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On the cooperativity of ouabain-binding to intact myocardium
j Mol Cell Cardiol 17, 1095-1104 (1985)
On the Cooperativlty of Ouabain-binding to Intact Myocardium Stefan Herzig, Heinz Liillmann* and Klaus Mohr Department of Pharmacology, University of Kiel, Hospitalstrasse 4-6, 2300 Kid 1, FRG (Received 15January 1985, acceptedin revisedform 11 June 1985) S. HERZ1G, H. L~TLLMANNANn K. MOHR. On the Cooperativity of Ouabain-binding to Intact Myocardlum.
Journal of Molecularand CellularCardiology (1985) 17, 1095-1104. A theoretical concept is presented which
proposes that binding of ouabain to intact myocardium should be positive cooperative. It is based on the assumption that the myocardial Na/K-ATPases expose the ouabain-bindingsite only at a particular conformation adopted during a turnover cycle. The turnover rate and thus the ouabain-bindingproperties are regulated by the cytosolic Na-ion-concentration Nai. Any occupation of cellular Na/K-ATPases should affect the ouabain-binding properties of the unoccupied Na/K-ATPases, because their turnover rate is increased via an elevated Na i . A computer model which takes into account the interrelationships of the Na/K-ATPases both with Na i and with the ouabain-concentrationpredicts that ouabain-bindingshould proceed in a concentratio~ proportional fashion as long as the Na-load can be counterbalanced by non-occupied Na/K-ATPase molecules. The concentration-proportional binding reflects a positive cooperativity. Experimental results reveal that (3H)ouabain-bindingto Na/K-ATPase of electrically stimulated guinea-pig left atria was in fact concentration proportional under certain experimental conditions. The biological significance of the proposed concept remains to be elucidated. KEY WoRDs: Cooperativity; Ouabain-binding; Na/K-ATPase; Intact myocardium.
Introduction W h e n the i n t e r a c t i o n of o u a b a i n with its r e c e p t o r site, the N a / K - A T P a s e , is studied in vitro u s i n g for i n s t a n c e c r u d e m e m b r a n e or purified N a / K - A T P a s e - p r e p a r a t i o n s , ouab a i n b i n d i n g is often f o u n d to o c c u r at a single p o p u l a t i o n of i n d e p e n d e n t b i n d i n g sites [9, 10, 12, 13, 31, 32]. T h e b i n d i n g sites are called ' i n d e p e n d e n t ' , because the o c c u p a t i o n of a f r a c t i o n of these sites does n o t i n f l u e n c e the b i n d i n g characteristics of the u n o c c u p i e d sites. I n this c o m m e n t a r y , the a t t e m p t is m a d e to p r e d i c t the characteristics of o u a b a i n - b i n d i n g in i n t a c t m y o c a r d i a l tissue. T h e considerations are based o n g e n e r a l l y a c c e p t e d aspects of N a / K - A T P a s e - f u n c t i o n a n d of o u a b a i n N a / K - A T P a s e - i n t e r a c t i o n . T h e y lead to the c o n c l u s i o n t h a t o u a b a i n - b i n d i n g should be c o o p e r a t i v e in i n t a c t m y o c a r d i u m . F i n a l l y , some e x p e r i m e n t a l results are p r e s e n t e d w h i c h reveal t h a t the expected c o o p e r a t i v i t y
of o u a b a i n - b i n d i n g c a n in fact be observed u n d e r c e r t a i n e x p e r i m e n t a l c o n d i t i o n s i n isolated g u i n e a - p i g atria. T h e i n t e r a c t i o n b e t w e e n o u a b a i n a n d its r e c e p t o r is d e s c r i b e d b y m e a n s of two concepts: one, the essential features of w h i c h h a v e b e e n described p r e v i o u s l y [3, 20, 21] a n d w h i c h is e x t e n d e d here (designated as ' c o n c e p t of m o d i f i e d b i n d i n g site d e n s i t y ' ) ; a n d a novel one ( ' c o n c e p t of modified affinity'), w h i c h m i g h t h a v e the a d v a n t a g e t h a t the events d e t e r m i n i n g o u a b a i n - b i n d i n g are illustrated in the m o r e f a m i l i a r terms comm o n l y used to c h a r a c t e r i z e r e c e p t o r - l i g a n d interactions.
Theoretical considerations Aspects of Na/ K A TPase function D u r i n g a t u r n o v e r cycle a N a / K - A T P a s e molecule is t h o u g h t to u n d e r g o c o n f o r m a t i o n a l c h a n g e s (Fig. 1) : s t a r t i n g from a 'resting' con-
* To whom correspondence should be sent at the above address. 0022 2828/85/111095 + 10 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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~
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F I G U R E I. Transport cycle of the Na/K-ATPase; note: the ouabain binding site is only exposed at a particular conformation. For details see text.
formation, the phosphorylated enzyme is activated by Na-ions bound to its cytosolic site to translocate these Na-ions into the extracellular space; thereafter, the enzyme can bind K-ions at the extracellular site and translocate them into the cytosol. After dissociation of these K-ions, the 'resting' conformation is adopted again [1, 18, 20, 26]. The concentration for half-maximum enzyme-activation by cytosolic Na-ions has been determined to lie in the range of Na-ion-concentrations, which occur physiologically in the cytosol [-22, 25, 27]. The concentration for half maximum activation by extracellular K-ions was reported to lie below the physiological extracellular K-ion-concentration [1, 19]. Hence, the activation exerted by K-ions in the range of physiological concentrations shall be considered to be near maximum, and the cytosolic Na-ion-concentration is taken to be the determinant of the turnover rate. The extraordinary feature of the interaction between ouabain and its receptor is that ouabain can only associate with high affinity to that particular Na/K-ATPaseconformation, which is exposed during the p u m p cycle before attachment of K-ions. For this reason, the turnover rate of Na/KATPase or the cytosolic Na-ion-concentration, respectively, essentially determines the extent of ouabain-binding. This can be illustrated by the well-established dependency of ouabain-binding on the beat frequency of myocardial preparations [-3, 20, 21, 28].
Dependency of ouabain-binding on the beat-frequency A synopsis of the interrelationship between heart rate, Na/K-ATPase transport rate and ouabain-binding is schematically depicted in Figure 2. As illustrated in Figure 2(a) and
2(b), the equilibrium binding of ouabain is enhanced with increasing beat frequency. The explanation for this finding is presented in Figures 2(c) (e): the increased stimulation frequency necessitates an enhanced N a / K ATPase transport rate in order to counterbalance the elevated Na- and K-ion fluxes. 'Transport rate' is defined as the amount of Na/K-ions transported per unit time by all Na/K-ATPases of a cell, while 'turnover rate' means the transport velocity of a single N a / K ATPase molecule. Since the turnover rate is probably regulated by the cytosolic Na-ionconcentration Nal, the enhanced transport rate should correspond to an increased Na i. In fact, Cohen et al. [6] found Na i to be elevated at increased beat frequencies. To illustrate the general relationship between transport rate and Nal, in vitro data reported by Philipson and Nishimoto [22] were used to evaluate Nal-values for three transport rates arbitrarily chosen [Fig. 2(c)]. In turn, the elevated Na/K-ATPase transport rate leads to a more frequent appearance of the ouabain-binding enzyme-conformation. The time interval, in which this particular conformation exists during a transport cycle, should be constant and independent of the turnover rate, if the activation of the Na/KATPase by extracellular K-ions is assumed to be constant. So, when the enzyme undergoes a higher number of turnover cycles per unit of time, then it stays for a longer fraction of this time in the ouabain-binding conformation. Formally, the dependency ofouabain-binding on transport rate can be analysed in terms of (a): an increased actual density of binding sites, and (b): an increased actual affinity of ouabain receptors. (a) Concept of modified binding site density [-Fig. 2(d)]: Since only a particular conformation of Na/K-ATPase is capable to bind ouabain with a given and constant high affinity, only a certain fraction of the total number of Na/K-ATPases is actually available as ouabain binding sites at any moment. Thus, each transport rate is connected with a certain actual density of available binding sites. With an altered Na i the ratio between the Na/K-ATPases available as binding sites (filled circles) and unaccessible for ouabain (open circles)--and hence ouabain-binding, is modified. It should be noted that an instant
Cooperativity of Ouabain-binding
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F I G U R E 2. Dependency of ouabain-binding on the beat frequency. (a): Isolated guinea-pig atria were electrically stimulated at the beat frequencies of 0.5, 3 and 6 Hz (rectangular pulses, 5 ms duration, intensity about 30% above threshold). Tyrode solution in m ~ : NaC1 136.8, KC1 5.4, N a H C O 3 11.9, NaH2PO 4 0.21, CaCI 2 1.8, MgC12 1.05, glucose 5.5; 95% 0 2 / 5 % CO2; 32~ (b) : Equilibrium values of specific (3H)ouabain-binding to guinea-pig left atria were determined as described previously [3, 15]. In short, after an equilibration period of 1 h, the atria were exposed to 1 nM (3H)ouabain; after different time intervals up to 5 h, atria were removed from the organ baths, blotted, weighed and processed for liquid scintillation counting. Specific (3H)ouabain-binding was calculated as the difference between the total tissue-bound radioactivity and the amount of (3H)ouabain present in the extracellular space (30% of tissue wet weight) ; (c) : Dependency of Na/K-ATPase transport rate on the cytosolic Na-ion-concentration. The data are adapted from in vitro experiments carried out with dog sarcolemmal vesicles by Philipson and Nishimoto [22]. The arrows indicate points, which are assumed to correspond to the respective beat frequency; (d): Illustration of the concept of modified binding site density. Circles represent N a / K - A T P a s e molecules of the cell in the ouabain-binding (filled) or in other (open) conformations; (e) ; Illustration of the concept of modified affinity. The intensity of hatching indicates the apparent ouabain-binding affinity of the Na/K-ATPases.
1098
S. Herzig et aL
view is depicted; since all Na/K-ATPase molecules take part in the ion transport, all enzyme molecules are randomly present in the ouabain-binding conformation. The ratio, however, between the two conformational states is constant at any moment of a given condition. (b) Concept of modified affinity [Fig. 2 (e)] : Each Na/K-ATPase molecule is considered to represent a receptor irrespective of the actual transport rate; i.e. the density of binding sites equals the number of Na/K-ATPases under all conditions. The affinity of the receptors, however, is modified by the transport rate. The more often the ouabain-binding conformation occurs per unit time, the more is the probability increase of the association of a ouabain molecule to the enzyme. Therefore, the association rate constant k + 1 of ouabain-binding is variable and is a function of the turnover rate of the N a / K - A T P a s e molecules and thus a function of Na i. It is assumed that the dissociation of the complex formed between ouabain and the Na/KATPase is independent of Na i. So, the rate constant of dissociation k - 1 shall be considered constant. Accordingly, the affinity k + l / k 1 is a function ofNai. With altered Na i the affinity of the receptor sites (intensity of hatching) and hence ouabain-binding, is modified. This time-independent 'overall' affinity of the Na/K-ATPase molecules shall be designated 'apparent affinity' in order to differentiate it from the constant affinity of the ouabain-binding enzyme conformation used in the concept of modified binding site density. Irrespective of the theoretical concept applied, different amounts of ouabain have to be expected to become bound at a given ouabain-concentration at different beatfrequencies. Generally, an alteration of the cytosolic Na-ion-concentration seems to be associated with an altered ouabain-binding; e.g. the sodium-ionophore monensin enhances ouabain-binding [15, 30], a reduction of the extracellular Na-ion-concentration diminishes ouabain-binding [8, 15, 30].
Concentration-dependencyof ouabain-binding in intact myocardium The dependency of ouabain-binding on the cytosolic Na-ion-concentration should have
bearings upon the concentration-dependency of ouabain-binding in intact myocardium. When a cell is exposed to a non-toxic ouabain-concentration, ouabain will occupy a certain fraction of the Na/K-ATPases, depending on the actual binding site density or the actual apparent affinity, respectively. Since the occupied Na/K-ATPases are inhibited, the unoccupied are driven at a higher turnover rate in order to counterbalance the Na-load. This activation is brought about by an elevated cytosolic Na-ion-concentration. The increment o f N a i in turn leads to an additional ouabain-binding at the given ouabainconcentration, analogously to the above described dependency of ouabain-binding on Na i . The equilibrium binding of ouabain will be attained, when (1) the transport activity provided by the unoccupied Na/K-ATPases balances the Na-load and (2) the amount of ouabain bound corresponds to the new Na i . The situations before addition of ouabain and after attainment of the binding equilibrium at 50% occupancy of Na/K-ATPases are schematically depicted in Figure 3. The transport rate required for the compensation of Na- and K-ion-fluxes is arbitrarily chosen to amount to 30% of the maximum. In this example, an increment of Na i by about 4 mM is required to maintain the transport rate [Fig. 3(a)]. In the lower part of Figure 3 the example is illustrated with respect to both concepts. (a) Concept of modified binding site density [Fig. 3(b)]: under control conditions, randomly three out of ten Na/K-ATPases are in the ouabain-binding conformation at any moment. At equilibrium binding of ouabain, five out of ten Na/K-ATPases are occupied and inhibited. Because the remaining unoccupied Na/K-ATPases still counterbalance the Na-load, the number of unoccupied Na/K-ATPases being in the ouabain-binding conformation at any moment again amounts to three. Accordingly, eight out of ten Na/KATPases represent binding sites actually taking part in the ouabain-Na/K-ATPaseinteraction at any moment, instead of three under control conditions. (b) Concept of modified affinity [-Fig. 3(c)]: In the presence of ouabain, the unoccupied Na/K-ATPases expose the ouabainbinding conformation more frequently due to
Cooperativity o f O u a b a i n - b i n d i n g
1099
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FIGURE 3. (a): Effect of a 50% occupancy of the cellular Na/K-ATPases by ouabain on the relationship between Na/K-ATPase transport rate and the cytosolic Na-ion-concentration. Illustration of the consequences of the inhibition of Na/K-ATPase molecules and the accompanying increment of Na i on the cellular ouabain-binding characteristics; (b): Concept of modified binding site density: in spite of the occupied Na/K-ATPases (crossed filled circles), the number of available unoccupied binding sites (filled circles) remains unaltered; (c) : Concept of modified affinity: the apparent affinity is increased as a result of the occupation of Na/K-ATPases.
their increased t u r n o v e r rate; this leads to an increased p r o b a b i l i t y o f ouabain-association. Therefore, the resulting affinity of the receptors exceeds the control affinity. Both concepts reveal that a n y o c c u p a t i o n of N a / K - A T P a s e s by o u a b a i n should affect the o u a b a i n - b i n d i n g properties of the unoccupied enzyme molecules, because the turnover rate of these N a / K - A T P a s e s is increased. This means that the receptor sites of o u a b a i n are not i n d e p e n d e n t from each other: in contrast, they are cooperative. T h e i n t e r d e p e n d e n c e of the receptors is m e d i a t e d b y the cytosolic Naion-concentration. In o r d e r to predict o u a b a i n - b i n d i n g curves
taking into account these prerequisites, a c o m p u t e r model was a p p l i e d [13]: for each o u a b a i n - c o n c e n t r a t i o n , the a p p r o p r i a t e N a i to m a i n t a i n the t r a n s p o r t rate and the corres p o n d i n g level of o u a b a i n - b i n d i n g was calculated. I n short, the m o d e l combines the s i g m o i d - s h a p e d interrelationships of the N a / K - A T P a s e with (1) the cytosolic N a - i o n concentration, and (2) the o u a b a i n - c o n centration. This model is a p p l i c a b l e for nontoxic conditions, i.e. for the o u a b a i n c o n c e n t r a t i o n range, in which the inhibition of N a / K - A T P a s e s can be effectively c o m p e n sated for by an elevated N a i. At toxic o u a b a i n - c o n c e n t r a t i o n s the unoccupied N a /
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FIGURE 4. Ouabain binding curve predicted by means of a computer model, which combinesthe interrelationships of the Na/K-ATPase with Nai and the ouabain-concentration. The calculations refer only to non-toxic ouabainconcentrations, because toxic concentrations induce a progressive accumulation of Nai; (a): Dependency of ouabainbinding and Nai on the ouabain-concentration. For sake of comparison, the dotted line indicates a sigmoid-shaped binding-curve, which would result, if the dependency of ouabain-binding on Nai were not considered, i.e. in the absence of cooperativity; (b) : Scatchard plot of the binding curve shown in (a) : the horizontal line means that binding is concentration proportional. The thin lines were constructed according to de Meyts and Roth [7]. The slope is a measure of the respective apparent affinity at the indicated fractional receptor occupancies; (c) : Plot of the apparent affinityv. the fractional receptor occupancy: the ascendingline means that binding is positivecooperative.
K-ATPases c a n n o t c o u n t e r b a l a n c e the Naload. Therefore, Na-ions will progressively a c c u m u l a t e within the cell, i.e. equilibrium values o f N a i and also of o u a b a i n - b i n d i n g will no longer be attained. Figure 4(a) depicts an e x e m p l a r y calculation based on a required transport rate of 26% of the m a x i m u m transport capacity. Some aspects of these theoretically predicted relationships are in accordance with experimental findings reported in the litera t u r e : the m a i n t a i n e d N a / K - A T P a s e transport rate is reflected by the observation that 8 6 R b + - u p t a k e is not inhibited in guinea pig atria treated with non-toxic o u a b a i n concentrations [4]; the o u a b a i n - i n d u c e d elevation of Na i has been recorded in the m y o c a r d i u m of several species by means of ion-sensitive microelectrodes [16, 17, 29]; the steepness of the Nai-curve is similar to a curve shown by Lee et al. [16] for the d e p e n d e n c y of N a i on the d i h y d r o o u a b a i n - c o n c e n t r a t i o n in sheep Purkinje fibers. T h e steepness of the o u a b a i n - b i n d i n g curve corresponds to the experimentally evaluated curve shown in Figure 5. T h e steep, n a r r o w concentrationi n o t r o p y curves usually obtained with cardiac glycosides p r o b a b l y reflect both the steep b i n d i n g curve a n d the fact that only a certain
fraction of the cellular N a / K - A T P a s e s can be occupied w i t h o u t intoxication. I n order to analyse the p a t t e r n of o u a b a i n binding, the calculated b i n d i n g data were plotted according to Scatchard [24] [Fig. 4(b)]. T h e horizontal line indicates a proportionality between the b i n d i n g a n d the concentration of o u a b a i n in the non-toxic range, suggesting that b i n d i n g were not saturable at these concentrations. This result is not surprising with respect to the concept of modified b i n d i n g site density: it includes that o u a b a i n b i n d i n g does not induce a reduction of the n u m b e r of actually available unoccupied b i n d i n g sites in the non-toxic concentration range. W h e n the b i n d i n g data are analysed according to de Meyts a n d R o t h [7] in order to determine the a p p a r e n t affinity of binding, it becomes obvious that the increased fractional o c c u p a n c y is paralleled by an augmented a p p a r e n t affinity [Fig. 4(c)]: this indicates a positive cooperativity.
Experimental
results
I n an a t t e m p t to test whether the outlined theoretical predictions could be experimentally substantiated, ( 3 H ) o u a b a i n - b i n d i n g was
Cooperativity of Ouabain-bindlng
1101
low concentrations a small s a t u r a t i n g comp o n e n t was seen, the extent of which is repu cu .resented by the difference between the ~5o~ continuous a n d the h a t c h e d line in Figure .E ----500 " ~ o o .," o9~ E ,.5(a). W h e n the d a t a are plotted according to o S c a t c h a r d [Fig. 5(b)], the s a t u r a t i n g com. . . . . , , , . p o n e n t is reflected by the initial descending 50O I000 1500 line. T h e c o n c e n t r a t i o n - p r o p o r t i o n a l binding 0u0boin-c0ncentrofion [ nM] is i n d i c a t e d by the horizontal line ( b o u n d / free = constant!), which has been expected a c c o r d i n g to the theoretical considerations (cf Fig. 4(b)]. T h e u n e x p e c t e d initial descending line could represent the presence of an additional p o p u l a t i o n of b i n d i n g sites not being i II I J l l l II I i i subject to the discussed r e g u l a t o r y mecha500 tO00 nisms; alternatively, the descending line Bound [nmol/kg cell] would indicate t h a t the a p p a r e n t affinity of FIGURE 5. Dependency of specific ouabain-binding in isolated guinea-pig atria on the ouabain-con- the m y o c a r d i a l N a / K - A T P a s e s did not reveal centration. The atria were stimulated at a frequency of the expected increase with o u a b a i n - c o n 0.5 Hz in the presence of 1 to 3 nM (SH)ouabain and centrations up to 100 nM. A t present it c a n n o t various concentrations of unlabelled ouabain under be decided between these two possibilities, b u t experimental conditions as described in the legend of it should be p o i n t e d out that this initial comFigure 2. The atria were removed from the organ baths after different time intervals up to 3 h. (a): Specific p o n e n t is e x a g g e r a t e d by the S c a t c h a r d plot binding represents the difference between the equilibrium a n d that the biological significance is obscure, values of total (3H)ouabain-binding and (3H)ouabain because an i n o t r o p i c effect could not be accumulated in the extracellular space, which amounted detected at these concentrations. to 30% of the tissue wet weight. The indicated data refer T h e c o n c e n t r a t i o n - p r o p o r t i o n a l compoto non-toxic ouabain-concentrations; at toxic concentranent reflects a positive c o o p e r a t i v i t y u n d e r the tions a stable equilibrium binding was not attained. The hatched line shows the concentration-proportional com- assumption that b i n d i n g takes place at a ponent of binding. (b): Scatchard plot of the binding s a t u r a b l e p o p u l a t i o n of specific b i n d i n g sites, data. The shaded area includes the range of receptor i.e. the o u a b a i n - r e c e p t o r of m y o c a r d i a l N a / K occupancy, in which a positive inotropic effect was detectable, when the isometric force of contraction was record- ATPases. T h a t this condition is actually fuled under conditions as applied in the binding filled, becomes evident from the following experiments. observations ( d a t a not shown); ( 1 ) I n the presence of 10 . 4 M unlabelled ouabain, the c o n c e n t r a t i o n - p r o p o r t i o n a l b i n d i n g of (all) m e a s u r e d in i s o l a t e d guinea-pig left atria [2], o u a b a i n was abolished, the r a d i o a c t i v i t y a c c o r d i n g to the m e t h o d described by Bent- r e t a i n e d by the a t r i a m a t c h i n g the a m o u n t of feld et al. [3]. T h e atria were m o u n t e d in ( a H ) o u a b a i n a c c u m u l a t e d in the extracellular o r g a n - b a t h s containing a modified T y r o d e space. Thus, the c o n c e n t r a t i o n - p r o p o r t i o n a l solution (cflegend Fig. 2) a n d were stimulated b i n d i n g occurred at a s a t u r a b l e p o p u l a t i o n of at t h e beat-frequencies of 0.5 a n d 3 Hz, b i n d i n g sites. (2) Binding was potassiumrespectively. After an equilibration period of sensitive: when the K - i o n - c o n c e n t r a t i o n in 1 h, ( a H ) o u a b a i n was a d d e d and its the organ b a t h was raised from 5.4 mR to e q u i l i b r i u m - b i n d i n g was d e t e r m i n e d (cf 10.8 mM, the concentration-proportional legends Fig. 2 a n d Fig: 5). T h e isometric force c o m p o n e n t was r e d u c e d (the bound/free of c o n t r a c t i o n was separately recorded u n d e r value was decreased by a b o u t 50%). T h e K i o n - i n d u c e d inhibition is a typical feature of identical e x p e r i m e n t a l conditions. T h e o u a b a i n - b i n d i n g curve o b t a i n e d at a specific o u a b a i n - b i n d i n g . (3) T h e b i n d i n g was s t i m u l a t i o n frequency of 0.5 Hz is depicted in s o d i u m - l o a d d e p e n d e n t : a reduction of the Na-ion-concentration to F i g u r e 5. O v e r a wide range of o u a b a i n - extracellular concentrations, the b i n d i n g p r o c e e d e d in a 83.5 mR d i m i n i s h e d the c o n c e n t r a t i o n c o n c e n t r a t i o n - p r o p o r t i o n a l m a n n e r . O n l y at p r o p o r t i o n a l b i n d i n g (the bound/free value
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was decreased by about 20%). The dependence on the sodium-load is indicative of ouabain-Na/K-ATPase-interaction (cf Fig. 2). (4) The concentration range, in which positive inotropic effects could be measured, matched the concentration range, in which the concentration-proportional binding was obvious [shaded area in Fig. 5(b)]. The time courses of the inotropic response and of the ouabain-binding were similar. In conclusion, the concentration-proportional binding took place at those receptors, which mediate the positive inotropic response. Accordingly, the concentration-proportional component of ouabain-binding does not represent unspecific binding, but the expected positive cooperative ouabain-binding to the Na/K-ATPases of intact myocardium. At a stimulation frequency of 3 Hz, however, specific binding of ouabain did not reveal a concentratio~proportional component in the investigated non-toxic concentration range up to 300 nM. At higher concentrations a stable equilibrium binding could not be attained, because the ouabainbinding declined after a temporary maximum. In the Scatchard-plot (Fig. 6, upper curve), the points could be connected by a descending straight line, formally indicating that binding followed a simple law of mass action. A concentration-proportional corn-
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3Hz
"6 =
~,~
"--O--"O---'O [ N~ =83.5 mM
i
=
I
I
I
I
i
I
I
I
500 1000 Bound [nmol/kg cell]
F I G U R E 6. Equilibrium values of specific ouabainbinding at non-toxic concentrations at a beat frequency of 3 Hz, plotted according to Scatchard. Methodical details as described above. The upper line represents binding at a normal Na-ion-concentration in the organ bath. The lower line indicates ouabain-binding at a reduced Na-ion-concentration of 83.5 mM, NaG1 having been isoosmotically replaced by sucrose.
ponent of binding became, however, obvious even at 3 Hz, when the extracellular Na-ionconcentration was reduced to 83.5 mM (Fig. 6, lower curve). Under this condition, the non-toxic range of ouabain-concentrations was extended up to 600 nM. The theoretically predicted concentrationproportional binding of ouabain was demonstrable at 'conditions, which allowed to investigate a wide range of non-toxic ouabainconcentrations. Under all investigated conditions, however, a saturating component of binding was detected, which was reflected by the initial descending parts of the Scatchard plots. Since only a limited range of nontoxic ouabain-concentrations could be applied at 3 Hz under normal conditions, the saturating component might have had the predominant influence on the pattern of the binding curve. The mechanism underlying the saturating component of ouabain-binding cannot be readily explained at present. Two speculative mechanisms might be offered: ( 1 ) a small population of high-affinity binding sites not being involved in the regulatory events could have been present; the saturating binding to this population would then add to the concentration-proportional ouabain-binding. (2) Binding could have taken place at a homogeneous population of myocardial Na/ K-ATPases, but parameters not being considered by the proposed model could have modulated the pattern of ouabain-binding: apart from Nal, additional influences could determine the Na/K-ATPase transport rate or the ouabain-binding affinity of the Na/KATPases at a given turnover rate (e.g. alterations of the extracellular cleft K-ionconcentration, of the membrane potential, or of cytosolic determinants of Na/K-ATPase function [9, 23]). Both explanations do not exclude that the general regulatory mechanisms described in the theoretical part, which became obvious as a concentration-proportional binding at other conditions, could still have been present at 3 Hz. In summary, the expected concentrationproportional pattern of ouabain-binding has been demonstrated under certain experimental conditions. Although the majority of studies dealing with the binding of ouabain to intact myocardial preparations revealed
Cooperativity of Ouabain.binding b i n d i n g c u r v e s w i t h o u t signs o f a positive c o o p e r a t i v i t y , s o m e reports [5, 11] s h o w d a t a s u g g e s t i n g the p r e s e n c e o f a c o n c e n t r a t i o n p r o p o r t i o n a l c o m p o n e n t of o u a b a i n - b i n d i n g , yet h a v i n g b e e n i n t e r p r e t e d by the a u t h o r s as unspecific b i n d i n g . T h u s , the p r e s e n t experim e n t a l results are n o t in f u n d a m e n t a l conflict w i t h findings p u b l i s h e d previously.
Conclusion T h e o r e t i c a l c o n s i d e r a t i o n s as well as experimental observations have been presented, w h i c h m a y f a v o u r the i d e a o f a c o o p e r a t i v i t y o f o u a b a i n - b i n d i n g in i n t a c t m y o c a r d i u m .
1103
O c c u p a t i o n a n d i n h i b i t i o n of a f r a c t i o n of the c e l l u l a r N a / K - A T P a s e s by o u a b a i n is ass u m e d to a l t e r the o u a b a i n - b i n d i n g c h a r a c teristics of the N a / K - A T P a s e s via a n e l e v a t i o n of the cytosolic N a - i o n - c o n c e n t r a t i o n . B i n d i n g data indicating a non-cooperativity of ouabain-binding may only apparently contradict the p r o p o s e d theory. I n a n y case, a discussion of this a l t e r n a t i v e v i e w c o u l d p r o v i d e f u r t h e r i n s i g h t i n t o the m e c h a n i s m s i n v o l v e d in o u a b a i n - r e c e p t o r - i n t e r a c t i o n s in i n t a c t m y o c a r d i u m , w h i c h are c o m p l i c a t e d by the fact t h a t N a / K - A T P a s e in vivo is subject to physiological r e g u l a t i o n s - - i n c o n t r a s t to N a / K A T P a s e of c r u d e m e m b r a n e - or p u r i f i e d enzyme preparations.
References 1 AKERA,T., BRODY,T. M. The role of Na +, K+-ATPase in the inotropic action of digitalis. Pharmacol Rev 29, 187-220 (1978). 2 BENECKE,W., HERmO, S. Characteristics of (3H)ouabain binding and ouabain-induced positive inotropism in guinea-pig atria under various conditions [Abstract]. Naunyn-Schmiedeberg's Arch Pharmaco1329, R56 (1985). 3 BENTFEL~,M., L/JI~L~ANN,H-, PETERS,T., PROPPE, D. Interdependence of ion transport and the action ofouabain in heart muscle. BrJ Pharmaco161, 19-27 (1977). 4 BRowN,L., WE~DAN,K., ERDMANN,E. Consequences of specific (3H)ouabain binding to guinea pig left atria and cardiac cell membranes. Biochem Pharmacol 32, 423-435 (1983). 5 BussE, F., LiJLLNANN,I't., PETERS,Y. Concentration-dependence of the binding ofouabain to isolated guinea-pig atria.J Cardiovasc Pharmacol 1,687 698 (1979). 6 COHEN,C. J., FOZZARD,H. A., SHEU, S.-S. Increase in intracellular sodium ion activity during stimulation in mammalian cardiac muscle. Circ Res 50, 651-662 (1982). 7 DE MEVTS, P., Ror~I, J. Cooperativity in ligand binding: a new graphic analysis. Biochem Biophys Res Commun 66, 1118-1126 (1975). 8 DUT'rA, S., MARKS,B. H. Factors that regulate ouabain (3H)-accumulation by the isolated guinea pig heart. J Pharmacol Exp Ther 170, 318-325 (t969). 9 ERI~AN~, E., SCnONER, W. Ouabain-receptor interactions in (Na + + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants. Biochim Biophys Acta 330, 302-315 (1973). 10 ERDMANN~E., SCnONER,W. Ouabain-receptor interactions in (Na + + K +)-ATPase preparations. IV. The molecular structure of different cardioactive steroids and their affinity to the glycoside receptor. Naunyn-Schmiedeberg's Arch Pharmaco12113, 335-356 (1974). 11 GOOFRA~ND,T., LESNE,M. The uptake of cardiac glycosides in relation to their actions in isolated cardiac muscle. BrJ Pharmaco146, 488 497 (1972). 12 HANSON,O., SKOU,J. C. A study on the influence of the concentration of Mg 2+, Pi, K +, Na +, and tris on (Mg2+ + Pi)-supported g-strophanthin binding to (Na++ K+)-activated ATPase from ox brain. Biochim Biophys Acta 311, 51 66 (1973). 13 HERZIG,S. An attempt to describe by a mathematical model the relationship between cardiac glycoside binding, Na-K-ATPase activity and cytosolic sodium ion activity [Abstract]. Naunyn-Schmiedeberg's Arch Pharmacol 325, R50 (1984). 14 HERzm, S., MOHR, K. Action of ouabain on rat heart: comparison with its effect on guinea pig heart. Br J Pharmaco182, 135 142 (1984). 15 HERZm, S., MOnR, K. Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue. BrJ Pharmaco1114, 685-688 (1985). 16 LEE, C. O., KANO,D. H., So~oL, J. H., LE~, K. S. Relation between intracellular Na ion activity and tension of sheep cardiac Purkinj e fibres exposed to dihydro-ouabain. BiophysJ 29, 3 l 5-330 (1980). 17 LEE, C. O., DAGOSTINO,M. Effect of strophanthidin on intracellular Na ion activity and twitch tension of constantly driven canine cardiac Purkinje fibres. BiophysJ 4tl, 185 198 (1982). 18 LEE, K. S., KLAUS,W. The subcellular basis for the mechanism ofinotropic action of cardiac glycosides. Pharmacol Rev 23, 193-261 (I971).
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19 LINDENMAYER,G. E. Mechanism of action of digitalis glycosides at the subcellular level. Pharmacol Ther B 2, 20 21 22 23 24 25 26 27 28 29 30
31 32
843 861 (1976). L/JLLMANN,H., PETERS, T. Action of cardiac glycosides on the excitation-contraction coupling in heart muscle. Prog Pharmacol 2(2), 1 53 (1979). L/.iLLMANN,H., PETERS, T., ZmGLER, A. Kinetic events determining the effects of cardiac glycosides. TIPS l, 102-106 (1979). PHILIPSON,K. D., NISHIMOTO, A. Y. ATP-dependent Na + transport in cardiac sarcolemmal vesicles. Biochim Biophys Acta 733, 133-141 (1983). REPKE,K. R. H., HERRMANN,I., PORTIUS,H.J. Interaction of cardiac glycosides and Na, K-ATPase is regulated by effector-controlled equilibrium between two limit enzyme conformers. Biochem Pharmacol 33, 2089-2099 (1984). SCATCHARD,G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51,660~672 (1949). SCHONER,W., von ILBERG, C., KRAMER, R.; SLUBERT, W. On the mechanism of Na +- and K+-stimulated hydrolysis of adenosine triphosphatase I. Purification and properties of a Na+- and K § ATPase from ox brain. EurJ Biochem l, 334 343 (1967). SCHWARTZ,A., LINDENMAYER,G. E., ALLEN,J. C. The sodium-potassium adenosine triphosphatase: pharmacological, physiological and biochemical aspects. Pharmacol Rev 27, 3=134 (1975). SOLTOFF,S. P., MANBEL, L.J. Active ion transport in the renal proximal tubule. IL Ionic dependence of the Na pump. J Gen Physio184, 623-642 (1984). TEMMA,K., AKERA, T. Enhancement of cardiac actions of ouabain and its binding to Na+,K§ triphosphatase by increased sodium influx in isolated guinea-pig heart. J Pharmacot Exp Ther 223, 490-496 (1982). WASSERSTROM,J. A., SCHWARTZ,D.J., FOZZARI),H. A. Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides: Circ Res 52, 697 705 (1983). YAMAMOTO,S., AKERA,T., BROBY, T. M. Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria. Biochim Biophys Acta 555, 270 284 (1979). YAMAMOTO,S., AKERA,T., KIM, D.-H., BRODY,T. M. Tissue concentration ofNa+,K+-adenosine triphosphatase and the positive inotropic action ofouabain in guinea pig heart:J Pharmacol Exp Ther 217, 701 707 (1981a). YAMAMOTO,S., Fox, A. A. L., GREEFF,K. Inotropic effects and Na +,K+-ATPase inhibition ofouabain in isolated guinea-pig atria and diaphragm. EurJ Pharmaco171,437-446 (198 lb).
On the Cooperativlty of Ouabain-binding to Intact Myocardium Stefan Herzig, Heinz Liillmann* and Klaus Mohr Department of Pharmacology, University of Kiel, Hospitalstrasse 4-6, 2300 Kid 1, FRG (Received 15January 1985, acceptedin revisedform 11 June 1985) S. HERZ1G, H. L~TLLMANNANn K. MOHR. On the Cooperativity of Ouabain-binding to Intact Myocardlum.
Journal of Molecularand CellularCardiology (1985) 17, 1095-1104. A theoretical concept is presented which
proposes that binding of ouabain to intact myocardium should be positive cooperative. It is based on the assumption that the myocardial Na/K-ATPases expose the ouabain-bindingsite only at a particular conformation adopted during a turnover cycle. The turnover rate and thus the ouabain-bindingproperties are regulated by the cytosolic Na-ion-concentration Nai. Any occupation of cellular Na/K-ATPases should affect the ouabain-binding properties of the unoccupied Na/K-ATPases, because their turnover rate is increased via an elevated Na i . A computer model which takes into account the interrelationships of the Na/K-ATPases both with Na i and with the ouabain-concentrationpredicts that ouabain-bindingshould proceed in a concentratio~ proportional fashion as long as the Na-load can be counterbalanced by non-occupied Na/K-ATPase molecules. The concentration-proportional binding reflects a positive cooperativity. Experimental results reveal that (3H)ouabain-bindingto Na/K-ATPase of electrically stimulated guinea-pig left atria was in fact concentration proportional under certain experimental conditions. The biological significance of the proposed concept remains to be elucidated. KEY WoRDs: Cooperativity; Ouabain-binding; Na/K-ATPase; Intact myocardium.
Introduction W h e n the i n t e r a c t i o n of o u a b a i n with its r e c e p t o r site, the N a / K - A T P a s e , is studied in vitro u s i n g for i n s t a n c e c r u d e m e m b r a n e or purified N a / K - A T P a s e - p r e p a r a t i o n s , ouab a i n b i n d i n g is often f o u n d to o c c u r at a single p o p u l a t i o n of i n d e p e n d e n t b i n d i n g sites [9, 10, 12, 13, 31, 32]. T h e b i n d i n g sites are called ' i n d e p e n d e n t ' , because the o c c u p a t i o n of a f r a c t i o n of these sites does n o t i n f l u e n c e the b i n d i n g characteristics of the u n o c c u p i e d sites. I n this c o m m e n t a r y , the a t t e m p t is m a d e to p r e d i c t the characteristics of o u a b a i n - b i n d i n g in i n t a c t m y o c a r d i a l tissue. T h e considerations are based o n g e n e r a l l y a c c e p t e d aspects of N a / K - A T P a s e - f u n c t i o n a n d of o u a b a i n N a / K - A T P a s e - i n t e r a c t i o n . T h e y lead to the c o n c l u s i o n t h a t o u a b a i n - b i n d i n g should be c o o p e r a t i v e in i n t a c t m y o c a r d i u m . F i n a l l y , some e x p e r i m e n t a l results are p r e s e n t e d w h i c h reveal t h a t the expected c o o p e r a t i v i t y
of o u a b a i n - b i n d i n g c a n in fact be observed u n d e r c e r t a i n e x p e r i m e n t a l c o n d i t i o n s i n isolated g u i n e a - p i g atria. T h e i n t e r a c t i o n b e t w e e n o u a b a i n a n d its r e c e p t o r is d e s c r i b e d b y m e a n s of two concepts: one, the essential features of w h i c h h a v e b e e n described p r e v i o u s l y [3, 20, 21] a n d w h i c h is e x t e n d e d here (designated as ' c o n c e p t of m o d i f i e d b i n d i n g site d e n s i t y ' ) ; a n d a novel one ( ' c o n c e p t of modified affinity'), w h i c h m i g h t h a v e the a d v a n t a g e t h a t the events d e t e r m i n i n g o u a b a i n - b i n d i n g are illustrated in the m o r e f a m i l i a r terms comm o n l y used to c h a r a c t e r i z e r e c e p t o r - l i g a n d interactions.
Theoretical considerations Aspects of Na/ K A TPase function D u r i n g a t u r n o v e r cycle a N a / K - A T P a s e molecule is t h o u g h t to u n d e r g o c o n f o r m a t i o n a l c h a n g e s (Fig. 1) : s t a r t i n g from a 'resting' con-
* To whom correspondence should be sent at the above address. 0022 2828/85/111095 + 10 $03.00/0
9 1985 Academic Press Inc. (London) Limited
1096
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~
Extracelhdar space
,~u
r
Ouabain Na +
r
r
K +
Cyfosol
F I G U R E I. Transport cycle of the Na/K-ATPase; note: the ouabain binding site is only exposed at a particular conformation. For details see text.
formation, the phosphorylated enzyme is activated by Na-ions bound to its cytosolic site to translocate these Na-ions into the extracellular space; thereafter, the enzyme can bind K-ions at the extracellular site and translocate them into the cytosol. After dissociation of these K-ions, the 'resting' conformation is adopted again [1, 18, 20, 26]. The concentration for half-maximum enzyme-activation by cytosolic Na-ions has been determined to lie in the range of Na-ion-concentrations, which occur physiologically in the cytosol [-22, 25, 27]. The concentration for half maximum activation by extracellular K-ions was reported to lie below the physiological extracellular K-ion-concentration [1, 19]. Hence, the activation exerted by K-ions in the range of physiological concentrations shall be considered to be near maximum, and the cytosolic Na-ion-concentration is taken to be the determinant of the turnover rate. The extraordinary feature of the interaction between ouabain and its receptor is that ouabain can only associate with high affinity to that particular Na/K-ATPaseconformation, which is exposed during the p u m p cycle before attachment of K-ions. For this reason, the turnover rate of Na/KATPase or the cytosolic Na-ion-concentration, respectively, essentially determines the extent of ouabain-binding. This can be illustrated by the well-established dependency of ouabain-binding on the beat frequency of myocardial preparations [-3, 20, 21, 28].
Dependency of ouabain-binding on the beat-frequency A synopsis of the interrelationship between heart rate, Na/K-ATPase transport rate and ouabain-binding is schematically depicted in Figure 2. As illustrated in Figure 2(a) and
2(b), the equilibrium binding of ouabain is enhanced with increasing beat frequency. The explanation for this finding is presented in Figures 2(c) (e): the increased stimulation frequency necessitates an enhanced N a / K ATPase transport rate in order to counterbalance the elevated Na- and K-ion fluxes. 'Transport rate' is defined as the amount of Na/K-ions transported per unit time by all Na/K-ATPases of a cell, while 'turnover rate' means the transport velocity of a single N a / K ATPase molecule. Since the turnover rate is probably regulated by the cytosolic Na-ionconcentration Nal, the enhanced transport rate should correspond to an increased Na i. In fact, Cohen et al. [6] found Na i to be elevated at increased beat frequencies. To illustrate the general relationship between transport rate and Nal, in vitro data reported by Philipson and Nishimoto [22] were used to evaluate Nal-values for three transport rates arbitrarily chosen [Fig. 2(c)]. In turn, the elevated Na/K-ATPase transport rate leads to a more frequent appearance of the ouabain-binding enzyme-conformation. The time interval, in which this particular conformation exists during a transport cycle, should be constant and independent of the turnover rate, if the activation of the Na/KATPase by extracellular K-ions is assumed to be constant. So, when the enzyme undergoes a higher number of turnover cycles per unit of time, then it stays for a longer fraction of this time in the ouabain-binding conformation. Formally, the dependency ofouabain-binding on transport rate can be analysed in terms of (a): an increased actual density of binding sites, and (b): an increased actual affinity of ouabain receptors. (a) Concept of modified binding site density [-Fig. 2(d)]: Since only a particular conformation of Na/K-ATPase is capable to bind ouabain with a given and constant high affinity, only a certain fraction of the total number of Na/K-ATPases is actually available as ouabain binding sites at any moment. Thus, each transport rate is connected with a certain actual density of available binding sites. With an altered Na i the ratio between the Na/K-ATPases available as binding sites (filled circles) and unaccessible for ouabain (open circles)--and hence ouabain-binding, is modified. It should be noted that an instant
Cooperativity of Ouabain-binding
1097
(a) t Beats/min
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F I G U R E 2. Dependency of ouabain-binding on the beat frequency. (a): Isolated guinea-pig atria were electrically stimulated at the beat frequencies of 0.5, 3 and 6 Hz (rectangular pulses, 5 ms duration, intensity about 30% above threshold). Tyrode solution in m ~ : NaC1 136.8, KC1 5.4, N a H C O 3 11.9, NaH2PO 4 0.21, CaCI 2 1.8, MgC12 1.05, glucose 5.5; 95% 0 2 / 5 % CO2; 32~ (b) : Equilibrium values of specific (3H)ouabain-binding to guinea-pig left atria were determined as described previously [3, 15]. In short, after an equilibration period of 1 h, the atria were exposed to 1 nM (3H)ouabain; after different time intervals up to 5 h, atria were removed from the organ baths, blotted, weighed and processed for liquid scintillation counting. Specific (3H)ouabain-binding was calculated as the difference between the total tissue-bound radioactivity and the amount of (3H)ouabain present in the extracellular space (30% of tissue wet weight) ; (c) : Dependency of Na/K-ATPase transport rate on the cytosolic Na-ion-concentration. The data are adapted from in vitro experiments carried out with dog sarcolemmal vesicles by Philipson and Nishimoto [22]. The arrows indicate points, which are assumed to correspond to the respective beat frequency; (d): Illustration of the concept of modified binding site density. Circles represent N a / K - A T P a s e molecules of the cell in the ouabain-binding (filled) or in other (open) conformations; (e) ; Illustration of the concept of modified affinity. The intensity of hatching indicates the apparent ouabain-binding affinity of the Na/K-ATPases.
1098
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view is depicted; since all Na/K-ATPase molecules take part in the ion transport, all enzyme molecules are randomly present in the ouabain-binding conformation. The ratio, however, between the two conformational states is constant at any moment of a given condition. (b) Concept of modified affinity [Fig. 2 (e)] : Each Na/K-ATPase molecule is considered to represent a receptor irrespective of the actual transport rate; i.e. the density of binding sites equals the number of Na/K-ATPases under all conditions. The affinity of the receptors, however, is modified by the transport rate. The more often the ouabain-binding conformation occurs per unit time, the more is the probability increase of the association of a ouabain molecule to the enzyme. Therefore, the association rate constant k + 1 of ouabain-binding is variable and is a function of the turnover rate of the N a / K - A T P a s e molecules and thus a function of Na i. It is assumed that the dissociation of the complex formed between ouabain and the Na/KATPase is independent of Na i. So, the rate constant of dissociation k - 1 shall be considered constant. Accordingly, the affinity k + l / k 1 is a function ofNai. With altered Na i the affinity of the receptor sites (intensity of hatching) and hence ouabain-binding, is modified. This time-independent 'overall' affinity of the Na/K-ATPase molecules shall be designated 'apparent affinity' in order to differentiate it from the constant affinity of the ouabain-binding enzyme conformation used in the concept of modified binding site density. Irrespective of the theoretical concept applied, different amounts of ouabain have to be expected to become bound at a given ouabain-concentration at different beatfrequencies. Generally, an alteration of the cytosolic Na-ion-concentration seems to be associated with an altered ouabain-binding; e.g. the sodium-ionophore monensin enhances ouabain-binding [15, 30], a reduction of the extracellular Na-ion-concentration diminishes ouabain-binding [8, 15, 30].
Concentration-dependencyof ouabain-binding in intact myocardium The dependency of ouabain-binding on the cytosolic Na-ion-concentration should have
bearings upon the concentration-dependency of ouabain-binding in intact myocardium. When a cell is exposed to a non-toxic ouabain-concentration, ouabain will occupy a certain fraction of the Na/K-ATPases, depending on the actual binding site density or the actual apparent affinity, respectively. Since the occupied Na/K-ATPases are inhibited, the unoccupied are driven at a higher turnover rate in order to counterbalance the Na-load. This activation is brought about by an elevated cytosolic Na-ion-concentration. The increment o f N a i in turn leads to an additional ouabain-binding at the given ouabainconcentration, analogously to the above described dependency of ouabain-binding on Na i . The equilibrium binding of ouabain will be attained, when (1) the transport activity provided by the unoccupied Na/K-ATPases balances the Na-load and (2) the amount of ouabain bound corresponds to the new Na i . The situations before addition of ouabain and after attainment of the binding equilibrium at 50% occupancy of Na/K-ATPases are schematically depicted in Figure 3. The transport rate required for the compensation of Na- and K-ion-fluxes is arbitrarily chosen to amount to 30% of the maximum. In this example, an increment of Na i by about 4 mM is required to maintain the transport rate [Fig. 3(a)]. In the lower part of Figure 3 the example is illustrated with respect to both concepts. (a) Concept of modified binding site density [Fig. 3(b)]: under control conditions, randomly three out of ten Na/K-ATPases are in the ouabain-binding conformation at any moment. At equilibrium binding of ouabain, five out of ten Na/K-ATPases are occupied and inhibited. Because the remaining unoccupied Na/K-ATPases still counterbalance the Na-load, the number of unoccupied Na/K-ATPases being in the ouabain-binding conformation at any moment again amounts to three. Accordingly, eight out of ten Na/KATPases represent binding sites actually taking part in the ouabain-Na/K-ATPaseinteraction at any moment, instead of three under control conditions. (b) Concept of modified affinity [-Fig. 3(c)]: In the presence of ouabain, the unoccupied Na/K-ATPases expose the ouabainbinding conformation more frequently due to
Cooperativity o f O u a b a i n - b i n d i n g
1099
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io
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o.
C _
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FIGURE 3. (a): Effect of a 50% occupancy of the cellular Na/K-ATPases by ouabain on the relationship between Na/K-ATPase transport rate and the cytosolic Na-ion-concentration. Illustration of the consequences of the inhibition of Na/K-ATPase molecules and the accompanying increment of Na i on the cellular ouabain-binding characteristics; (b): Concept of modified binding site density: in spite of the occupied Na/K-ATPases (crossed filled circles), the number of available unoccupied binding sites (filled circles) remains unaltered; (c) : Concept of modified affinity: the apparent affinity is increased as a result of the occupation of Na/K-ATPases.
their increased t u r n o v e r rate; this leads to an increased p r o b a b i l i t y o f ouabain-association. Therefore, the resulting affinity of the receptors exceeds the control affinity. Both concepts reveal that a n y o c c u p a t i o n of N a / K - A T P a s e s by o u a b a i n should affect the o u a b a i n - b i n d i n g properties of the unoccupied enzyme molecules, because the turnover rate of these N a / K - A T P a s e s is increased. This means that the receptor sites of o u a b a i n are not i n d e p e n d e n t from each other: in contrast, they are cooperative. T h e i n t e r d e p e n d e n c e of the receptors is m e d i a t e d b y the cytosolic Naion-concentration. In o r d e r to predict o u a b a i n - b i n d i n g curves
taking into account these prerequisites, a c o m p u t e r model was a p p l i e d [13]: for each o u a b a i n - c o n c e n t r a t i o n , the a p p r o p r i a t e N a i to m a i n t a i n the t r a n s p o r t rate and the corres p o n d i n g level of o u a b a i n - b i n d i n g was calculated. I n short, the m o d e l combines the s i g m o i d - s h a p e d interrelationships of the N a / K - A T P a s e with (1) the cytosolic N a - i o n concentration, and (2) the o u a b a i n - c o n centration. This model is a p p l i c a b l e for nontoxic conditions, i.e. for the o u a b a i n c o n c e n t r a t i o n range, in which the inhibition of N a / K - A T P a s e s can be effectively c o m p e n sated for by an elevated N a i. At toxic o u a b a i n - c o n c e n t r a t i o n s the unoccupied N a /
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10
I I00
I 1000 [nMl
Oua ba in-concentrotion
0
50 Bound [% ]
IO 0
FIGURE 4. Ouabain binding curve predicted by means of a computer model, which combinesthe interrelationships of the Na/K-ATPase with Nai and the ouabain-concentration. The calculations refer only to non-toxic ouabainconcentrations, because toxic concentrations induce a progressive accumulation of Nai; (a): Dependency of ouabainbinding and Nai on the ouabain-concentration. For sake of comparison, the dotted line indicates a sigmoid-shaped binding-curve, which would result, if the dependency of ouabain-binding on Nai were not considered, i.e. in the absence of cooperativity; (b) : Scatchard plot of the binding curve shown in (a) : the horizontal line means that binding is concentration proportional. The thin lines were constructed according to de Meyts and Roth [7]. The slope is a measure of the respective apparent affinity at the indicated fractional receptor occupancies; (c) : Plot of the apparent affinityv. the fractional receptor occupancy: the ascendingline means that binding is positivecooperative.
K-ATPases c a n n o t c o u n t e r b a l a n c e the Naload. Therefore, Na-ions will progressively a c c u m u l a t e within the cell, i.e. equilibrium values o f N a i and also of o u a b a i n - b i n d i n g will no longer be attained. Figure 4(a) depicts an e x e m p l a r y calculation based on a required transport rate of 26% of the m a x i m u m transport capacity. Some aspects of these theoretically predicted relationships are in accordance with experimental findings reported in the litera t u r e : the m a i n t a i n e d N a / K - A T P a s e transport rate is reflected by the observation that 8 6 R b + - u p t a k e is not inhibited in guinea pig atria treated with non-toxic o u a b a i n concentrations [4]; the o u a b a i n - i n d u c e d elevation of Na i has been recorded in the m y o c a r d i u m of several species by means of ion-sensitive microelectrodes [16, 17, 29]; the steepness of the Nai-curve is similar to a curve shown by Lee et al. [16] for the d e p e n d e n c y of N a i on the d i h y d r o o u a b a i n - c o n c e n t r a t i o n in sheep Purkinje fibers. T h e steepness of the o u a b a i n - b i n d i n g curve corresponds to the experimentally evaluated curve shown in Figure 5. T h e steep, n a r r o w concentrationi n o t r o p y curves usually obtained with cardiac glycosides p r o b a b l y reflect both the steep b i n d i n g curve a n d the fact that only a certain
fraction of the cellular N a / K - A T P a s e s can be occupied w i t h o u t intoxication. I n order to analyse the p a t t e r n of o u a b a i n binding, the calculated b i n d i n g data were plotted according to Scatchard [24] [Fig. 4(b)]. T h e horizontal line indicates a proportionality between the b i n d i n g a n d the concentration of o u a b a i n in the non-toxic range, suggesting that b i n d i n g were not saturable at these concentrations. This result is not surprising with respect to the concept of modified b i n d i n g site density: it includes that o u a b a i n b i n d i n g does not induce a reduction of the n u m b e r of actually available unoccupied b i n d i n g sites in the non-toxic concentration range. W h e n the b i n d i n g data are analysed according to de Meyts a n d R o t h [7] in order to determine the a p p a r e n t affinity of binding, it becomes obvious that the increased fractional o c c u p a n c y is paralleled by an augmented a p p a r e n t affinity [Fig. 4(c)]: this indicates a positive cooperativity.
Experimental
results
I n an a t t e m p t to test whether the outlined theoretical predictions could be experimentally substantiated, ( 3 H ) o u a b a i n - b i n d i n g was
Cooperativity of Ouabain-bindlng
1101
low concentrations a small s a t u r a t i n g comp o n e n t was seen, the extent of which is repu cu .resented by the difference between the ~5o~ continuous a n d the h a t c h e d line in Figure .E ----500 " ~ o o .," o9~ E ,.5(a). W h e n the d a t a are plotted according to o S c a t c h a r d [Fig. 5(b)], the s a t u r a t i n g com. . . . . , , , . p o n e n t is reflected by the initial descending 50O I000 1500 line. T h e c o n c e n t r a t i o n - p r o p o r t i o n a l binding 0u0boin-c0ncentrofion [ nM] is i n d i c a t e d by the horizontal line ( b o u n d / free = constant!), which has been expected a c c o r d i n g to the theoretical considerations (cf Fig. 4(b)]. T h e u n e x p e c t e d initial descending line could represent the presence of an additional p o p u l a t i o n of b i n d i n g sites not being i II I J l l l II I i i subject to the discussed r e g u l a t o r y mecha500 tO00 nisms; alternatively, the descending line Bound [nmol/kg cell] would indicate t h a t the a p p a r e n t affinity of FIGURE 5. Dependency of specific ouabain-binding in isolated guinea-pig atria on the ouabain-con- the m y o c a r d i a l N a / K - A T P a s e s did not reveal centration. The atria were stimulated at a frequency of the expected increase with o u a b a i n - c o n 0.5 Hz in the presence of 1 to 3 nM (SH)ouabain and centrations up to 100 nM. A t present it c a n n o t various concentrations of unlabelled ouabain under be decided between these two possibilities, b u t experimental conditions as described in the legend of it should be p o i n t e d out that this initial comFigure 2. The atria were removed from the organ baths after different time intervals up to 3 h. (a): Specific p o n e n t is e x a g g e r a t e d by the S c a t c h a r d plot binding represents the difference between the equilibrium a n d that the biological significance is obscure, values of total (3H)ouabain-binding and (3H)ouabain because an i n o t r o p i c effect could not be accumulated in the extracellular space, which amounted detected at these concentrations. to 30% of the tissue wet weight. The indicated data refer T h e c o n c e n t r a t i o n - p r o p o r t i o n a l compoto non-toxic ouabain-concentrations; at toxic concentranent reflects a positive c o o p e r a t i v i t y u n d e r the tions a stable equilibrium binding was not attained. The hatched line shows the concentration-proportional com- assumption that b i n d i n g takes place at a ponent of binding. (b): Scatchard plot of the binding s a t u r a b l e p o p u l a t i o n of specific b i n d i n g sites, data. The shaded area includes the range of receptor i.e. the o u a b a i n - r e c e p t o r of m y o c a r d i a l N a / K occupancy, in which a positive inotropic effect was detectable, when the isometric force of contraction was record- ATPases. T h a t this condition is actually fuled under conditions as applied in the binding filled, becomes evident from the following experiments. observations ( d a t a not shown); ( 1 ) I n the presence of 10 . 4 M unlabelled ouabain, the c o n c e n t r a t i o n - p r o p o r t i o n a l b i n d i n g of (all) m e a s u r e d in i s o l a t e d guinea-pig left atria [2], o u a b a i n was abolished, the r a d i o a c t i v i t y a c c o r d i n g to the m e t h o d described by Bent- r e t a i n e d by the a t r i a m a t c h i n g the a m o u n t of feld et al. [3]. T h e atria were m o u n t e d in ( a H ) o u a b a i n a c c u m u l a t e d in the extracellular o r g a n - b a t h s containing a modified T y r o d e space. Thus, the c o n c e n t r a t i o n - p r o p o r t i o n a l solution (cflegend Fig. 2) a n d were stimulated b i n d i n g occurred at a s a t u r a b l e p o p u l a t i o n of at t h e beat-frequencies of 0.5 a n d 3 Hz, b i n d i n g sites. (2) Binding was potassiumrespectively. After an equilibration period of sensitive: when the K - i o n - c o n c e n t r a t i o n in 1 h, ( a H ) o u a b a i n was a d d e d and its the organ b a t h was raised from 5.4 mR to e q u i l i b r i u m - b i n d i n g was d e t e r m i n e d (cf 10.8 mM, the concentration-proportional legends Fig. 2 a n d Fig: 5). T h e isometric force c o m p o n e n t was r e d u c e d (the bound/free of c o n t r a c t i o n was separately recorded u n d e r value was decreased by a b o u t 50%). T h e K i o n - i n d u c e d inhibition is a typical feature of identical e x p e r i m e n t a l conditions. T h e o u a b a i n - b i n d i n g curve o b t a i n e d at a specific o u a b a i n - b i n d i n g . (3) T h e b i n d i n g was s t i m u l a t i o n frequency of 0.5 Hz is depicted in s o d i u m - l o a d d e p e n d e n t : a reduction of the Na-ion-concentration to F i g u r e 5. O v e r a wide range of o u a b a i n - extracellular concentrations, the b i n d i n g p r o c e e d e d in a 83.5 mR d i m i n i s h e d the c o n c e n t r a t i o n c o n c e n t r a t i o n - p r o p o r t i o n a l m a n n e r . O n l y at p r o p o r t i o n a l b i n d i n g (the bound/free value
~ ,,0oo~
Z
1102
S. H e r z i g et aL
was decreased by about 20%). The dependence on the sodium-load is indicative of ouabain-Na/K-ATPase-interaction (cf Fig. 2). (4) The concentration range, in which positive inotropic effects could be measured, matched the concentration range, in which the concentration-proportional binding was obvious [shaded area in Fig. 5(b)]. The time courses of the inotropic response and of the ouabain-binding were similar. In conclusion, the concentration-proportional binding took place at those receptors, which mediate the positive inotropic response. Accordingly, the concentration-proportional component of ouabain-binding does not represent unspecific binding, but the expected positive cooperative ouabain-binding to the Na/K-ATPases of intact myocardium. At a stimulation frequency of 3 Hz, however, specific binding of ouabain did not reveal a concentratio~proportional component in the investigated non-toxic concentration range up to 300 nM. At higher concentrations a stable equilibrium binding could not be attained, because the ouabainbinding declined after a temporary maximum. In the Scatchard-plot (Fig. 6, upper curve), the points could be connected by a descending straight line, formally indicating that binding followed a simple law of mass action. A concentration-proportional corn-
3~
3Hz
"6 =
~,~
"--O--"O---'O [ N~ =83.5 mM
i
=
I
I
I
I
i
I
I
I
500 1000 Bound [nmol/kg cell]
F I G U R E 6. Equilibrium values of specific ouabainbinding at non-toxic concentrations at a beat frequency of 3 Hz, plotted according to Scatchard. Methodical details as described above. The upper line represents binding at a normal Na-ion-concentration in the organ bath. The lower line indicates ouabain-binding at a reduced Na-ion-concentration of 83.5 mM, NaG1 having been isoosmotically replaced by sucrose.
ponent of binding became, however, obvious even at 3 Hz, when the extracellular Na-ionconcentration was reduced to 83.5 mM (Fig. 6, lower curve). Under this condition, the non-toxic range of ouabain-concentrations was extended up to 600 nM. The theoretically predicted concentrationproportional binding of ouabain was demonstrable at 'conditions, which allowed to investigate a wide range of non-toxic ouabainconcentrations. Under all investigated conditions, however, a saturating component of binding was detected, which was reflected by the initial descending parts of the Scatchard plots. Since only a limited range of nontoxic ouabain-concentrations could be applied at 3 Hz under normal conditions, the saturating component might have had the predominant influence on the pattern of the binding curve. The mechanism underlying the saturating component of ouabain-binding cannot be readily explained at present. Two speculative mechanisms might be offered: ( 1 ) a small population of high-affinity binding sites not being involved in the regulatory events could have been present; the saturating binding to this population would then add to the concentration-proportional ouabain-binding. (2) Binding could have taken place at a homogeneous population of myocardial Na/ K-ATPases, but parameters not being considered by the proposed model could have modulated the pattern of ouabain-binding: apart from Nal, additional influences could determine the Na/K-ATPase transport rate or the ouabain-binding affinity of the Na/KATPases at a given turnover rate (e.g. alterations of the extracellular cleft K-ionconcentration, of the membrane potential, or of cytosolic determinants of Na/K-ATPase function [9, 23]). Both explanations do not exclude that the general regulatory mechanisms described in the theoretical part, which became obvious as a concentration-proportional binding at other conditions, could still have been present at 3 Hz. In summary, the expected concentrationproportional pattern of ouabain-binding has been demonstrated under certain experimental conditions. Although the majority of studies dealing with the binding of ouabain to intact myocardial preparations revealed
Cooperativity of Ouabain.binding b i n d i n g c u r v e s w i t h o u t signs o f a positive c o o p e r a t i v i t y , s o m e reports [5, 11] s h o w d a t a s u g g e s t i n g the p r e s e n c e o f a c o n c e n t r a t i o n p r o p o r t i o n a l c o m p o n e n t of o u a b a i n - b i n d i n g , yet h a v i n g b e e n i n t e r p r e t e d by the a u t h o r s as unspecific b i n d i n g . T h u s , the p r e s e n t experim e n t a l results are n o t in f u n d a m e n t a l conflict w i t h findings p u b l i s h e d previously.
Conclusion T h e o r e t i c a l c o n s i d e r a t i o n s as well as experimental observations have been presented, w h i c h m a y f a v o u r the i d e a o f a c o o p e r a t i v i t y o f o u a b a i n - b i n d i n g in i n t a c t m y o c a r d i u m .
1103
O c c u p a t i o n a n d i n h i b i t i o n of a f r a c t i o n of the c e l l u l a r N a / K - A T P a s e s by o u a b a i n is ass u m e d to a l t e r the o u a b a i n - b i n d i n g c h a r a c teristics of the N a / K - A T P a s e s via a n e l e v a t i o n of the cytosolic N a - i o n - c o n c e n t r a t i o n . B i n d i n g data indicating a non-cooperativity of ouabain-binding may only apparently contradict the p r o p o s e d theory. I n a n y case, a discussion of this a l t e r n a t i v e v i e w c o u l d p r o v i d e f u r t h e r i n s i g h t i n t o the m e c h a n i s m s i n v o l v e d in o u a b a i n - r e c e p t o r - i n t e r a c t i o n s in i n t a c t m y o c a r d i u m , w h i c h are c o m p l i c a t e d by the fact t h a t N a / K - A T P a s e in vivo is subject to physiological r e g u l a t i o n s - - i n c o n t r a s t to N a / K A T P a s e of c r u d e m e m b r a n e - or p u r i f i e d enzyme preparations.
References 1 AKERA,T., BRODY,T. M. The role of Na +, K+-ATPase in the inotropic action of digitalis. Pharmacol Rev 29, 187-220 (1978). 2 BENECKE,W., HERmO, S. Characteristics of (3H)ouabain binding and ouabain-induced positive inotropism in guinea-pig atria under various conditions [Abstract]. Naunyn-Schmiedeberg's Arch Pharmaco1329, R56 (1985). 3 BENTFEL~,M., L/JI~L~ANN,H-, PETERS,T., PROPPE, D. Interdependence of ion transport and the action ofouabain in heart muscle. BrJ Pharmaco161, 19-27 (1977). 4 BRowN,L., WE~DAN,K., ERDMANN,E. Consequences of specific (3H)ouabain binding to guinea pig left atria and cardiac cell membranes. Biochem Pharmacol 32, 423-435 (1983). 5 BussE, F., LiJLLNANN,I't., PETERS,Y. Concentration-dependence of the binding ofouabain to isolated guinea-pig atria.J Cardiovasc Pharmacol 1,687 698 (1979). 6 COHEN,C. J., FOZZARD,H. A., SHEU, S.-S. Increase in intracellular sodium ion activity during stimulation in mammalian cardiac muscle. Circ Res 50, 651-662 (1982). 7 DE MEVTS, P., Ror~I, J. Cooperativity in ligand binding: a new graphic analysis. Biochem Biophys Res Commun 66, 1118-1126 (1975). 8 DUT'rA, S., MARKS,B. H. Factors that regulate ouabain (3H)-accumulation by the isolated guinea pig heart. J Pharmacol Exp Ther 170, 318-325 (t969). 9 ERI~AN~, E., SCnONER, W. Ouabain-receptor interactions in (Na + + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants. Biochim Biophys Acta 330, 302-315 (1973). 10 ERDMANN~E., SCnONER,W. Ouabain-receptor interactions in (Na + + K +)-ATPase preparations. IV. The molecular structure of different cardioactive steroids and their affinity to the glycoside receptor. Naunyn-Schmiedeberg's Arch Pharmaco12113, 335-356 (1974). 11 GOOFRA~ND,T., LESNE,M. The uptake of cardiac glycosides in relation to their actions in isolated cardiac muscle. BrJ Pharmaco146, 488 497 (1972). 12 HANSON,O., SKOU,J. C. A study on the influence of the concentration of Mg 2+, Pi, K +, Na +, and tris on (Mg2+ + Pi)-supported g-strophanthin binding to (Na++ K+)-activated ATPase from ox brain. Biochim Biophys Acta 311, 51 66 (1973). 13 HERZIG,S. An attempt to describe by a mathematical model the relationship between cardiac glycoside binding, Na-K-ATPase activity and cytosolic sodium ion activity [Abstract]. Naunyn-Schmiedeberg's Arch Pharmacol 325, R50 (1984). 14 HERzm, S., MOHR, K. Action of ouabain on rat heart: comparison with its effect on guinea pig heart. Br J Pharmaco182, 135 142 (1984). 15 HERZm, S., MOnR, K. Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue. BrJ Pharmaco1114, 685-688 (1985). 16 LEE, C. O., KANO,D. H., So~oL, J. H., LE~, K. S. Relation between intracellular Na ion activity and tension of sheep cardiac Purkinj e fibres exposed to dihydro-ouabain. BiophysJ 29, 3 l 5-330 (1980). 17 LEE, C. O., DAGOSTINO,M. Effect of strophanthidin on intracellular Na ion activity and twitch tension of constantly driven canine cardiac Purkinje fibres. BiophysJ 4tl, 185 198 (1982). 18 LEE, K. S., KLAUS,W. The subcellular basis for the mechanism ofinotropic action of cardiac glycosides. Pharmacol Rev 23, 193-261 (I971).
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S. Herzig etaL
19 LINDENMAYER,G. E. Mechanism of action of digitalis glycosides at the subcellular level. Pharmacol Ther B 2, 20 21 22 23 24 25 26 27 28 29 30
31 32
843 861 (1976). L/JLLMANN,H., PETERS, T. Action of cardiac glycosides on the excitation-contraction coupling in heart muscle. Prog Pharmacol 2(2), 1 53 (1979). L/.iLLMANN,H., PETERS, T., ZmGLER, A. Kinetic events determining the effects of cardiac glycosides. TIPS l, 102-106 (1979). PHILIPSON,K. D., NISHIMOTO, A. Y. ATP-dependent Na + transport in cardiac sarcolemmal vesicles. Biochim Biophys Acta 733, 133-141 (1983). REPKE,K. R. H., HERRMANN,I., PORTIUS,H.J. Interaction of cardiac glycosides and Na, K-ATPase is regulated by effector-controlled equilibrium between two limit enzyme conformers. Biochem Pharmacol 33, 2089-2099 (1984). SCATCHARD,G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51,660~672 (1949). SCHONER,W., von ILBERG, C., KRAMER, R.; SLUBERT, W. On the mechanism of Na +- and K+-stimulated hydrolysis of adenosine triphosphatase I. Purification and properties of a Na+- and K § ATPase from ox brain. EurJ Biochem l, 334 343 (1967). SCHWARTZ,A., LINDENMAYER,G. E., ALLEN,J. C. The sodium-potassium adenosine triphosphatase: pharmacological, physiological and biochemical aspects. Pharmacol Rev 27, 3=134 (1975). SOLTOFF,S. P., MANBEL, L.J. Active ion transport in the renal proximal tubule. IL Ionic dependence of the Na pump. J Gen Physio184, 623-642 (1984). TEMMA,K., AKERA, T. Enhancement of cardiac actions of ouabain and its binding to Na+,K§ triphosphatase by increased sodium influx in isolated guinea-pig heart. J Pharmacot Exp Ther 223, 490-496 (1982). WASSERSTROM,J. A., SCHWARTZ,D.J., FOZZARI),H. A. Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides: Circ Res 52, 697 705 (1983). YAMAMOTO,S., AKERA,T., BROBY, T. M. Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria. Biochim Biophys Acta 555, 270 284 (1979). YAMAMOTO,S., AKERA,T., KIM, D.-H., BRODY,T. M. Tissue concentration ofNa+,K+-adenosine triphosphatase and the positive inotropic action ofouabain in guinea pig heart:J Pharmacol Exp Ther 217, 701 707 (1981a). YAMAMOTO,S., Fox, A. A. L., GREEFF,K. Inotropic effects and Na +,K+-ATPase inhibition ofouabain in isolated guinea-pig atria and diaphragm. EurJ Pharmaco171,437-446 (198 lb).