of active K+transport as a function of exposure time of myocytes to HIF (2units/ml). n = 4 determinations at each point.

(Fig. 2, Inset). Neonatal rat myocytes appear to have arelatively high affinity for HIF; the ID50 = 0.55 unit/mlcontrasts with an ID50 = 15 units/ml found in active-transport-inhibition studies with porcine renal tubular cells inculture (19).HIF Effects on Myocyte [Ca2+]j. Because cardiac glycoside

inhibition of the Na+ pump in myocytes is associated withincreases in [Ca2+]i, leading to the positive inotropic effectscaused by these drugs, it was of interest to determine whetherHIF-induced Na+/K+-ATPase inhibition in the cardiac cellswas accompanied by parallel changes in [Ca2+]i. Fig. 3 showssteady-state fluorescence measurements in myocytes loadedwith fura-2 during a control period and at 30 and 60 min afteradding HIF at 1 unit/ml to the bathing medium. The mea-surements correspond to an increase in [Ca2+], from 138 nM(control) to 250 nM (30 min) and 432 nM (60 min). The onsetof change in [Ca2 ]i caused by HIF occurred within 15 min,reached a new steady-state concentration by 60 min, andremained stable at this level for at least 2 hr. Table 1 showsincreases in steady-state [Ca2+], induced by various doses ofHIF. A dose-response relationship was found with HIF at 0.5unit/ml increasing [Ca2+], in the myocytes to a level (303 ±15 nM) similar to that caused by 1 ,uM ouabain (287 ± 15 nM)when tested on cells from the same culture under the sameexperimental conditions. Levels of [Ca2+]J induced by 1 ,uMouabain in the current studies agreed with our more detaileddose-response data for ouabain found previously with quin-2as fluorescence indicator (26). HIF at 1 unit/ml raised [Ca2+]j

Table 1. Changes in steady-state [Ca2+] in cardiac myocytestreated with ouabain or various concentrations of HIF

[Ca2 ], nM

Ouabain HIFControl (10-6 M) 0.25 U/ml 0.5 U/ml 1.0 U/ml138 ± 3 287 ± 15 197 9 303 ± 15 432 ± 18Values are mean ± SEM; n = three determinations at each

concentration. U, units.

to a level significantly greater (432 ± 18 nM) than that inducedby 1 ,uM ouabain. Thus, like ouabain, inhibition of Na+/K+-ATPase activity in these cardiac cells was accompaniedby progressive elevation, in [Ca2+]i.HIF Effects on Myocyte Contractility. The effects of differ-

ent doses ofHIF on cell contractility, measured as ASM, andbeating frequency were measured using the phase-contrastvideo motion-detection system and compared to the effects ofouabain. Results of a typical experiment are shown in Fig. 4.Ouabain (5 x 10-7 M) (Fig. 4B), a maximally inotropic butnontoxic dose -in these cells (26), increased ASM by 41% anddecreased beating frequency compared to the same cells inthe control period (Fig. 4A). There was no change in theposition of maximal relaxation, indicating "therapeuticrange" but nontoxic effects with this dose of the cardiacglycoside. Ouabain at 1 AuM (Fig. 4C) showed a decrease inASM, an elevation in the position of maximal relaxation, andan increase in beating frequency, all indicating "toxic-range"effects of this higher dose on the contracting myocytes. HIFat 1 unit/ml (Fig. 4E) increased ASM 39% compared tocontrol (Fig. 4D), with a decrease in beating frequency and nochange in the position of maximal relaxation. After a 1-min"washout," during which the same cell was perfused withHIF-free buffer, ASM and beating frequency returned tocontrol levels (Fig. 4F), indicating rapid reversibility of thepositive inotropic effects caused by HIF. Table 2 summarizeschanges- in ASM and beating frequency as a function ofvarious HIF doses. Parallel to changes in [Ca2+], (Table 1),increased HIF concentrations caused progressive increasesin ASM and decreases in beating rate. Maximal increase inASM occurred at an HIF concentration of 0.5 unit/ml (39 ±6%), a level equal to the maximal nontoxic dose of ouabain(0.5 ,uM, Table 2). It was striking that at the maximal dose ofHIF tested (1.0 unit/ml), elevation of [Ca2+], well in excessof the level induced by a toxic dose of ouabain (1 ,M)occurred (Table 1, Fig. 4C), although the cells showed nomanifestations of toxicity at this HIF dose (Table 1, Fig. 4E).

DISCUSSIONThe HIF possesses some of the characteristics ascribed to anendogenous "digitalis-like" compound that has been pro-posed to act as a natriuretic hormone and a pathogeneticfactor in human essential hypertension and, perhaps, as aneven more general regulator of Na+/K+-ATPase activity inmammalian cells (for review, see ref. 29). HIF satisfiesseveral essential biochemical criteria for a physiologic regu-lator of the Na+ pump, including high-binding affinity, re-

HIF(WU/ml)

840H380H

40LLI_

After 30 Minutes After 60 Minutes

FIG. 3. Effect of HIF (1 unit/ml) on[Ca2+]i in cultured cardiac myocytes.Fluorescence of fura-2-loaded myocyteswas recorded continuously. Controlvalue (138 nM) is for cells before additionof HIF (arrow). Recordings are shownafter 30-min (250 nM) and 60-min (432nM) exposure to HIF.

I' '7'1

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Proc. Natl. Acad. Sci. USA 86 (1989)

III -, A ALI 4A.,AA IMr -RI . 11911'. r I'll It j I -I r, -1

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FIG. 4. Effects of ouabain (Upper) and HIF (Lower) on cardiacmyocyte contractility. Contractile responses are measured as theASM in single cells by video motion detection. Segments fromcontinuous recording of the same cells are shown. Ouabain at 5 x10-7 M increased ASM by 40% (B) compared to control (A). Ouabainat 1 x 10-6 M produced toxicity, indicated by decrease in ASM,elevation of position of maximal relaxation (AMR), and increase inbeating rate (C). HIF at 1 unit (U)/ml enhanced ASM by 40o (E)compared to its control (D). One-minute perfusion of HIF-free bufferreturned ASM and beating frequency to control levels (F).

versibility of effects, and specificity for the Na+/K+-ATPasein the plasma membrane (14, 18). Studies of the mechanismof inhibition of Na+/K+-ATPase by HIF showed similaritiesand important differences when compared with the effects ofthe cardiac glycoside ouabain. Like ouabain, HIF binds tothe extracellular domain of the enzyme, stabilizes it in anE2-like conformation as judged by fluorescence labeling, andinhibits p-nitrophenylphosphatase activity, a partial reactionof the Na+/K+-ATPase (14). Significantly different fromouabain, HIF does not support phosphorylation of the Na+/K+-ATPase from inorganic phosphate and magnesium (14)and has ligand requirements for optimal activity quite differ-ent from those of the cardiac glycosides (18). Based on theavailable biochemical data, the hypothesis has been ad-vanced that HIF may inhibit Na+/K+-ATPase by influencingthe phosphate-binding site on the enzyme and may alteractive Na' transport by preventing the cycling of the enzymebetween the E1 and E2 conformations, thus preventing thetranslocation of Na+ and K+ ions across the cell membrane(30).Thus, while differences in mechanism of action exist for

HIF and ouabain, ultimate biological effects on cation trans-port in blood cells and ion-transporting epithelia are sharedby the two substances. The extent to which HIF might betruly digitalis-like in its biological effects in vivo remaineduncertain because HIF actions in cardiac tissues, the primarytarget for digitalis glycosides, were unknown. The presentwork addresses this question by studying the effects of HIFon Na+ pump activity, [Ca2+]i accumulation, and contractile

Table 2. Effects on ASM and beating rate in cardiac myocytesby various concentrations of HIF and ouabain

HIF, units/ml ASM, % increase Rate, % decrease

0.2 15 ± 2 12 ± 10.25 22 ± 2 15 ± 10.33 32 ± 9 25 ± 70.5 39 ± 6 40 ± 21.0 37 ± 3 49 ± 6Ouabain (0.5 ,uM) 41 ± 3 23 ± 2

Values are mean ± SEM; n .3 for each concentration.

responses in spontaneously beating neonatal rat myocytes inculture.Na' pump activity was measured in the myocytes using

86Rb+ as a tracer for active K+ transport into the cells. HIFsignificantly decreased ouabain-inhibitable K+ transport intothe cells, and the combination of HIF and ouabain producedno additive inhibition of K+ transport, indicating HIF inhib-itory effects to be specific for K+ transport through theNa+/K+-ATPase (Fig. 1). The specificity of HIF acting onlyvia pump-mediated cation transport in the myocytes parallelsprevious findings in human erythrocytes (14)- and culturedrenal tubular cells (19).HIF-mediated Na+ pump inhibition was dose-dependent

and related to exposure time of myocytes to inhibitor (Fig. 2).Ninety percent of Na+ pump activity in the myocytes wasinhibited by the maximal dose of HIF used in these studies.The neonatal rat myocytes appear to have a relatively highaffinity for HIF. Thus, the ID50 for these cells was =30-foldless than that for cultured porcine renal tubular cells (Fig. 2;ref. 19).The shape of the dose-response curve for pump inhibition

in the myocytes suggests a single population of HIF-bindingsites, closely resembling the hyperbolic curve characterizingouabain binding and 86Rb+ transport inhibition in culturedchicken embryo myocytes (31). This is of interest, as itsuggests another significant difference between HIF andouabain because the ouabain-binding curve in neonatal ratmyocytes is much flatter and bimodal, reflecting two bindingsites of different affinities (31).Na+ pump inhibition in the myocytes was accompanied by

increases in [Ca2+],. [Ca2+], increased from a baseline of 138nM to 250 nM and 432 nM after 30- and 60-min exposure toHIF, respectively (Fig. 3). As with Na+ pump inhibition,HIF-induced increase in steady-state [Ca2+ concentrationswas dose-related (Table 1). The sensitivity of the myocytes torelatively low concentrations of HIF is again illustrated withHIF at 0.5 unit/ml, where [Ca2+], was elevated to a levelcomparable to that produced by 1 ,uM ouabain (Table 1).

Inhibition of Na+ pump activity and increases in [Ca2+j] inthe myocytes were accompanied by enhanced contractility.By use of cell monolayers identical to those used for mea-suring [Ca2]1j, the contraction amplitude was measured insingle beating myocytes as the ASM by using a phase-contrast video motion-detection system. Effects ofHIF werecompared with those of ouabain. HIF at 1 unit/ml caused a37 ± 3% increase in ASM, an augmentation in contractilityequal to that of the maximal nontoxic dose of ouabain forthese cells (5 x 10-7 M, producing 41 ± 3% increase in ASM,Fig. 4). At these doses of HIF and ouabain no manifestationsof toxicity occurred in the cells. Cells treated with 1 ,uMouabain, however, showed marked toxic effects, indicated bydecrease in ASM, increase in the position of maximal relax-ation, and increase in beating frequency (Fig. 4C). Increasein ASM caused by HIF showed a dose-dependent relation-ship, with maximal effects occurring at an HIF concentrationof 0.5 unit/ml (Table 2). Positive inotropic effects of HIFwere accompanied by a dose-related decrease in beatingfrequency. The mechanism for the decrease in rate caused byHIF is unknown. Our findings with HIF would support thoseof Shimoni and coworkers (32), who found partially purifiedextracts of sheep brain to enhance contractile tension inisolated sheep ventricular trabeculum. Effects on Na+ pumpactivity and Ca2+ metabolism in the tissue were not reported.The biochemical events underlying cardiac glycoside tox-

icity and the narrow therapeutic index characteristic of thesedrugs is incompletely understood, although excessive [Ca2+]Isecondary to persistently elevated intracellular Na+ associ-ated with tonic pump inhibition has been postulated as havinga central role (33). The work reported here does not addressthe question of the mechanism by which HIF elevates [Ca2"]1

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in the myocytes. But the results ofour studies suggest that thetoxicity induced by ouabain in the neonatal rat myocytes isnot simply due to progressive elevation of total [Ca2+]j, atleast in the pool measured by fura-2 fluorescence. Ouabain at1 ,M in these cells raised [Ca2+], to 287 ± 15 nM (Table 1)and was clearly toxic (Fig. 4C), confirming toxic effects atthese levels of [Ca2+], caused by 1 ,M ouabain found inprevious studies (26). HIF at 1 unit/ml raised [Ca2+], in thesame cells to 432 + 18 nM but was accompanied by a stablemaximal inotropic effect with no sign of toxicity (Fig. 4E).These findings suggest that the toxicity caused by ouabain inthe myocytes must involve drug interactions with the cell thatare more complex than simply inhibition of the Na' pump orthat abnormalities in Ca2" metabolism within the cell areoccurring in compartments not selectively discernible by thefura-2 method. In any case, HIF, at a dose producing similarpump inhibition [1 ,uM ouabain inhibits the Na' pump by 30%during a 20-min incubation in these cells (24)] and greaterelevations in [Ca2+], than the toxic dose of 1 AM ouabain, didnot produce this toxicity.The positive inotropic effect induced by HIF was readily

reversible. A 1-min perfusion with HIF-free buffer sufficed tocompletely reverse HIF-induced steady-state augmentationof contractility, returning the cell to baseline ASM andbeating frequency (Fig. 4E). HIF effects appear to be morereadily reversible than those of ouabain because a 5-minwashout period in ouabain-treated neonatal rat myocytes isrequired to return enhanced contractility to control levels(34). The ready reversibility ofHIF effects on the contractileresponse in the cardiac myocytes recalls the rapid revers-ibility in HIF-induced Na+/K+-ATPase inhibition that oc-curs in sided (intact cell) preparations (19) but not isolatedenzyme (18), and argues for the possibility that, in contrast tothe cardiac glycosides, HIF could be a physiologic regulatorof Na+/K+-ATPase activity in vivo.

In summary, HIF potently inhibits the Na+ pump incultured neonatal rat myocytes. The Na+ pump inhibitioninduced by HIF in these cells is dose-dependent and asso-ciated with augmentation of [Ca2+], content and an increasein contractile force equivalent to that caused by maximalnontoxic doses of the cardiac glycoside ouabain. Compara-tive studies with HIF and ouabain of pump inhibition andcalcium content parameters in treated myocytes suggest thatglycoside-mediated cardiac toxicity is more complex thansimple elevation of fura-2-measurable increases in [Ca2+],.Further studies of the mechanism by which HIF increasesmyocyte contractility should provide new insight into thebiochemical events associated with positive inotropy and therole of specific endogenous Na+/K+-ATPase inhibition inthe physiologic regulation of cardiac function.

The authors gratefully acknowledge Thomas W. Smith, M.D., forproviding access to the video motion-detection system in his labo-ratory, and Joseph V. Bonventre, M.D., Ph.D., for assistance incalcium measurements. This work was supported, in part, by a grantfrom the Squibb Institute for Medical Research (G.T.H.) and by theTrustees of the Massachusetts General Hospital and was done duringthe tenure of a Research Fellowship of the American Heart Asso-ciation, Massachusetts Affiliate (H.A.H.).

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