Download Actions of bufalin and cinobufotalin, two bufadienolides respectively

Clinical Science (1990)78,169-174
169
Actions of bufalin and cinobufotalin, two bufadienolides
respectively more active and less active than ouabain, on
ouabain binding and 86Rbuptake by human erythrocytes
A. A. BROWNLEE, PAUL JOHNSON AND NOR H. MILLS
Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K.
(Received 31 July/l4 September 1989; accepted 20 September 1989)
SUMMARY
1. We have reported that the bufadienolide, bufalin
(purified from toad skin), was more potent than ouabain
in inhibiting the sodium/potassium-dependent adenosine
triphosphatase from canine kidney (Sigma)[Brownlee, A.
A., Lee, G. & Mills, 1. H. J. Physiol. (London) 1987; 390,
94PI.
2. The activities of bufalin and cinobufotalin were
compared with ouabain in the [JHIouabain binding assay
and on x6Rbuptake in human erythrocytes.
3. When the percentage binding of ouabain-sensitive
[3H]ouabainwas plotted against the log of the concentration of drug in mol/l, it was shown that the bufalin curve
was shifted to the left of that of ouabain and that of cinobufotalin was to the right.
4. Linear regression lines were fitted to the data transformed as the log of ( p / l - p ) plotted against the log of the
drug concentration, where p is the proportion of maximal
ouabain-sensitive activity at the drug concentration being
considered. The ICs,, (the concentration of drug producing a 50% change in the maximal ouabain-sensitive
mol/ 1 for bufalin, 9.7 x
response) was 1.4 X
mol/l for ouabain and 1.70x lo-’ mol/l for cinobufotalin.
5. The introduction of bufalin 1 h before ouabain
reduced the binding of [JHIouabain to 23.4 f 1.5%
(P<O.OOl). Bufalin added in the second hour reduced the
ouabain-sensitive binding from 100 +_ 1.9% to
87.4k2.97’0 (P<0.01).
6. The oubain-sensitive x6Rb uptake curves showed
that that of bufalin was to the left of ouabain and that of
cinobufotalin was to the right.
7. Regression lines were fitted to the x6Rbuptake data
as described for the ouabain-binding data. The lCs,,was
1 . 0 5 1QdX
~
mol/l for bufalin, 4.4x
mol/l for
ouabain and 4.3 x lo-’ mol/l for cinobufotalin.
Correspondence: Professor Ivor H. Mills, Department of
Surgery, University of Cambridge, Douglas House, Trumpington
Road, Cambridge CB2 2AH, U.K.
8. The differences in potencies are not all explained by
what is known of the effects of structure on the cardiac
toxicity of cardenolides and bufadienolides.
Key words: bufadienolides, bufalin, cinobufotalin,
ouabain, X6Rbuptake.
Abbreviations: 1CS(,,concentration of a drug producing a
50% change in the maximal ouabain-sensitive response;
Na+/K+-ATPase, sodium/potassium-dependent adenosine triphosphatase.
INTRODUCTION
For many years ouabain has been the standard cardiac
glycoside against which other inhibitors of the enzyme
sodium/potassium-dependent adenosine triphosphatase
(Na+/K+-ATPase)have been assessed. In a preliminary
publication we reported that bufalin is more potent than
ouabain in inhibiting canine renal Na+/K+-ATPase
(Sigma) [l]. Bufalin is one of a group of compounds
known as bufadienolides. They are extracted from the
skin and parotid secretion of the toad and were first
isolated in active form by Abel & Macht in 1911 [2]. For
centuries the Chinese used dried, powdered toad skin in a
preparation known as Ch’an Su for the treatment of
‘dropsy’, and in the London Dispensatory of 1702
Salmon states that dried toad would cure ‘incurable’
dropsy. It was overtaken by Withering’s discovery in 1775
of the active principle of the foxglove [3]. Abel & Macht,
having isolated ‘bufagin’ in crystalline form, then showed
that in cats and rabbits it increased urine flow rate, noting
that it was 50-100 times as potent as digitalis.
The structure of the bufadienolides resembles the
steroid structure of the plant cardiac-active cardenolides,
except that in the toad the bufadienolides have no sugars
attached to C-3 in ring A and they have a six-membered
lactone ring instead of a five-membered ring attached to
C-17.
Toad venom acts on the heart in a similar way to that of
digitalis and the bufadienolides extracted from toad skin
170
A. A. Brownlee et al.
have been shown to have an effect on ion transport in the
amphibia [4]. However, little work has been carried out to
compare the activity of the animal bufadienolides in pure
form with the cardenolides of plant origin.
Studies in recent years have indicated the presence of a
substance in animals which reacts with digoxin antibodies
and which may be associated with natriuresis and, when
present in plasma, may be related to hypertension. The
study by de Wardener & MacGregor [5] suggested that a
primary defect in sodium excretion might lead to
increased production of a natriuretic substance which led
to hypertension, perhaps by inhibition of Na+/K+ATPase in smooth muscle. Some of the known inhibitors
of Na+/K+-ATPase which react with digitalis receptors
have a steroid molecule, as have bufadienolides and
cardenolides [6].
The present study was carried out to characterize the
activities of the two bufadienolides, bufalin and cinobufotalin (Sigma), in relation to that of ouabain (Sigma) in
the [3H]ouabain (Amersham)-binding assay on human
erythrocytes and in inhibition of the uptake of X6Rb
(Amersham) also in human erythrocytes. In both tests
bufalin proved to be more potent than ouabain and cinobufotalin was less potent.
Competitive effect of bufalin on 13H]ouabainbinding
METHODS
The technique was exactly as described by De Luise et
al. [7]. The tests were carried out on the same nine
samples of blood as for the ouabain-binding assay. After
the initial dilution of erythrocytes in saline, 1 ml of cells
was washed three times with 5 ml of saline and finally
resuspended in saline/lO mmol/l glucose/ 10 mmol/l
Tris/2 mmol/l RbCl at pH 7.4. Each tube contained 0.15
pCi of x6Rb.The final packed cell volume was 8.4 f0.2%
and the erythrocyte volume was 16.7 f0.5 pl. Incubation
was for 1 h at 37°C. Then 1 ml of saline at 4°C was added
before spinning down the cells in a temperaturecontrolled centrifuge at 4°C. This was repeated three
times in all and the cells were resuspended in 0.05 ml of
saline and 0.2 ml of 10% (v/v) trichloroacetic acid and
spun at 4°C for 5 min. The supernatant was taken for
counting.
Nine samples of heparinized blood were obtained from
five healthy adults (four males and one female), aged
18-37 years, and each was tested separately.
[3H]ouabain-binding assay
The assay technique used was as described by De Luke
et al. [7], with the exception that incubation was continued
for 2 h to allow binding to reach a plateau (see the Results
section). After removal of plasma and buffy coat, 1 ml of
the cells was resuspended in an equal volume of saline
(150 mmol/l NaCI). The cells were washed three times
with 5 ml of 140 mmol/l choline chloride and finally
suspended in a buffer consisting of saline/l0 mmol/l
glucose/Hepes 30 mmol/l (pH 7.4). The final mean
( fSEM) cell concentration was 7.85 f0.27% measured in
a microhaematocrit apparatus. The mean ( fSEM) volume
of erythrocytes was 15.7f0.54 pl. The binding was
assessed with 9 x
mol of [3H]ouabain in each tube
in a final total volume of 0.25 ml, making a final concentration of 3.64x lo-' rnol/l ouabain. They were incubated at 37°C for 2 h. The cells were spun down with 1ml
of ice-cold saline three times and then suspended in 0.05
ml of choline chloride (140 mmol/l) and 0.2 ml of 10%
(v/v) trichloroacetic acid. After centrifuging (10 000 g) for
5 min, the supernatant was taken for counting.
The binding with only ['Hlouabain was taken as control
and all other measurements were calculated as a
percentage of the control value. All determinations were
carried out in triplicate. Non-specific (ouabain nonsensitive) binding was defined as that in the presence of
mol/l non-radioactive ouabain: it was 0.9 f0.3% of
the total binding (mean fSEM, ii = 9).
A series of experiments was carried out with three
separate blood samples (all from males) as described for
the [3H]ouabain-binding assay. Each point was determined in sextuplicate. Ouabain and bufalin were added in
equimolar amounts, namely 3.64 x lo-' mol/l. Total
incubation was for 2 h at 37°C and counting took place
only at the end of the incubations. The pattern of addition
of ouabain and bufalin was as follows:
(1)Group A: set 1,first hour [3H]ouabain,second hour
continuation;
set 2, first hour [3H]ouabain,second hour
bufalin added;
set 3, first hour [3H]ouabain,second hour
cold ouabain added.
(2) Group B: set 1,first hour buffer only, second hour
[3H]ouabain added;
set 2, first hour bufalin, second hour
[3H]ouabain added;
set 3, first hour cold ouabain, second
h~ur[~H]ouabain
added.
%b uptake
Analysis of data
To obtain the concentration of each drug which
produces a 50% change in the maximal ouabain-sensitive
response, the ICsc,,the log of (p/l - p ) was plotted ( y )
against the log of the drug concentration in mol/l (x). In
each case p is the proportion of the maximal ouabainsensitive response caused by the concentration of the
drug being considered. This plot produces a straight line
and the 1Cs0is given by the drug concentration where the
log of (p/l- p ) is equal to zero.
RESULTS
U
[3H]Ouabainbinding
The effects of duration of incubation on the percentage
of ['Hlouabain bound for two studies were as follows:
Effect of bufadienolides compared with ouabain on erythrocytes
Study 1
Time(min)...
%Bound ...
Study 2
Time(min)...
%Bound ...
15
0.68
30
1.55
60
90
2.22 '2.61
45
2.27
105
2.82
60
2.64
120
3.25
75
3.06
90
3.55
135
3.25
As a result of this brief study it was decided to carry
out all experimental studies on ouabain binding with incubation for 2 h.
The results of the ouabain-sensitive binding assays with
the addition of non-labelled ouabain, bufalin and cinobufotalin are shown in Fig. 1.All nine blood samples were
tested separately over the range 10-9-10-6 mol/l for
ouabain and cinobufotalin and 10- l o - l O - h mol/l for
bufalin. The curve with bufalin is shifted to the left
compared with that of ouabain alone and the curve with
cinobufotalin is shifted to the right.
The log of ( p / l - p ) plotted against the log of the drug
concentration for all three drugs is shown in Fig. 2, the
legend of which contains the three equations. The goodness of fit in each case is highly significant and the slopes
of the lines are not significantly different. The lCsovalues
are shown in Table 1. From these it is seen that bufalin is
almost seven times as potent as ouabain and cinobufotalin
is less than one-tenth as active as ouabain.
The results of the competitive binding assay are shown
in Table 2. Thq mean ouabain-sensitive percentage
binding with ['Hlouabain alone was taken as 100% in
both groups A and B. The presence of bufalin in the
second hour in group A reduced the binding to
87.4 f2.9% (P<0.01), whereas the addition of an
equimolar amount of non-radioactive ouabain did not
produce a significant change. In group B the presence of
bufalin in the first hour in set 2 markedly reduced ouabain
binding to 23.43~1.5% of that in set 1 (P<0.001).
Allowing unlabelled ouabain to be present in the first
hour limited exchange with the ["Hlouabainin the second
hour so that binding was 65.1 k4.170 of that in set 1, but it
was still highly significantly greater than when bufalin was
present in the first hour ( P < 0.001).
This confirms that binding of bufalin to erythrocytes
must have a higher affinity than oubain, allowing bufalin
to displace or prevent ouabain binding.
86Rbuptake
The ouabain-sensitive '(>Rb uptake was tested
separately on nine samples of blood from the five adults.
The inhibitory effects of ouabain and cinobufotalin were
tested over the range 10-"-10-6 mol/l and bufalin was
tested over the range 10-"'-10-6 mol/l. Each determination was in triplicate. The ouabain-insensitive XhRbuptake
was assessed in the presence of
mol/l ouabain. The
mean ( k SEM) in these experiments was 16.4 f0.67%
( I I = 9) of the total uptake. The mean ( f SEM) "'b
uptake
with no inhibitor present was 5.94 f 0.68% (range
8.56-4.60%) of the rubidium present. The x6Rbuptake in
the presence of ouabain or a bufadienolide was calculated
-8
v
171
100-
80-
on
E
5
.E 6 0
D
c:
'Z 40-
s
-6I
I
c
.
200- 10
I
-9
-8
-7
-6
log[Drug](mol/l)
Fig. 1. Ouabain binding to erythrocytes. Ouabainsensitive ["Hlouabain binding is plotted against the log of
the molarity of the inhibitors. Results are means fSEM. B,
Bufalin; 0,ouabain; C, cinobufotalin.
i-1.61
.
log[Drug] (mol/l)
Fig. 2. Ouabain-sensitive ["Hlouabainbinding to erythrocytes. The log of ( p / l - p ) is plotted against the log of the
molarity of the inhibitors, where p is the proportion of the
maximum ouabain-sensitive response at the drug concentration being considered. The regression lines were fitted
by least squares, the equations being: bufalin (B),
y = -6.880-0.777~ (goodness of fit, P<O.OOl); ouabain
(0),y = -6.360-0.794~ (goodness of fit, P<0.001);
cinobufotalin (C), y= - 4.10-0.606~ (goodness of fit,
P<0.001).
as a percentage of the ouabain-sensitive uptake with the
same blood with no inhibitor present. The mean ( ~ S E M )
ouabain-sensitive '"Rb uptake results are given in Table 3.
In Fig. 3 are shown the regression lines for log (p/l - p )
for ouabain, bufalin and cinobufotalin against the log of
the drug concentration in mol/l. The legend to Fig. 3
contains the equations of the regression lines and the
goodness of fit is highly significant in each case. The
slopes of the three lines are not significantly different. The
values, where y = 0, are shown in Table 1.Bufalin in
this test was 4.2 times as active as ouabain but cinobufotalin was only one-tenth as active as ouabain. The
sequence of potencies is the same as that for the
['Hlouabain-binding assays but the ICso values for
A. A. Brownlee et al.
172
recently cinobufotalin has been reported to be less active
than ouabain on this enzyme [8].
Ouabain is not the most toxic of the cardiac glycosides,
but many studies have related activity of other molecules
to that of ouabain, especially in relation to inhibition of
Na+/K+-ATPase and the inhibition of the sodium pump
or xhRbuptake. In the studies of the interaction between
bufalin and ouabain shown in Table 2, bufalin was able to
displace some of the bound [3H]ouabain (added an hour
previously), whereas competition with an equimolar
amount of cold ouabain had no effect. When bufalin was
added first it markedly reduced the ability of [3H]ouabain
added an hour later to bind to the erythrocytes. These
facts suggest a higher affinity of bufalin for the receptors
on the erythocytes. Presumably the same applies to the
XhRbuptake in the erythrocytes on which bufalin had 4.2
times the potency of ouabain.
Cinobufotalin has a 5a-OH group which is thought to
decrease biological activity and a 14,15-epoxide which
decreases activity relative to the 14p-OH possessed by
ouabain and bufalin. Ouabain has a 5p-OH which
increases activity relative to the 5p-H in bufalin. Why
bufalin with hydroxyls only at C-3 and C-14 should be
more potent than ouabain is not clear on the basis of
structure, although it is suggested by Fieser & Fieser [3]
that the 1p-OH on ouabain may have sufficient inhibitory
action to overcome the potentiating effects of the 5p-OH
and the 11a-OH also present on the ouabain molecule.
T h e animal bufadienolides differ from the plant cardiac
glycosides, not only in having a six- instead of a fivemembered lactone ring, but especially because the
Table 1. IC,, values for ouabain binding and s6Rb uptake
for the three drugs
G,,(moI/l)
Drug
Ouabain binding
X"Rbuptake
1.40 X lo-'
9.77 x 10-9
1.71 x 1 0 - 7
1.05 X
4.37 x
4.27 x 1 0 - 7
Bufalin
Ouabain
Cinobufotalin
inhibiting xhRb uptake are much higher than those for
inhibiting ouabain binding. The ratios of IC5,, for XhRb
uptake to IC,, for ouabain binding are, respectively, 7.5
for bufalin, 4.5 for ouabain and 2.5 for cinobufotalin.
DISCUSSION
These studies show that in two tests the potency of bufalin
is greater than that of ouabain and that of cinobufotalin is
less. Binding to human erythrocytes may well not be so
precise an assessment of function as the uptake of
rubidium which can replace potassium in the sodium/
potassium pump. The greater activity of bufalin in the
ouabain-binding assay could be because not all the
binding sites represent functional activity, in spite of not
being displaced by
mol/l ouabain. T h e order of
potencies of the three drugs tested was the same in both
tests. The greater potency of bufalin than ouabain in
inhibiting canine renal Na+/K+-ATPase has been
previously reported in preliminary form [ l ] and more
Table 2. Competitive effect of bufalin on [3H]ouabain binding to erythrocytes
[3H]Ouabain binding is expressed as a percentage at the end of the second hour. Results are
mean sf^^^. Ouabain and bufalin were used at a concentration of 3.64 x lo-' mol/l. Group A:
set 2 vs set 3, P< 0.02; group B: set 2 vs set 3, mean difference ( k SEM)41.76 f 4.37 (P<0.001).
NS, Not significant.
Group A
Set 1
Set 2
1st hour
2nd hour
[.'H]Ouabain
The same
1 O O f 1.9
( n = 18)
1st hour
["HIOuabain
Set 3
2nd hour
Bufalin added
87.4 k 2.9
1st hour
["HIOuabain
2nd hour
Unlabelled
ouabain added
97.9 f 2.7
( n = 18,*t=3.58, P<O.Ol)
( n= 18, * t = 0.623, NS)
Set 2
Set 3
*Compared with control set.
Group B
Set 1
1st hour
Buffer only
2nd hour
['HIOuabain
added
100k3.1
(n=17)
*Compared with control set.
1st hour
Bufalin
2nd hour
[3H]Ouabain
added
23.4 f 1.5
( n = 18,*f=22.88, P<O.OOl)
1st hour
Unlabelled
ouabin
2nd hour
[3H]Ouabain
added
65.1 f 4 . 1
( n = 18,*r=6.755, P<O.OOl)
Effect of bufadienolides compared with ouabain on erythrocytes
-1.1
- 2.0
,
, ,
, ,,,
,
, ,,,
,
, ,
\=-
- 10
-9
-8
-7
-6
log[Dwl (mol/l)
Fig. 3. Ouabain-sensitive xhRb uptake by erythrocytes.
The log of (p/l-p) is plotted against the log of the
molarity of the inhibitors, where p is the proportion of the
maximum ouabain-sensitive response at the drug concentration being considered. The regression lines were fitted
by least squares, the equations being: bufalin (B),
y = - 7.620-0.955~(goodness of fit, P< 0.001); ouabain
(0),y = - 5.943-0.807~ (goodness of fit, P<O.OOl);
cinobufotalin (C), y = - 4.203-0.660~ (goodness of fit,
P<O.OOl).
bufadienolides are not glycosylated at C-3. According to
Fieser & Fieser [3],it is the attachment to a sugar molecule
which greatly potentiates the uptake by cardiac tissues
and so determines the lethality in the Chen test (K. K.
Chen estimated the toxicity of cardiac glycosides and
bufadienolides by the lethal dose which led to the death of
an anaesthetized cat when given by slow intravenous
infusion; the data produced by Chen have been analysed
by Fieser & Fieser [3]). It may be that molecules
173
resembling bufalin (which has not been reported on in the
Chen test) may be less likely to have such a high uptake by
the heart and thus less likely to be lethal in the Chen test,
because they are not glycosylated.They may nevertheless
have appreciable activity in inhibiting the sodium/
potassium pump as the studies on x6Rb uptake here
suggest.
The structural requirements of cardenolides and
bufadienolides for action on the sodium/potassium pump
of tissues other than the heart have not been defined in
detail. However, Glynn [9]established that a lactone on its
own was largely inactive, the 17a-configuration of the
lactone is important in cardenolides and so also is the
double bond in the lactone ring. The two bufadienolides
conform to these rules. Cinobufotalin was much less
active than ouabain in the tests reported here, whereas
our unpublished data on cinobufagin show that replacing
the 5a-OH on cinobufotalin by 5B-H in cinobufagin
restored the activity in inhibiting canine renal Na+/K+ATPase to that comparable with ouabain.
It is well known that the rat is insensitive to cardiac
glycosides and even to the plant bufadienolide, scillaren A
(studied by Glynn [9]). However, the closely related
scillaroside (from the red squill plant) is highly toxic to the
rat and unique in that regard. The only difference
between the scillaren A and scillaroside (apart from the
sugars on C-3) is that the lethal compound has an acetoxy
function on C-6 131. We have confirmed that the rat is
insensitive to the bufadienolide, bufalin, and, though
unable to affect the natriuretic response to atrial natriuretic peptide when superimposed on it [lo], it did
potentiate the natriuretic response to angiotensin when
superimposed on it in the rat [ 111.
It may be of interest that neither of the bufadienolides
used here reacted with digoxin antibodies (A. A. Brownlee & I. H. Mills, unpublished work). Studies on digoxinlike substances in animals in relation to sodium
metabolism may not, therefore need to be limited to those
which react with digoxin antibodies.
Table 3. Effect of inhibitors on ouabain-sensitive8aRbuptake
Results are means k SEM. n =number of blood samples, each tested in triplicate.
x6Rbuptake (%)
Concn. (mol/l)
Bufalin
10-lIl
10-9
2 x 10-9
4X lo-'
10-8
2 x 10-8
4X
6X
8 x 10-8
10-7
4X
6X
lo-"
2 x 10-6
99.0f0.8 (n= 5)
85.4f4.3(n=5)
90.0f5.8(n=4)
79.7 f 8.8 (n= 4)
48.4 f 2.3 (n= 9)
-
8.OIt1.1(n=5)
-
Ouabain
94.8f 2.9(n= 5)
-
-
81.4f 1.4 (n= 5)
68.0f2.1 (n=4)
51.7 f 5.2( n= 4)
41.3 f 2.9 (n= 4)
34.0f 3.3(n= 4)
34.6f 0.9 ( n= 5)
-
Cinobufotalin
-
99.8f 3.8(n'= 5)
-
100.0f2.0( n = 5 )
-
81.0 f2.6( n= 5)
56.0 f 6.0( n= 4)
46.0f 4.6(n= 4)
36.7f 1.4 ( n= 9)
23.3 f 3.8( n= 3)
174
A. A. Brownlee et al.
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