856
Inhibition of Myocardial Monovalent Cation Active
Transport by Subtoxic Doses of Ouabain
in the Dog
THOMAS J. HOUGEN AND THOMAS W. SMITH
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SUMMARY We studied the effects of sustained subtoxic doses of ouabain on left ventricular myocardial
monovalent cation active transport and contractility in open-chest dogs. Using a serial biopsy technique, fullthickness left ventricular myocardial samples were obtained prior to and 1 and 2 hours after ouabain infusion,
according to a loading dose and infusion schedule demonstrated to produce stable plasma and left ventricular
myocardial ouabain concentrations. Ouabain-inhibitable uptake of the potassium analogue 86 Rb + was measured in
vitro in these biopsy samples and compared with transport activity in biopsies taken prior to ouabain administration
and values in biopsies from dogs receiving vehicle alone. Left ventricular maximum dP/dt increased above baseline
values by 29 ± 3 = % (SKM) and 46 ± 9% in ouabain-treated dogs at the 1- and 2-hour biopsy times, respectively,
and active transport of 86 Rb + was significantly reduced by 21 ± 6% at 1 hour and 33 ± 5% at 2 hours. Continuous
ECG monitoring did not show any arrhythmias. Control dogs receiving vehicle alone had unchanged values for
contractility and monovalent cation transport. In another group of dogs, significant inhibition of active ""Rb+
transport was demonstrated at the time a sustained ouabain-induced positive inotropic effect was first clearly
demonstrable (30 minutes). In contrast, norepinephrine in doses sufficient to produce comparable increases in dP/dt
for 30-120 minutes did not change active 80 Rb + transport. These results demonstrate that subtoxic doses of ouabain
sufficient to induce a positive inotropic effect are associated with significant reduction of myocardial monovalent
cation active transport in a cardiac glycoside-sensitive species. Although a causal relationship cannot be established
on the basis of these data, our findings are consistent with the hypothesis that cardiac glycoside-induced inotropy is
related to inhibition of myocardial NaK-ATPase-mediated monovalent cation transport.
THE ROLE of altered monovalent cation transport in
causing the therapeutic and toxic effects of cardiac glycosides has been studied extensively and debated widely
over the past 2 decades.'"5 It is now generally agreed that
toxic doses of cardiac glycosides are associated with inhibition of sodium- and potassium-activated adenosine triphosphatase (NaK-ATPase) and consequently with reduced transmembrane active transport of Na+ and K"1".6""
However, subtoxic doses of cardiac glycosides, sufficient
to cause an increase in contractility without the eventual
development of arrhythmias, have not been reported
uniformly to be accompanied by decreased monovalent
cation active transport or NaK-ATPase inhibition.12"16
Current theories of the mechanism of inotropic action
of digitalis as proposed by Langer,r>t" Schwartz and
colleagues,2-4- " and Akera and Brody3 are critically dependent on the correctness of the inference that some
degree of monovalent cation active transport inhibition
From the Cardiovascular Division. Peter Bent Brigham Hospital, the
Department of Cardiology, Children's Hospital Medical Center, and the
Departments of Medicine and Pediatrics. Harvard Medical School. Boston.
Massachusetts.
Presented in part at the 69th Annual Meeting of the American Society
for Clinical Investigation. Washington, D C , May 2, 1977.
Supported by National Institutes of Health Grants HL-18003 and HL05855, and the King Trust Fund.
Dr. Hougen is a Charles A. Janeway Scholar.
Address for reprints: Dr. Thomas W. Smith, Cardiovascular Division.
Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston. Massachusetts 02115.
Received July 5, 1977; accepted for publication January 27. 1978.
occurs at positively inotropic but subtoxic doses and
plasma and myocardial concentrations of cardiac glycosides. The experiments to be described in this communication were, therefore, designed to approach in the most
direct way possible the question of whether and to what
extent monovalent cation transport is altered by positively
inotropic but subtoxic digitalis doses in the intact animal.
Accordingly, we investigated the effects of doses of ouabain sufficient to produce sustained subtoxic plasma and
myocardial concentrations on left ventricular contractility
and myocardial monovalent cation active transport as
reflected by the active uptake of the potassium analogue
86
Rb + .
Methods
Hemodynamic Measurements
Adult mongrel dogs (17-26 kg) of either sex were
anesthetized with intravenous pentobarbital (42 mg/kg),
maintained at 37°C on a heating blanket, intubated with a
cuffed endotracheal tube, and ventilated with 100% oxygen using a Harvard respirator. The chest was opened by
a midline sternotomy. Hemodynamic data were recorded
continuously on a Hewlett-Packard model 7700 multichannel oscillograph at paper speeds of 0.5-100 mm/sec.
The left ventricular (LV) pressure and maximum rate of
change in pressure (dP/dt) were monitored with a Hewlett-Packard model 1280 transducer connected to a 5-cm
stiff-walled catheter, which directly punctured the apex of
OUABAIN INHIBITION OF MYOCARDIAL
the LV. The fluid-filled system had a natural frequency
greater than 200 Hz and a damping ratio of 0.06, as
measured at the output of the carrier amplifier. A resistance-capacitance differentiating circuit was used to record
dP/dt. Heart rate was maintained constant at baseline
values in each experiment by electrically stimulating the
right atrial appendage with an American Optical model
2002 pacemaker. Femoral artery pressure was recorded
with a short polyethylene intra-arterial catheter connected
to a Hewlett-Packard model 1280 transducer. Standard
limb leads were applied for continuous electrocardiographic monitoring.
Arterial blood samples were withdrawn from the femoral artery for measurement of initial pH, Po2, Pco2,
hematocrit, and potassium concentrations and, at 5- to
30-minute intervals, for ouabain concentrations as indicated in experimental protocols.
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Ki;
Rb* Transport Measurements
When dogs were stable hemodynamically after completion of surgical procedures, and at subsequent times as
indicated in individual experimental protocols, pursestring
sutures were placed in the LV free wall avoiding major
coronary vessels. Using a standard 4-mm i.d. cork borer
attached to a hand-held power drill, transmural biopsies
weighing approximately 50 mg were obtained. They were
rinsed briefly in oxygenated buffer at 30°C, then immediately sliced lengthwise into four to eight strips about 1 mm
by I mm in cross-sectional area and 5-20 mm in length,
weighing 3-18 mg each.
The LV biopsy samples were placed in physiological
buffer containing, in IDM concentrations: KC1, 4.0; NaCl,
120; NaHCO,, 24; MgCl2, 2.0; CaCl2, 2.5: glucose, 5.6;
and Na phosphate buffer, 1.1, adjusted to pH 7.4, and
equilibrated with 95% oxygen, 5% CO2 at 30°C.
After a 5-minute equilibration period, myocardia! samples were transferred to flasks containing the same buffer
medium (5-ml final volume) except that the KG concentration was changed to 2.0 HIM and ""RbCI (New England
Nuclear) was added to give count rates of 10" dpm per
milliliter of medium. Unlabeled RbCI was added to give a
final Rb+ concentration of 0.1 ITIM. The flasks were gassed
with 95% O2-5% CO2. The tissues were incubated in the
presence or absence of ouabain (10~:! M) for 15 minutes at
30cC. At the comletion of this incubation, slices were
rinsed in four successive beakers containing 300 ml of
physiological buffer for 5 seconds each. Cerenkov radiation from individual slices was counted immediately in 1
ml of physiological buffer in a Beckman model LS-133
scintillation counter. Tissue samples then were blotted
and weighed. Active Rb+ uptake, in nmoles Rb+/mg wet
weight tissue per min was calculated as the difference
between uptake in the presence and absence of 10~:i M
ouabain.
The response of the preparation to various concentrations of ouabain in vitro was determined to assess the
sensitivity and precision of the method used. Three dogs
were anesthetized with pentobarbital, intubated, and ventilated as described above. Thirty minutes later, multiple
left ventricular drill biopsies were obtained from the
8li
Rb+ TRANSPORT/Wougen and Smith
857
beating heart. Active uptake of 8"Rb+ was determined as
described earlier in the presence of concentrations of
ouabain varying from 10~9 to 10":) M.
Experimental Protocol
Ouabain was given intravenously essentially according
to the schedule devised by Rhee et al."' to maintain stable
plasma concentrations in the subtoxic range. :iH-Ouabain
(New England Nuclear) was added to give a final specific
activity of 59.0 Ci/mol in all experiments. After an initial
control LV biopsy had been taken and stable hemodynamic conditions recorded for 30 minutes, ouabain in
saline was given intravenously in a dose of 30 pig/kg over
2 minutes, to six dogs. A constant intravenous infusion of
0.036 /xg/kg per min of ouabain, including ''H-ouabain as
above, immediately followed, using a Harvard Apparatus
model 2681 infusion pump. Additional left ventricular
biopsies were obtained 1 and 2 hours after the initiation
of ouabain infusion. Another control group of six dogs
was given saline in the same volume and rate, and LV
biopsies were taken at the same times as in the ouabaintreated group.
Plasma samples for determination of ouabain conentration were obtained at 0, 10, 30, 60, 90, and 120
minutes following onset of ouabain infusion.
No dog developed any atrial. atrioventricular junctional
or ventricular premature beats, nor did any conduction
abnormalities or other electrocardiographic evidence
of digitalis toxicity occur during the 2-hour infusion period. Toxicity would not be expected at the doses given or
the plasma levels maintained, based on our prior experience.18- '"
To examine the effect of ouabain on monovalent cation
active transport at the onset of inotropy, five additional
dogs were given ouabain by the dosage schedule outlined
previously. Hemodynamic data, arterial blood gases, hematocrit, and serum potassium concentration measurements were made as in other experiments. At the onset of
a sustained 26 ± 5% increase in contractility (average 30
minutes after initial ouabain bolus), a left ventricular
biopsy was obtained and active s"Rb+ uptake measured as
described above.
To exclude the possibility that transport inhibition
would result nonspecifically from any positively inotropic
agent, five dogs received a continuous infusion of norepinephrine (average 0.50 /u.g/kg per min) adjusted to produce a sustained positive inotropic effect similar to that
observed in the ouabain-treated group. Heart rate was
maintained constant and hemodynamic variables, together
with arterial blood gases, serum potassium concentration,
and hematocrit determinations, were measured. Left ventricular biopsies were obtained for transport measurements prior to norepinephrine, at the onset of sustained
positive inotropy, and after 2 hours of sustained positive
inotropy.
Plasma and Tissue Ouabain Determinations
Plasma ouabain content was measured by quantifying
:1
H counts. Myocardial samples were digested overnight at
40°C in Soluene (Packard Instrument Corp.), followed by
858
CIRCULATION RESEARCH
VOL. 42, No. 6, JUNE 1978
0.7,-
addition of glacial acetic acid (40 /xl/ml of Soluene) to
reduce chemiluminescence. Quenching was corrected by
the use of internal standards for both plasma and myocardial samples. Samples were counted until stable count
rates wee obtained, using a Packard model 3300 scintillation counter with appropriate windows and correction
for 8tiRb+ spill into the :lH channel.
Data were analyzed by paired Mest, each dog serving as
its own control, and differences in paired values were
considered significant when P values were less than 0.05.
i
Results
Uptake of si;Rb' by Myocardial Samples as a Function of
Time
Initial experiments demonstrated that active uptake of
Rb+ by canine myocardial samples was linear at 30°C
over the 15-minute incubation period employed, as illustrated in Figure 1. Active wiRb+ uptake averaged 0.200
± 0.010 nmoles Rb+/mg wet weight tissue per 15 minutes
(n = 8). Tissue exposed to 10"3 M ouabain showed no
further wlRb+ accumulation after 10 minutes. Active uptake remained linear for at least 30 minutes, after which
some deviation from linearity was usually observed at 60
minutes, as shown in Figure 1.
8li
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Concentration-Effect Curve for Ouabain Inhibition of
Rb+ Transport
The active transport of sliRb+ by myocardial biopsy
samples as a function of concentration of ouabain is shown
in Figure 2. Tissue samples were incubated for 15 minutes
at 30°C. The assay system was highly sensitive to ouabain,
with 10"" M concentrations significantly reducing 86Rb+
active transport by 5 ± 1%, (P < 0.05) compared to
active transport in the absence of ouabain. Half-maximal
inhibition occurred at 8 2 10~7 M ouabain. The sigmoid
shape of the log concentration-effect curve is similar to
results reported in numerous studies of cardiac glycoside
inhibition of monovalent cation transport or NaK-ATPase
activity (see Schwartz et al.2 for review).
8(i
Experimental Animals
As summarized in Table 1, there were no significant
differences in mean body weight, hematocrit, serum potassium concentration, arterial blood Po2, Pco2, or pH
between control and experimental groups of dogs. Heart
rate and mean arterial pressure did not vary significantly
from the initial values at any point in the various experimental groups.
Plasma and Myocardial Ouabain Concentration
Serial plasma and myocardial ouabain concentrations
are summarized in Figure 3 for dogs receiving the ouabain
infusion regimen previously described and intended to
establish and sustain positively inotropic but subtoxic
ouabain concentrations. The mean plasma ouabain concentration was 6.0 ± 0.4 ng/ml (1.0 x 10"8 M) at 1 hour
and fell slightly to 4.6 ± 0.3 ng/ml (7.7 x 10"9 M) at 2
hours (P < 0.05) after initial ouabain administration. The
15
MINUTES
FIGURE 1 mRb+ transport in myocardial biopsy samples as a
function of time. Left ventricular biopsy samples were incubated at
30°C for 5-60 minutes in 86/?fc+ with (i\) and without (O) /0" : l M
ouabain. Values given represent total Rb+ taken up per milligram
wet weight of tissue at the times indicated on the horizontal axis.
Active Rb+ transport, calculated as the difference between uptake
in the presence and absence of ouabain, is shown by filled circles
(%). Each point represents the mean value for 4-8 tissue samples.
Horizontal lines indicate 1 SEM, which lies within the dimensions of
the symbol for groups incubated with I0'3 M ouabain.
myocardial ouabain content remained essentially constant
at the 1 -hour (168 ± 23 ng/g wet weight) and 2-hour (173
± 20 ng/g wet weight) biopsy times.
Effects of Ouabain on Myocardial Contractility
The ouabain infusion regimen used produced readily
demonstrable sustained increases in myocardial contractility. As shown in Figure 4, the maximum rate of change of
left ventricular pressure (dP/dt) increased above pre-ouabain control values by an average of 29 ± 3% at 1 hour
and 46 ± 9% at 2 hours. Contractility did not change
significantly in control dogs, and no evidence of digitalis
toxicity occurred in any dog during the course of these
experiments.
Effects of Ouabain on Myocardial Active Transport of
8f, R b +
As shown in Figure 5, active transport of 8(1Rb+ into
myocardium from ouabain-treated dogs was reduced significantly below pre-ouabain control values by 21 ± 6%
at 1 hour and by 33 ± 5% at 2 hours. In contrast, control
dogs receiving saline infusions showed unchanged 8(iRb+
transport at these times. These results clearly demonstrate, by a direct assay technique, significant reduction of
myocardial monovalent cation active transport by sustained positively inotropic but subtoxic cardiac glycoside
doses.
OUABAIN INHIBITION OF MYOCARDIAL
86
Rb+ TRANSPORT/Hougen and Smith
859
16
00
80 12
10
60 -
Ian
CD
A
J
40 -
W(/3)
20 -
Jt"6'
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JIU9)
0
10
" T/ibj
i
.
o
n—
io's io'°
OUABAIN
m
IO
io'6
IO" 3
CONCENTRATION
IO
( M )
FIGURE 2 Rb transport: ouabain concentration-effect curve.
Left ventricular biopsy samples obtained from three pentobarbitalanesthetized dogs were incubated for 15 minutes at 30°C in the
presence of the ouabain concentrations shown (horizontal axis).
Complete inhibition of active transport was defined as that observed in the presence of /0" :1 M ouabain. Statistically significant
transport inhibition was observed at concentrations of 10~9 M
ouabain or greater. Half-maximal inhibition occurred at 8 x /0~ 7
M. Numbers in parentheses indicate numbers of individual tissue
samples assayed; brackets indicate 1 SEM above and below the
mean.
Effects of Ouabain and Norepinephrine on Myocardial
Rb + Transport
81
In the groups of dogs studied at the onset of inotropy,
both norepinephrine in low doses (average 0.50 /xg/kg per
min) and ouabain (infusion regimen as previously described) produced comparable inotropic responses at 30
minutes. As summarized in Figure 6, the response to
norepinephrine was a 42 ± 13% increase in maximum LV
dP/dt, which was not significantly different from the 26 ±
5% increase in response to ouabain. The norepinephrine
infusion was adjusted to maintain LV dP/dt at a value
TABLE 1 Initial Measurements for Control and OuabainTreated Dogs
Each value is the mean ± SEM.
60
Control group
(n = 6)
Ouabain-treated
23.5 ± 0 . 6
132 ± 9
21.9 ± 1.2
128 ± 4
175 ± 12
39 ± 1.2
3.1 ± 0.2
185 ± 5
42 ± 0.2
3.3 ± 0.2
7.45 ± 0.02
465 ± 23
26 ± 4
7.46 ± 0.02
461 ± 18
31 ± 4
group (n = 6)
90
120
MINUTES
io's
+
Weight (kg)
Mean arterial pressure
(mm Hg)
Heart rate (beats/min)
Hcmatocrit (%)
Serum potassium
(mEq/liter)
Arterial pH
Arterial Po2 (mm Hg)
Arterial Pco2 (mm Hg)
30
FIGURE 3 Plasma (vertical axis to left) and myocardial (vertical
axis to right) 3H-ouabain content after administration of 20 yglkg
followed by constant infusion at a rate of 0.036 ng/kg per min.
Plasma ouabain concentrations, measured in triplicate, are indicated by points representing the mean ± SEM for six dogs.
Myocardial ouabain concentration was measured in triplicate in 1and 2-hour biopsy samples as described in the text. Each point
represents the mean value ± SEM from six dogs.
I 60
X*
I 50
^
Q.
140
"°
I 30
OUABAIN
_J
—
I 20
Z
MO
100
~ - 5 CONTROL
90 80 0
I
2
HOURS
FIGURE 4 The effect of nontoxic doses of ouabain on left
ventricular contractility. Dogs were given 3H-ouabain, 30 fj-g/kg,
followed by a constant infusion of 0.036 tag/kg per min. The mean
and standard error of the mean of absolute values ofdP/dt in the
control state were determined, and the mean expressed as 100%.
Left ventricular maximum dP/dt, expressed as percent of preouabain values, is shown on the vertical axis. The horizontal axis
refers to hours after the start of ouabain administration. Each
value represents the mean ± 1 SEM for six dogs. * = P < 0.001
compared with zero-time value; ** = P < 0.01 compared with
zero-time value.
860
CIRCULATION RESEARCH
,_
120
a:
2 i 10
v>
z
<
CONTROL
100
90
<D
CD
_l
80
mm
70
z
OUABAIN
u.
o
60
J«
50
0
I
2
HOURS
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FIGURE 5 Effect of ouabain administration on active transport of
Rb+ into canine myocardial biopsy samples incubated in vitro.
Rb+ uptake was measured in serial biopsy samples removed prior
to and 1 and 2 hours after the start of ouabain administration as
describedin the text. The mean and standard error of the mean of
absolute values of Rb+ active transport in the control state were
determined, and the mean expressed as 100%. Active transport of
Rb+, expressed as percent of the pre-ouabain value, is shown on
the vertical axis. Control dogs given saline showed unchanged
active transport at I and 2 hours. Each point represents the mean
± SEM of six dogs. * = P < 0.02 compared with zero-time value;
** = P < 0.001 compared with mean zero-time value.
similar to that produced by ouabain over a 2-hour interval.
As also summarized in Figure 6, there was no change in
8<i
Rb+ active transport compared to control values in the
norepinephrine-treated group, either at the onset of inotropy (99 ± 4% of control sllRb+ active transport) or after
a 2-hour period of sustained positive inotropy (101 ± 5%
of control). In contrast, at the time a definite increase in
myocardial contractility was first evident after onset of
ouabain administration, there was a 31 ± 9% decrease in
active 8liRb+ transport (P < 0.05) compared to control
values. Thus, the reduction in 8tiRb+ transport observed
after ouabain infusion does not appear to be a nonspecific
response to an enhanced contractile state.
Discussion
The observation by Calhoun and Harrison20 in 1931
that cardiac glycosides diminished myocardial K+ concentration first suggested that alteration in cardiac monovalent cation balance might underlie at least some of the
actions of digitalis. Subsequent studies of effects of cardiac
glycosides on myocardial monovalent cation active transport have resulted in conflicting conclusions. Most investigators have reported diminished myocardial potassium
uptake in the presence of large and potentially or overtly
toxic doses of cardiac glycosides.''2-2I Although some
studies have reported nontoxic concentrations of cardiac
glycosides to be associated with diminished potassium
uptake,22~'-4 Tuttle and colleagues2' reported unchanged
potassium uptake at subtoxic ouabain doses. Interpretation of the results of some of these studies is further
VOL. 42, No. 6, JUNE 1978
complicated by the use of animals (rabbit, guinea pig)
with intermediate sensitivity9'25 to cardiac glycosides. The
voluminous earlier literature regarding cardiac glycoside
effects on myocardial monovalent cation balance has been
reviewed in detail by Lee and Klaus.'
Since the observation by Schatzmann26 that cardiac
glycosides inhibit active transport of monovalent cations
across the cell membrane, studies as cited above showing
altered myocardial cation balance have been interpreted
as reflecting this inhibitory effect of cardiac glycosides on
transport. Binding to, and inhibition of, the monovalent
cation transport enzyme complex NaK-ATPase has been
postulated by a number of workers to be the initial event
in the production of the positive inotropic effect of cardiac
glycosides, and an impressive body of circumstantial evidence consistent with this hypothesis has accumulated.2'6'
7. K-ii.27 jfojg interpretation has by no means been universal, however, and others have presented evidence suggesting a lack of correlation between NaK-ATPase inhibition
and positive inotropy.12"14 Additional recent studies by
Rhee et al.16 did not demonstrate significant inhibition of
NaK-ATPase at positively inotropic but subtoxic glycoside
doses. Furthermore, Cohen et al.15 studied sheep Purkinje
fibers exposed to concentrations of ouabain thought to be
positively inotropic and obtained results consistent with
stimulation rather than inhibition of potassium transport
using voltage clamp techniques.
The present studies were designed to approach as
directly as possible the question of whether and to what
extent monovalent cation transport is altered by sustained
subtoxic, positively inotropic, and clinically relevant doses
of cardiac glycosides. The following criteria were felt to
be of particular importance in the design of these studies.
(1) A cardiac glycoside-sensitive species should be studied, i.e., one in which sustained positive inotropic effects
result from doses and plasma concentrations comparable
to those known to produce therapeutic effects in man.28-2il
(2) Animals with intact circulation and intact neural
pathways should be studied because of pharmacokinetic
considerations and the potential importance of neurally
mediated cardiac glycoside responses.30-3I (3) A cardiac
glycoside infusion regimen should be used that would
establish and maintain at least quasi-steady state plasma
and myocardial drug concentrations during the period of
study. (4) Plasma and myocardial glycoside levels should
be measured, and there should be no evidence of toxicity
accompanying the positive inotropic effect. (5) A sensitive
and precise method capable of detecting modest changes
in monovalent cation active transport at clinically relevant
digitalis concentrations should be used. This method
should be as direct as possible to avoid problems in
interpretation due to possible dissociation of cardiac glycoside from receptor during preparative procedures.2' '4
(6) The method of assessing monovalent cation transport
ideally should allow serial measurements in the same
animal, permitting each animal to serve as its own control
and enhancing the power of statistical correlations. (7)
The methods used should allow correlation of ion transport (or some counterpart thereof) with a simultaneous
measurement of the contractile state of the intact heart.
The informative study of Grupp and Charles22 meets
OUABAIN INHIBITION OF MYOCARDIAL
|
8(i
Rb+ TRANSPORT/Hougen and Smith
OUABAIN
|CONTROL
861
NOREPINEPHRINE
LV dP/dt
ACTIVE TRANSPORT
xx
XX
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(5) (51
30
120
I
15) (3)
120
MINUTES
FIGURE 6 Effecls ofouabain and norepinephrine on myocardial contractility (LV dP/dt) and mRb+ active transport. Dogs were given 3Houabain (as described in the text) or norepinephrine (mean infusion rate, 0.5 /xg/kg per min), intravenously. At the onset of sustained
inotropy, a mean of 30 minutes after drug administration, LV biopsies were obtained and mRb+ transport measured. The norepinephrine
infusion was continued and *"Rb+ active transport assayed at the completion of 2 hours of sustained inotropy. * = P < 0.01 compared with
zero-time values; ** = P < 0.05 compared with zero-time values.
most of these criteria, but did not use a steady state
infusion regimen, and plasma and myocardial cardenolide
levels were not documented. The study of Rhee and coworkers,16 further commented upon subsequently, carefully defined these items but reported a lack of significant
inhibition of myocardial NaK-ATPase at positively inotropic but subtoxic ouabain doses.
The methods used in the present study fulfill each of the
above-stated criteria. The canine preparation used is
comparable to man in digitalis sensitivity.2'9 The ouabain
infusion regimen used maintained subtoxic but positively
inotropic plasma and myocardial concentrations, as shown
in Figure 3, and has been shown by Rhee et al.18 to
maintain a positive inotropic state in the canine myocardium with no evidence of toxicity even when sustained for
periods up to 5 hours. Examination of transport in vivo
was approximated as nearly as possible by assay of transport in serial biopsy samples with intact cellular architecture within 5 minutes of removal from the beating heart.
Use of a pentobarbital-anesthetized, open-chest preparation with resulting tachycardia could alter the complex
relationships among arrhythmias, inotropy, and monovalent cation transport, but was necessitated by the serial
biopsy protocol.
The potassium analogue 86Rb+ has been shown to be
similar to K+ in its transport properties32 and has been
used as an indicator of NaK-ATPase-dependent coupled
ion transport in red cells,32"34 rat brain,32 guinea pig left
atrium,34- M and guinea pig ventricular slices.27
Our assay of monovalent cation active transport in
canine myocardium was highly sensitive, demonstrating
significant inhibition at 10"u M ouabain, and thus permitted the detection of alterations in active transport caused
by clinically relevant doses and plasma concentrations.
The major finding from the experiments reported here
was unequivocal reduction in myocardial monovalent cation active transport by sustained positively inotropic but
subtoxic doses ofouabain. These data confirm and extend
the observations of Ku et al.,27 who demonstrated monovalent cation pump inhibition at positively inotropic concentrations of cardiac glycosides in Langendorff-perfused
guinea pig hearts. The increases in left ventricular maximum dP/dt of 29% and 46% and decreases in active
86
Rb+ transport of 21% and 33% at the 1- and 2-hour
biopsy times, respectively, in the presence of essentially
constant mean myocardial ouabain concentrations (168
and 173 ng/g wet weight), are of interest. These progressive changes presumably reflect the fact that myocardial
ouabain content, as measured, included nonspecifically
bound ouabain as well as drug bound to NaK-ATPase or
other cellular receptors. These findings, then, suggest an
increase with time in the ratio of specific to nonspecific
862
CIRCULATION RESEARCH
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binding. A similar increase in contractility at nearly constant myocardial total ouabain concentrations was shown
by Luchi et al.36 Although the present data cannot be
regarded as proving a cause and effect relationship, the
association of increasing transport inhibition with increasing inotropy is consistent with the hypothesis that the
positive inotropic response to cardiac glycosides is related
to monovalent cation active transport inhibition.
Lack of change in active 86Rb+ transport at the onset or
after 2 hours of sustained norepinephrine-induced positive
inotropy (Fig. 6) indicates that inhibition of transport was
not a consequence of the enhanced contractile state. This
absence of alteration in 86Rb+ transport by norepinephrine-induced inotropy is consistent with the results of
Akera et al.fi who showed that isoproterenol infusion or
stellate ganglion stimulation sufficient to cause increased
contractility had no effect on canine myocardial NaKATPase activity.
Our results do not support the conclusion reached by
Rhee et al.16 who administered ouabain in the same
nontoxic doses used in the present study to intact, openchest dogs and achieved similar plasma and myocardial
ouabain levels as well as comparable increases in left
ventricular contractility. Measuring NaK-ATPase activity
in a partially purified myocardial membrane fraction after
multiple preparative steps, they found no significant differences in enzyme activity between control and ouabaintreated animals. NaK-ATPase inhibition was shown only
when arrhythmogenic doses were given. These authors
pointed out that enzyme-glycoside dissociation during
preparative steps could not be excluded; we consider this
the most likely mechanism accounting for the lack of
significant enzyme inhibition at subtoxic drug concentrations. Schwartz et al.u have previously demonstrated that
dissociation of cardiac glycosides from NaK-ATPase during preparation for assay can create problems in correlating enzyme inhibition with inotropic effects. Animal-toanimal variation in baseline enzyme activity may also have
contributed to the absence of a statistically significant
difference between control and ouabain-treated groups in
the studies of Rhee et al.16
Another recent study by Peters et al.14 interpreted as
showing a lack of correlation between positive inotropy
and myocardial NaK-ATPase inhibition, used isolated
atrial muscle from a relatively cardiac glycoside-insensitive
species, the guinea pig. A reported stimulation rather than
inhibition of NaK-ATPase at 10~9 M cardiac glycoside
concentrations could be observed only during the first 2
days after enzyme preparation, and this finding was not
correlated with any direct measurement of monovalent
cation transport. This study does not meet five of the
seven criteria advanced above, and thus is difficult to
relate directly to the findings of the present study.
The electrophysiological studies of TenEick and associates 13 showed persistent reduction of resting membrane
potential consistent with reduced monovalent cation transport after washout of the positive inotropic effect of
strophanthidin-3-bromoacetate from isolated myocardium. Cohen et al.15 have recently interpreted their volt-
VOL. 42, No. 6, JUNE
1978
age clamp studies of the effects of ouabain on isolated
sheep Purkinje fibers as being most consistent with net
stimulation rather than inhibition of potassium transport.
These studies used isolated tissue preparations and techniques that, while providing information of considerable
fundamental interest, do not directly measure monovalent
cation active transport.
We cannot exclude the possibility of a transient stimulation of active transport by low concentrations of ouabain
in vivo, since our first measurements were made 30
minutes after the start of ouabain infusion. However, in
the in vitro concentration-effect study (Fig. 1), a 15minute exposure to 10"" M ouabain produced significant
inhibition of active 86Rb+ transport.
The data presented here clearly demonstrate reduced
myocardial monovalent cation active transport during the
positive inotropic effect produced by subtoxic digitalis
doses in the intact dog. Although a causal relationship
cannot be established on the basis of these data, our
findings are consistent with the hypothesis that cardiac
glycoside-induced inotropy is related to inhibition of myocardial NaK-ATPase-mediated monovalent cation transport.
Acknowledgments
We thank Thomas Manning and Edward Woodford for excellent
technical assistance, and Margaret Wall and Lucille Levesque for secretarial assistance.
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Circ Res. 1978;42:856-863
doi: 10.1161/01.RES.42.6.856
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