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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. REFERENCES 1. Brownlee, A.A., Lee, G. & Mills, 1.H. Marked inhibition of 2. 3. 4. 5. 6. canine renal Na/K ATPase by a bufadienolide but weak natriuretic activity in the rat. J. Physiol. (London) 1987; 390,941: Abel. J.J. & Macht, D.J. Two crystalline pharmacological agents obtained from the tropical toad, Euja q u a . J. Pharmacol. Exp. Ther. 1 9 1 I ;3 , 3 19-77. Fieser, L.F. & Fieser, M. Steroids. London: Chapman and Hall, 1969: 727-809. Flier, J.S. Ouabain-like activity in toad skin and its implications for endogenous regulation of ion transport. Nature (London) 1978; 274,285-6. de Wardener, H.E. & MacGregor, G.A. Dahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. Kidney Int. 1980; 18, 1-9. Kim, R.-S.S. & LaBella, F.S. Endogenous ligands and 7. 8. 9. 10. 11. modulators of the digitalis receptor: some candidates. PharmacoLTher. 1981; 14,391-409. De Luise, M., Blackburn, G.L. & Flier, J.S. Reduced activity of the red-cell sodium-potassium pump in human obesity. N. Engl. J. Med. 1980; 303,1017-22. Mills, I.H., Brownlee, A.A. & Tait, A.D. Comparison of effects of bufadienolides and ouabain on the Na/K pump. 31st Int. Congr. Physiol. Sci. P1238. Glynn, I.M. The action of cardiac glycosides on sodium and potassium movements in human red cells. J. Physiol. (London) 157; 136,148-73. Brownlee, A.A., Lee, G. & Mills, I.H. Changes in renal function produced by the Na/K ATPase inhibitor bufalin when superimposed on infusion of atrial natriuretic peptide (ANP)in rats. J. Physiol. (London) 1987; 396,109R Brownlee, A.A., Lee, G. & Mills, I.H. Potentiation of the natriuretic action of angiotensin by the Na/K ATPase inhibitor bufalin in the rat. J. Physiol. (London) 1987; 394, 521?