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Lack of correlation between [3H]ouabain binding and Na-K ATPase inhibition in rat aorta
European Journal of Pharmacology, 99 (1984) 45-55
45
Elsevier
LACK OF C O R R E L A T I O N B E T W E E N [3H]OUABAIN B I N D I N G A N D Na-K ATPase I N H I B I T I O N IN RAT AORTA RICHARD C. DETH *, JANEL L. SMART, CHRISTOPHERJ. LYNCH and RAYMONDWALSH Northeastern University, College of Pharmacy and Allied Health Professions, Section of Pharmacology, Boston, Massachusetts 02115, U.S.A.
Received 21 September 1983, accepted 13 December 1983
R.C. DETH, J.L. SMART, C.J. LYNCH and R. WALSH, Lack of correlation between [3H]ouabain binding and Na-K A TPase inhibition in rat aorta, European J. Pharmacol. 99 (1984) 45-55. The binding of [3 H]ouabain to intact strips of rat aorta was compared with the ability of ouabain to inhibit the uptake of 86Rb by the same preparation. When a cold temperature wash was used to process tissues after binding of [3H]ouabain, a class of relatively high affinity binding sites was found (K D = 1.2 X 10 - 7 M). Binding was saturable and sensitive to both ATP depletion and elevated potassium. Elevation of cytoplasmic Ca2+ levels by phenylephrine or c-AMP levels by theophylline and terbutaline had no influence on [3H]ouabain binding. Ouabain inhibition of 86Rb uptake progressed to 60% of the total 86Rb uptake at 2 x 10 - 3 M from a threshold of about 10 -5 M. Half-maximal inhibition by ouabain occurred at a concentration of 10 -4 M. The disparity between [3H]ouabain binding and inhibition of 86Rb uptake indicates that the high affinity binding site in the rat does not contribute to inhibition of Na-K ATPase function. Na-K ATPase
Ouabain
Glycoside
S6Rb uptake
1. Introduction The Na-K ATPase or Na-pump located in the plasma membrane of arterial smooth muscle cells has been hypothesized to be an important site for the regulation of arterial contractility (Leonard, 1957; Wolowyk et al., 1971; Reuter et al., 1973; Friedman and Friedman, 1974; for review see Jones, 1980). Most prominently, certain forms of hypertension (e.g. volume overload) may result from impaired functioning of the arterial Na-pump (Overbeck et al., 1976; Blaustein, 1977; Haddy et al., 1978; Haddy et al., 1980). A number of investigators have examined Na-K ATPase activity in isolated arterial membranes in an effort to further characterize the role of the Na-pump (Wolowyk et al., 1971; Preiss and Banaschak, 1976; Allen and Seidel, 19.77; Allen et al., 1981). Additional information can be gained from binding studies with * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
labelled cardiac glycosides and from measurements of glycoside-sensitive 86Rb fluxes'which provide measures of Na-pump density and function respectively. Moreover, each of the latter two approaches can be applied to intact tissues, allowing a study of the Na-pump in its cellular environment. In an earlier paper we measured the binding of [ 3H]ouabain to N a - K ATPase sites in the aorta of the rabbit (Deth and Lynch, 1980). From these studies a K D value for binding of the glycoside to the pump (8 x 10 -8 M) and an estimate of the maximum number of binding sites (27.9 x 10 -14 m o l / m g dry weight) were obtained. However, since the rat has been more commonly employed as a model for the hypertensive state, we have extended [3H]ouabain binding studies to that species as well, and have compared binding with inhibition of 86Rb uptake. Our results reveal the presence of high affinity binding sites for [3H]ouabain in rat aorta which
46
appear to lack the ability to provide inhibition of Na-K-ATPase transport activity.
2. Materials and methods
2.1. Tissue preparation Male Sprague-Dawley rats weighing approximately 300 g were decapitated and the thoracic and upper abdominal segments of aorta excised. Arteries were maintained at 37°C in oxygenated buffer containing 140 mM NaCI, 4.5 mM KC1, 10 mM d-glucose, 5 mM HEPES (pH 7.4), 1.5 mM CaC12 and 1.0 mM MgC12. After cleaning the aorta of fat and connective tissue, transverse ring segments weighing about 3 mg were prepared for binding and 86Rb uptake studies.
2.2. [3H]Ouabain binding Aortic rings were labelled at 37°C in aerated buffer containing specified concentrations of ouabain labelled with [3H]ouabain (13 Ci/mmol, New England Nuclear). At the conclusion of the labelling interval tissues were briefly rinsed in 400 ml of ouabain-free buffer to remove labelling buffer and then processed one of two ways, depending on whether the entire pattern of [3H]ouabain efflux was to be measured, or only the residual content after 50 min of cold temperature washout.
2.3. Efflux studies After the initial rinse each aortic segment was passed through a series of scintillation vials, each of which contained 5 ml of buffer oxygenated via a polyethylene catheter. At 5 rain intervals, tissues were transferred to succeeding vials containing buffer at either 2 or 37°C for the desired period of efflux after which the tissues were placed in plastic scintillation vials and dried overnight. The dry tissue weights were recorded, and the tissues dissolved in 2 ml of 0.1 M KOH at 90°C for 6 h (in capped vials). After cooling to room temperature 5 ml of water was added to the vials and, after shaking, 7.5 ml of a Triton X-100/toluene-based
scintillation cocktail. Scintillation fluid was also added to the 5 ml of buffer in each efflux vial and all samples were counted for their 3H-content. Efflux curves were constructed by successive additions of the amount of [3H]ouabain leaving the tissue to the amount present in the tissue at the end of the efflux interval.
2.4. Low temperature bulk wash As described for rabbit aorta (Deth and Lynch, 1980) estimates of bound [3H]ouabain could be made from the residual content of the labelled glycoside after a period of washout at 2°C in unlabelled K-free buffer. This procedure allowed more facile handling of a large number of tissues. Groups of tissues (i.e. 4-6) were washed for 20 min in 50 ml of K-free buffer and then transferred to fresh buffer for an additional 30 min. After this procedure tissues were processed as outlined above to determine [3H]ouabain content.
2.5. 86Rb uptake studies Aortic rings were incubated at 37°C in normal, K-contained buffer containing 5 # C i / m l of 86Rb with or without ouabain. After the desired interval, rings were washed in unlabelled buffer at 2°C for 1 min and then transferred to a test tube and dried in an oven at 90°C overnight. After being weighed, tissue radioactivity was measured in a gamma counter. Results were expressed as a percentage of the total uptake of 86Rb by control tissues, normalized to tissue dry weight. Significance of observed differences was evaluated using Student's t-test with P values of < 0.05 taken to be significant.
2.6. Drug sources Ouabain, digitoxin, digoxin, dihydro-ouabain, phenylephrine, iodoacetic acid and dinitrophenol were obtained from Sigma Chemical, terbutaline from Geigy Pharmaceutical and theophylline from J.T. Baker Chemical Co. [3H]Ouabain was obtained for New England Nuclear.
47
5oi 2.0'
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rat aorta. Tissues were labelled for 30 min with 10 -7 M [3H]ouabain under K-free conditions and then washed out in K-free buffer at either 37 or 2°C. The tissue content during the 37°C wash could be described by two exponential decay processes with [3H]ouabain content and T1/2 values for fast and slow components of 2.46 × 10 -13 mol/mg, 4.5 min and 9.57 × 10 -14 mol/mg, 23.3 min respectively. At 2°C, loss from the slower component was greatly retarded (T]/2 = 238 min) while the fast component was little affected (T1/2 = 3.5 min).
3.2. Time course of [3H]ouabain binding
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Fig. 1. Washout of [3H]ouabain from rat aorta strips at 2 or 37°C. Tissues were incubated K+-free buffer containing 10 -7 M [3H]ouabain for 30 min then moved at 5 min intervals through a saries of vials containing K+-free buffer at either 2 or 37°C. The tissue content of [3H]ouabain was calculated from the amount remaining in the tissue after 1 h of washout and the [3H]ouabain content of each vial. Each value is the average ( +_S.E.M.) of eight tissues.
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3.1. Effect of temperature on [3H]ouabain binding Our previous experience with rabbit aorta (Deth and Lynch, 1980) and the experience of other investigators (Landowne and Ritchie, 1970; Baker and Willis, 1972; Brading and Widdicombe, 1974; Liillmann et al., 1975) has indicated that [3H]ouabain equilibrates into two major tissue compartments which probably reflect extracellular and cellular compartments. These two can be differentiated on the basis of their rates of washout, and this difference can be further accentuated by reducing the temperature of the washout buffer. As shown in fig. 1, a similar pattern was found for
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Fig. 2. Time course of [3H]ouabain uptake by rat aorta strips. Tissues were incubated for various times in K+-containing buffer with 10 -7 M [3H]ouabain then washed out at 2°C for 50 min as in fig. 1. Rapid and slow exchanging components of [3H]ouabain content were determined by a curve peeling technique and are summarized in the inset. Each value is the average of 4 tissues ( 5: S.E.M. in the inset).
48
out tissues at 2°C after 5, 10, 20, 30, 60 and 90 rain of [3H]ouabain labelling at 1 0 - 7 M (fig. 2). In this experiment normal K ÷ (4.5 mM) was present during labelling while K-free buffer was used during washout. As can be seen in fig. 2, the residual amount of [3H]ouabain after 50 min of washout gradually and significantly increased from 0.16 + 0.01 to 0.51 + 0.04 x 10 -13 m o l / m g over the 5 to 90 min interval. The slow component of washout, calculated from the Y-axis intercept of a line extrapolated from the last 5 efflux values, similarly increased as shown in the inset to fig. 2. The rapid component of [3H]ouabain washout, calculated by subtracting the slow component and fitting the residual to an exponential function, reached a value at 10 rain which was not significantly different from its value at 90 rain. This behavior is quite similar to the pattern observed for rabbit aorta.
3.3. Influence of K + and ATP depletion Based on the knowledge of the Na-K ATPase transport function, binding of cardiac glycosides to the enzyme should be sensitive to the concentration of K + at the extracellular surface of the cell, and be dependent on the availability of ATP within the cell (Akera and Brody, 1978). This reflects the ability of K ÷ to reduce the availability of the E2-P form of the enzyme which is the preferred configuration for glycoside binding, and the essential requirement of ATP for generation o f the E : P form. In rat aorta, the amount of slow component [3H]ouabain binding after 30 min of labelling at 10 -7 M was increased by K-free conditions (30 min prior to and during [3H]ouabain labelling) and suppressed by elevated K ÷ (18 mM prior to and during [3H]ouabain labelling) as compared to 4.5 mM K ÷. The y-intercepts were 7.3, 6.3 and 4.3 × 10 -14 m o l / m g for 0, 4.5 and 18 mM K + respectively. While significant, the effect of K + removal was less pronounced than previously observed for rabbit aorta in which case the ratio of binding at 0 vs. 4.5 mM was 2.1 as compared to 1.2 for rat aorta. When cellular ATP levels were depleted by a 30 min exposure to 10 .4 M dinitrophenol and 10 -3 M iodoacetic acid, slow component [3H]ouabain binding at 2 x 10 .7 M was reduced by approxi-
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Fig. 3. Determination of 'specific' binding of oual~ainto rat aorta strips. Tissues were incubated for various times in [3H}ouabain (2.5 × 10 - 7 M) with (e) or without (A) unlabelled ouabain (10 - 4 M). After the binding period tissues were processed via a bulk wash procedure as described in Methods. 'Specific' binding ( © ) is defined as ' t o t a l ' - ' n o n - s p e c i f i c ' binding. Each point is the mean _+S.E.M. of 5 values.
mately 90% (1.5 × 10 -14 m o l / m g vs. 1.8 x 10 -~3 mol/mg).
3.4. Definition of 'specific binding" The slow-exchanging, temperature-sensitive component of [3H]ouabain binding presumably includes a fraction of drug bound to Na-K ATPase sites, but also fractions which reflect non-saturable sites such as delayed extraceUular compartment exchange or transmembrane uptake. In order to distinguish among these fractions, the binding of [3H]ouabain was compared in the presence and absence of a high concentration of unlabelled ouabain (10 -4 M) which should occupy saturable sites with sub-micromolar affinity constants (K D values), while non-saturable fractions would be expected to exhibit quasi-linear binding behavior. By subtracting the latter "non-specific" binding from 'total binding', a 'specific binding' fraction can be defined. In these experiments tissues were processed in
49
groups using a bulk wash procedure at 2°C as described in Methods to mimic the efflux procedure and retain only slow-exchanging [3H]ouabain. When rat aorta segments were allowed to bind [3H]ouabain at 2.5 x 10 -7 M, specific binding followed a complex time course including an apparent decrease during the 30-90 min interval followed by an increase at 120 min (fig. 3). Nonspecific binding comprised approximately 20-35% of total binding and appeared to follow a rather linear time course (fig. 3) which might account for the somewhat upward trend of slow component binding evident in the inset to fig. 2. From these results an incubation time of 30 rain was chosen for subsequent binding studies.
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Tissues were allowed to bind [3H]ouabain at concentrations from 10 -8 to 5 × 10 -7 M in the presence or absence of 10 -4 M unlabelled ouabain in K÷-containing buffer and then processed via a bulk wash procedure. Specifically bound [3H]ouabain exhibited saturability with a plateau evident at concentrations above 2 × 10 -7 M (fig. 4 upper). A Scatchard analysis of specific binding (fig. 4 lower) yielded a K D value of 7.87 x 10 -8 M and a Bmax (density of binding sites) of 1.26 x 10-13 m o l / m g dry tissue weight. The fitted straight line had a correlation coefficient of 0.97, suggesting the presence of a single class of sites with the above binding characteristics. Non-specific binding was an approximately linear function of [3H]ouabain concentration which therefore represented a larger portion of total binding at higher [3H]ouabain levels (fig. 5). Specific binding approximated the portion of total binding which was earlier found to be ATP-dependent. A Scatchard plot of total binding yielded a somewhat higher K D of 1.08 x 10 -7 M and a Bm,x of 1.68 X 10 -13 m o l / m g which could be compared to previously obtained values for total binding to rabbit aorta (Deth and Lynch, 1980).
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3.6. Influence of phenylephrine or c-AMP levels on [ 3H]ouabain binding It has been proposed that a c-AMP-mediated increase in the rat of activity of the N a - p u m p may
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Fig. 4. Saturation binding of [3H]ouabain to rat aorta strips. (Upper) Tissues were incubated for 30 min in various concentrations of [3H]ouabain with (I) or without (A) unlabelled ouabain (10 -4 M). Tissues were then processedvia a bulk wash procedure. 'Specific' binding (O) is defined as [total binding][non-specific binding]. (Lower) Scatchard plot of specifically bound [3H]ouabain. Each point is the mean (__+S.E.M. where indicated) of 5 values.
leads to relaxation of smooth muscle (Schied et al., 1979) and we have shown that N a - K ATPasemediated ion transport is increased during aadrenergic receptor activation (Deth et al., 1983). To test whether the contractile state of the artery influenced the rate of [3H]ouabain binding, we pretreated tissues with 10 -5 M phenylephrine for 10 min and compared their ability to bind
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['Glycoside] Fig. 5, Competition for [3H]ouabain binding sites by cardiac glycosides. Tissues were incubated for 30 rain in buffer containing 5×10 -7 M [3H]ouabain and various concentrations of unlabelled glycosides (10-8-10 -4 M). After a 50 rain washout in K+-free buffer at 2°C, residual [3H]ouabain content was measured and expressed as a percent of binding without unlabelled glycoside. Each value is the mean of four determinations.
centrations of unlabelled glycosides (10-8-10 -4 M). Results were expressed as a percent of binding in the absence of any competing glycosides (fig. 5). At 10-4 M, unlabelled glycosides displaced from 61 to 81% of bound [3H]ouabain. IC50 concentrations were based on the fraction of [3H]ouabain displaced by each unlabelled glycoside and Kt~ values were computed by applying the ChengPrusoff relationship (1973) (KD -----IC~0/(1 + L / K 0 ) where L = 5 x 10 -7 M and K 0 = 7.8 x 10 -8 M). K D values were: digoxin 2.0 x 10 -7 M; ouabain 6.1 × 10 -8 M; digitoxin 6.8 x 10 -8 M; and dihydro-ouabain 2.0 x 10-6 M. This potency order parallels the reported ability of glycosides to compete for Na-K-ATPase sites in other tissues (Dutta et al., 1968; Haustein and Hauptmann, 1974; Godfraind and Ghysel-Burton, 1980).
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[3H]ouabain (10 -7 M) during continued PE exposure with binding by relaxed tissues. Although PE-treated tissues b o u n d significantly less [3H]ouabain after 5 min (3.5 + 0.4 vs. 5.9 + 0.5 m o l / m g × 10 -14) subsequently there was no difference between the two groups. In the same experiment other tissues were treated with 10 -5 M terbutaline and 10 -4 M theophylline to elevate c-AMP levels prior to and during a period of [3H]ouabain binding. N o difference in the amount of binding was found. Comparisons at different concentrations of [3H]ouabain similarly showed no marked difference in binding associated with PE or terbutaline and theophylline treatment.
3. 7. Competition for [3H]ouabain binding sites by other glycosides To determine whether [3H]ouabain binding sites in rat aorta exhibited molecular requirements similar to Na-K ATPase sites in other tissues, we examined the ability of several unlabelled glycosides to compete with [3H]ouabain for binding. Tissues were incubated for 30 min in buffer containing 5 x 10 -7 M [3H]ouabain and various con-
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Fig. 6. S6Rb uptake by rat aorta strips. Two groups of tissues were incubated in K + containing, S6Rb labelled buffer for 2, 5, 10 or 20 rain. One group was treated with 10 -3 M ouabain for 30 rain prior to and during 86Rb uptake and the residual 86Rb content after a I rain rinse is designated 'ouabain-insensitive 86Rb uptake'. Uptake by the other group (without ouabain) is designated 'total 86Rb uptake'. The difference between these two values represents 'ouabain-sensitive 86Rb uptake'. Each value is the mean of five tissues.
51
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Log [Ouobain ] Fig. 7. (a) Concentration dependence of ouabain-induced inhibition of 86Rb uptake in rat aorta strips. 86Rb uptake during 10 rain of exposure was compared in K+-containing buffer with various concentrations of ouabain (10-6-10-3 M) and expressed as a percentage of the maximum ouabain-sensitive uptake. Each value is the average of five determinations ( + S.E.M.). (b) Concentration dependence of ouabain-induced inhibition of S6Rb uptake in rabbit aorta strips. Same as in (a) except ouabain concentrations were 3 × 10-1°-10- 5 M. Results are the average (_.+S.E.M.) of five determinations.
3.8. Ouabain inhibition of 86Rb uptake Because of the ability of Rb + to substitute for K ÷ in the N a - K - A T P a s e mechanism, the
ouabain-sensitive component of 86Rb uptake has been used to assess the function of N a - K ATPase in isolated arteries (Haddy et al., 1978, 1980; Pamnani et al., 1979). 86Rb uptake by rat aortic
52 rings was linear over the first 10 min of exposure (fig. 6). Incubation with increasing concentrations of ouabain from 10 -5 to 2 x 10 -3 M caused a reduction in the amount of 86Rb accumulated (fig. 7a). Because of ouabain's limited solubility, it was not possible to clearly establish the maximum suppression by ouabain. However, similar studies using vanadate (10 #M) and ATP depletion (10 -4 M DNP and 10 -3 M IAA) as a means of completely inhibiting the Na-pump resulted in a similar suppression of 86Rb uptake (i.e. 55% of the total 86Rb uptake). Assuming this value to be 100% of the Na-pump-related uptake, half-maximal inhibition occurred at a ouabain concentration of 8.5 × 10 -5 M. Any underestimation of the 100% value would result in a higher IC50 value. 86Rb uptake by rabbit aorta was more sensitive to ouabain (fig. 7b). Prior incubation in low concentrations of ouabain (5 x 10-10 ___,10-8 M) caused an increase in the ouabain-sensitive component of 86Rb uptake while high concentrations reduced uptake. Using the value for zero ouabain as 100%, half-maximal inhibition occurred at a ouabain concentration of 3 x 10 -7 M. Increased 86Rb uptake at low ouabain levels most likely reflects the stimulatory influence of norepinephrine released from adrenergic nerve endings (Deth et al., 1983).
4. Discussion
Cardiac glycoside binding to Na-K-ATPase sites and the influence of glycosides on ion transport have both been extensively employed to study the role of the Na-pump in cellular function. The specificity of glycoside binding is well-documented (Schatzmann, 1953; Schwartz et al., 1969; Akera and Brody, 1978), although there is disagreement as to the relationship between binding and the functional effects that glycosides cause (Daniel et al., 1970; Murthy et al., 1974; Okita, 1977; Godfraind and Ghysel-Burton, 1980). The current studies were undertaken to investigate this relationship in the rat aorta. Our results have established the existence of relatively high affinity binding sites for [3H]ouabain which exhibit a K D of about 0.1 #M. T h i s
affinity is similar to values from studies on other intact smooth muscle preparations such as rabbit aorta (Deth and Lynch, 1980) or guinea-pig taenia coli (Brading and Widdecombe, 1974). However, this finding is ostensibly at odds with the often described resistance of Na-K ATPase in the rat to inhibition of ouabain (Allen and Schwartz, 1969; Friedman and Friedman, 1974; Ku et al., 1976; Urquilla et al., 1978; Fedan et al., 1978; Periyasamy et al., 1979). Allen and Schwartz (1969) observed that Na-K ATPase activity from rat heart and kidney was relatively insensitive to ouabain as compared to bovine or canine preparations. Urquilla et al. (1978) noted that ouabain (8 x 10 -5 M) or K-free buffer caused depolarization in the guinea pig vas deferens, but not in the rat vas deferens. In an extension of this observation, the total Na-K ATPase activity was found to be similar in membranes prepared from vas deferens of both species ruling out a scarcity of the enzyme as a basis for reduced glycoside sensitivity. Ouabain (8 x 10 -5 M) produced a 30% inhibition of the rat vas deferens derived Na-K ATPase. In the 86Rb-uptake studies shown in fig. 7, it is apparent that in rat aorta higher concentrations of ouabain are needed to produce full inhibition of the Napump-related transport activity, and the value of 30% inhibition is in reasonable agreement with the influence of 8 x 10 -5 M on 86Rb uptake. In fact 10 -3 M seems necessary to produce 100% inhibition, based on the observation that ATP-depletion or vanadate treatment produced essentially the same extent of inhibition of 86Rb uptake as 10 -3 M ouabain. Given that the Na-K ATPase of the rat has a low affinity for ouabain, the relevance of the high affinity binding sites for [3H]ouabain must be questioned. Several behaviors indicate that the site is needed a classical Na-K ATPase locus: (1) binding is saturable, yielding a Bmax similar to that reported for other tissues; (2) the affinity is similar to that reported for other tissues; (3) binding is greatly reduced in the absence of ATP; (4) binding is reduced in the presence of high K+; (5) structural requirements for binding are similar to other Na-K-ATPase sites. On the other hand several points are at variance with a Na-K ATPase loca-
53 tion: (1) binding to these sites does not result in inhibition of 86RB uptake; (2) K t free buffer increases [3H]ouabain binding above levels obtained at 4.5 mM K t to only a modest extent; (3) binding is not greatly altered during a-adrenergic agonist-induced contraction, which is known to increase the ouabain-sensitive 86Rb uptake (Deth et al., 1983). While the basis for the lower ouabain sensitivity of the rat Na-K ATPase has not been fully established, it has been shown to be retained in relatively purified enzyme preparations (Periyasamy et al., 1979), suggesting that differences in the functional environment of the intact cell are not involved (although see Mansier and Lelievre, 1982). The most straightforward explanation would be that the configuration of the glycoside binding site is different in the rat vs. more sensitive species. Within this context, a less favorable configuration could be a stable feature of the protein binding site itself or alternatively its presence could depend on the physical properties of the membrane environment, the state of aggregation of subunits, or other complex factors which might be preserved during enzyme purification. For instance, Wellsmith and Lindenmayer (1980) have provided kinetic evidence for the existence of two forms of the ouabain receptor with widely divergent affinities in canine cardiac membranes. The proportion of the two forms was shown to be dependent on the presence of Mg 2+, Na t and ATP, such that for 5 mM Mg 2+, 100 mM Na t and 5 mM ATP, the ratio of high affinity to low affinity sites was about 2 : 1 while in their absence, no significant high affinity binding was detected. The lower affinity form had a K o of 1.9 × 10 -7 M, and binding to this site did not result in inhibition of Na-K ATPase activity. In the same study, the addition of small quantities of detergent sodium dodecylsulfate (SDS) shifted the proportion of sites in favor of the higher affinity form. Since the active functional unit of the enzyme is thought to be a dimer of the ( a f t ) 2 form (a and fl being high and low molecular weight protein subunits, respectively) Craig and Kyte, 1980), the propensity for aggregation may play a role in determining ouabain sensitivity. Allen and coworkers (1981) also noted that SDS addition was necessary to unmask a latent
ouabain sensitive Na-K ATPase activity in membranes prepared from canine aorta. An additional mechanism by which alterations in ouabain sensitivity may be produced was reported by Charlemagne et al., (1980). Treatment of membranes prepared from a murine plasmacytoma with EDTA increased ouabain sensitivity by 300-1000 fold, while the readdition of Ca 2t along with either tropomyosin or proteins derived from the plasma membrane restored the lower ouabain sensitivity. Thus, it is implied that Ca 2t, acting in conjunction with membrane proteins is able to modulate the configuration of the ouabain binding site. A recent confirmation of this relationship is provided by Mansier and Lelievre (1982) who found that cardiac sarcolemmal membranes from the rat displayed only low ouabain affinity when normal calcium (2 mM) was present prior to homogenization, but a bimodal affinity (K D = 1.2 × 10 -8 M and 6 × 10 -5 M) when hearts were perfused with Ca-free buffer. Moreover, the higher affinity form is found to be present in isolated myocytes (Adams et al., 1982) and binding to this site results in an inotropic response. The high affinity [3H]ouabain binding sites we identify in intact rat aorta strips are most likely similar to these sites identified in rat myocardium. However, we have not observed alterations in arterial contractility at these concentrations, so a further comparison seems warranted. The work of Wellsmith and Lindenmayer (1980), discussed earlier, suggests that heterogeneity of cardiac glycoside binding sites may be a general feature of all species, and the discrepancy in affinity observed in the rat may reflect a rather extreme expression of a commonly available regulatory mechanism. Since the Ca 2t-dependent differences in rat cardiac sarcolemmal binding of ouabain were preserved in isolated membranes, one may tentatively hypothesize that the normal low sensitivity of rat arteries visa vis rabbit arteries is a result of relatively high Ca 2t levels at a regulatory site in the former species (i.e. the rat). This possibility may serve to explain the greater reliance of rat arteries on the activity of the Na-K ATPase for relaxation as compared to rabbit arteries (Webb and Bohr, 1978; Deth et al., 1983),
54 s i n c e N a - C a c h a n g e is c h a r a c t e r i z e d b y a l o w e r K D for C a 2+ a n d w o u l d t h e r e f o r e b e m o r e e v i d e n t a t h i g h e r C a 2 + levels. A l t e r n a t i v e to a s p e c i e s - d e p e n d e n t d i f f e r e n c e i n C a 2 + levels, t h e r a t p l a s m a m e m b r a n e m a y c o n t a i n a p r o t e i n e l e m e n t (cf. C h a r l e m a g n e et al., 1 9 8 0 ) w h i c h is e s s e n t i a l f o r t h e e x p r e s s i o n o f c a l c i u m ' s r e g u l a t o r y role. R a b b i t t i s s u e s m a y l a c k o r h a v e less o f t h i s p u t a t i v e p r o t e i n , r e s u l t i n g i n a p r e dominance of the higher affinity form. I n s u m m a r y , [3 H ] o u a b a i n b i n d s t o h i g h a f f i n i t y sites in rat aorta strips which exhibit characteristics e x p e c t e d o f a n N a - K A T P a s e locus, b u t d o n o t m e d i a t e i n h i b i t i o n o f i o n t r a n s p o r t . T h e s e sites may be analogous to Ca2+-dependent ouabain binding sites observed in the rat sarcolemma. Given a r e g u l a t o r y r o l e o f C a 2+ i n d e t e r m i n i n g o u a b a i n affinity, the lower affinity of rat arteries may be r e l a t e d t o a s p e c i e s - d e p e n d e n t d i f f e r e n c e i n C a 2÷ homeostatis.
Acknowledgement This work was supported by a grant-in-aid from the American Heart Association (81-1180) with funds contributed in part by the Massachusetts Heart Association to whom thanks are due.
References Adams, R.J., A. Schwartz, G. Grupp, I. Grupp, S.-W. Lee, E.T. Wallick, T. Powell, V.W. Twist and P. Gothiram, 1982, High-affinity ouabain binding site and low-dose positive inotropic effect in rat myocardium, Nature 296, 167. Akera, T. and T.H. Brody, 1978, The role of Na÷,K+-ATPase in the inotropic action of digitalis, Pharmacol. Rev. 29 (3), 187. Allen, J.C. and A. Schwartz, 1969, A possible biochemical explanation for the insensitivity of the rat to cardiac glycosides, J. Pharmacol. Exp. Ther. 168, 42. Allen, J.C. and C.L. Seidel, 1977, EGTA-stimulated ouabain inhibited ATPase of vascular smooth muscle in excitationcontraction coupling in smooth muscle, eds. R. Casteels, T. Godfraind and J.C. Ruegg, p. 211, Elsevier/North-Holland Press, Amsterdam. Allen, J.C., D.J. Smith and W.T. Gerthoffer, 1981, Ca ++ accumulation and latent Na+,K+-ATPase activity of heterogeneous membrane fractions of vascular smooth muscle, Life Sci. 28, 945. Baker, P.F. and J.S. Willis, 1972, Binding of the cardiac glycoside ouabain to intact cells, J. Physiol. London 224, 444.
Blaustein, M.P., 1977, Sodium ions, calcium ions, blood pressure regulation and hypertension: a reassessment and a hypothesis, Am. J. Physiol. 232, C165. Brading, A.F. and J.H. Widdicombe, 1974, An estimate of sodium/potassium pump activity and the number of pump sites in the smooth muscle of the guinea-pig taenia coli using [3H]ouabain, J. Physiol. London 238, 235. Charlemagne, D., J. Leger, K. Schwartz, B. Geny, A. Zachowski and L. Lelievre, 1980, Involvement of tropomyosin in the sensitivity of Na+-K + ATPase to ouabain, Biochem. Pharmacol. 29, 297. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Craig, W.S. and J. Kyte, 1980, Stoichiometry and molecular weight of the minimum asymmetric of canine renal sodium and potassium ion-activated adenosine triphosphatase, J. Biol. Chem. 255, 6262. Daniel, E.E., R. Massingham and P.A. Nasmyth, 1970, The mechanism of contractile effects of ouabain and zinc on the rat uterus, J. Pharmacol. Exp. Ther. 173, 293. Deth, R.C. and C.J. Lymch, 1980, The binding of [3H]-ouabain to Na+-K + ATPase sites in arterial smooth muscle, Pharmacol. 21, 29. Deth, R.C., C.J. Lynch, J.L. Smart and R. Payne, 1983, Role of Na-K ATPase in al-adrenergic receptor events in arteries and liver, Fed. Proc. 42, 1364. Dutta, S., S. Goswani, D.K. Dutta, S.D. Lindower and B.H. Marks, 1968, The uptake and binding of six radiolabelled cardiac glycosides by guinea pig hearts and by isolated sarcoplasmic reticulum, J. Pharmacol. Exp. Ther. 164, 10. Fedan, J.S., D.P. Westfall and W.W. Fleming, 1978, Species differences in sodium-potassium adenosine triphosphatase activity in the smooth muscle of the guinea-pig and rat vas deferens, J. Pharmacol. Exp. Ther. 207, 356. Friedman, S.M. and C.L. Friedman, 1974, The action of ouabain on the smooth muscle cells of the rat tail artery, Proc. Soc. Exptl. Biol. Med. 146, 825. Godfraind, T. and J. Ghysel-Burton, 1980, Independence of the positive inotropic effect of ouabain from the inhibition of the heart N a ÷ / K + pumps, Proc. Natl. Acad. Sci. 77, 3067. Haddy, F., M. Pamnani and D. Clough, 1978, The sodiumpotassium pump in volume expanded hypertension, Clin. Exp. Hypertension 1,295. Haddy, F.J., M.G. Pamnani and D.L. Clough, 1980, Voluem overload hypertension: A defect in the sodium-potassium pump?, Cardiovasc. Rev. Rep. 1 (5), 376. Haustein, K.O. and J. Hauptmann, 1974, Studies on cardioactive steroids II. Structure-activity relationships in the isolated guinea-pig heart, Pharmacol. 11, 129. Jones, A.W., 1980, Content and fluxes of electrolytes, in: Handbook of Physiology, The Cardiovascular System, Vol. II, Vascular Smooth Muscle, p. 253, Williams and Wilkins, Baltimore, Md. Ku, D.D., T. Akera, T. Tobin, T.M. Brody, 1976, Comparative species studies on the effect of monovalent cations and
55 ouabain on cardiac Na÷-K÷-adenosine triphosphatase and contractile force, J. Pharmacol. Exp. Ther. 197 (2), 458. Landowne, D. and J.M. Ritchie, 1970, The binding of tritiated ouabain to mammalian non-myelineted nerve fibers, J. Physiol. London 207, 528. Leonard, E., 1957, Alteration of contractile response of artery strips by a potassium-free solution, cardiac glycosides and change in stimulation frequency, Am. J. Physiol. 189, 185. LiJllmann, H., T. Peters and U. Ravens, 1975, Studies on the kinetics of [ 3H]ouabain uptake and exchange in the isolated papillary muscle of the guinea-pig, Br. J. Pharmacol. 53, 99. Mansier, P. and L.G. Lelievre, 1982, Ca2+-free perfusion of rat heart reveals a (Na t + K +) ATPase form highly sensitive to ouabain, Nature 300, 535. Murthy, R.V., A.M. Kidwai and E.E. Daniel, 1974, Dissociation of contractile effect and binding and inhibition of Nat-Kt-adenosine triphosphatase by cardiac glycosides in rabbit myometrium, J. Pharmacol. Exp. Ther 188, 575. Okita, G.T., 1977, Dissociation of Na+,K+ATPase inhibition from digitalis inotropy, Fed. Proc. 36, 2225. Overbeck, H.W., M.B. Pamnani, T. Akera, T.M. Brody and F.J. Haddy, 1976, Depressed function of a ouabain-sensitive sodium-potassium pump in blood vessels from renal hypertensive dogs, Circ. Res. 38 (Suppl. II), 48. Pamnani, M.B., D.L. Clough and F.J. Haddy, 1979, N a t - K ÷ pump activity in tail arteries of spontaneously hypertensive rats, Jap. Heart J. 20 (Suppl. 1), 228. Periyasamy, S.M., L.K. Lane and A. Askari, 1979, Ouabain-insensitivity of highly active Na+-Kt-dependent adenosinetriphosphatase from rat kidney, Life Sci. 86, 742.
Preiss, R. and H. Banaschak, 1976, Characterization of MgATPase and (Na+-Kt)-stimulated Mg ATPase in smooth muscular cells of the sheep's common cartoid artery, Acta. Biol. Med. Germ. 35, 453. Reuter, H., M.P. Blaustein and G. Haeusler, 1973, Na-Ca exchange and tension development in arterial smooth muscle, Phil. Trans. R. Soc. London 265, 87. Schatzmann, H.J., 1953, Herzglykoside als Hemmstoffe fiir den aktiven Kalium-und Natriumtransport durch die Erythrocytenmembran, Helv. Physiol Pharmacol. Acta 11,346. Schied, C.R., T.W. Honeyman and F.S. Fay, 1979, Mechanism of fl-adrenergic relaxation of smooth muscle, Nature London 277, 32. Schwartz, A., J.C. Allen and S. Harigaya, 1969, Possible involvement of cardiac Na+,K+-adenosine triphosphatase in the mechanism of action of cardiac glycosides, J. Pharmacol. Exp. Ther. 168, 31. Urquilla, P.R., D.P. Westfall, K. Gato and W.W. Fleming, 1978, The effects of ouabain and alterations in potassium concentration on the sensitivity to drugs and the membrane potential of the smooth muscle of the guinea-pig and rat vas deferens, J. Pharmacol. Exp. Ther. 207, 347. Webb, R.C. and D.F. Bohr, 1978, Potassium-induced relaxation as an indicator of Na+-K + ATPase activity in vascular smooth muscle, Blood Vessels 15, 198. Wellsmith, N.V. and G.E. Lindenmayer, 1980, Two receptor forms for ouabain in sarcolemma-enriched preparations from canine ventricle, Circ. Res. 47, 710. Wolowyk, M.W., A.M. Kidevai and E.E. David, 1971, Na+,Kt-stimulated ATPase of vascular smooth muscle, Can. J. Biochem. 49, 376.
45
Elsevier
LACK OF C O R R E L A T I O N B E T W E E N [3H]OUABAIN B I N D I N G A N D Na-K ATPase I N H I B I T I O N IN RAT AORTA RICHARD C. DETH *, JANEL L. SMART, CHRISTOPHERJ. LYNCH and RAYMONDWALSH Northeastern University, College of Pharmacy and Allied Health Professions, Section of Pharmacology, Boston, Massachusetts 02115, U.S.A.
Received 21 September 1983, accepted 13 December 1983
R.C. DETH, J.L. SMART, C.J. LYNCH and R. WALSH, Lack of correlation between [3H]ouabain binding and Na-K A TPase inhibition in rat aorta, European J. Pharmacol. 99 (1984) 45-55. The binding of [3 H]ouabain to intact strips of rat aorta was compared with the ability of ouabain to inhibit the uptake of 86Rb by the same preparation. When a cold temperature wash was used to process tissues after binding of [3H]ouabain, a class of relatively high affinity binding sites was found (K D = 1.2 X 10 - 7 M). Binding was saturable and sensitive to both ATP depletion and elevated potassium. Elevation of cytoplasmic Ca2+ levels by phenylephrine or c-AMP levels by theophylline and terbutaline had no influence on [3H]ouabain binding. Ouabain inhibition of 86Rb uptake progressed to 60% of the total 86Rb uptake at 2 x 10 - 3 M from a threshold of about 10 -5 M. Half-maximal inhibition by ouabain occurred at a concentration of 10 -4 M. The disparity between [3H]ouabain binding and inhibition of 86Rb uptake indicates that the high affinity binding site in the rat does not contribute to inhibition of Na-K ATPase function. Na-K ATPase
Ouabain
Glycoside
S6Rb uptake
1. Introduction The Na-K ATPase or Na-pump located in the plasma membrane of arterial smooth muscle cells has been hypothesized to be an important site for the regulation of arterial contractility (Leonard, 1957; Wolowyk et al., 1971; Reuter et al., 1973; Friedman and Friedman, 1974; for review see Jones, 1980). Most prominently, certain forms of hypertension (e.g. volume overload) may result from impaired functioning of the arterial Na-pump (Overbeck et al., 1976; Blaustein, 1977; Haddy et al., 1978; Haddy et al., 1980). A number of investigators have examined Na-K ATPase activity in isolated arterial membranes in an effort to further characterize the role of the Na-pump (Wolowyk et al., 1971; Preiss and Banaschak, 1976; Allen and Seidel, 19.77; Allen et al., 1981). Additional information can be gained from binding studies with * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
labelled cardiac glycosides and from measurements of glycoside-sensitive 86Rb fluxes'which provide measures of Na-pump density and function respectively. Moreover, each of the latter two approaches can be applied to intact tissues, allowing a study of the Na-pump in its cellular environment. In an earlier paper we measured the binding of [ 3H]ouabain to N a - K ATPase sites in the aorta of the rabbit (Deth and Lynch, 1980). From these studies a K D value for binding of the glycoside to the pump (8 x 10 -8 M) and an estimate of the maximum number of binding sites (27.9 x 10 -14 m o l / m g dry weight) were obtained. However, since the rat has been more commonly employed as a model for the hypertensive state, we have extended [3H]ouabain binding studies to that species as well, and have compared binding with inhibition of 86Rb uptake. Our results reveal the presence of high affinity binding sites for [3H]ouabain in rat aorta which
46
appear to lack the ability to provide inhibition of Na-K-ATPase transport activity.
2. Materials and methods
2.1. Tissue preparation Male Sprague-Dawley rats weighing approximately 300 g were decapitated and the thoracic and upper abdominal segments of aorta excised. Arteries were maintained at 37°C in oxygenated buffer containing 140 mM NaCI, 4.5 mM KC1, 10 mM d-glucose, 5 mM HEPES (pH 7.4), 1.5 mM CaC12 and 1.0 mM MgC12. After cleaning the aorta of fat and connective tissue, transverse ring segments weighing about 3 mg were prepared for binding and 86Rb uptake studies.
2.2. [3H]Ouabain binding Aortic rings were labelled at 37°C in aerated buffer containing specified concentrations of ouabain labelled with [3H]ouabain (13 Ci/mmol, New England Nuclear). At the conclusion of the labelling interval tissues were briefly rinsed in 400 ml of ouabain-free buffer to remove labelling buffer and then processed one of two ways, depending on whether the entire pattern of [3H]ouabain efflux was to be measured, or only the residual content after 50 min of cold temperature washout.
2.3. Efflux studies After the initial rinse each aortic segment was passed through a series of scintillation vials, each of which contained 5 ml of buffer oxygenated via a polyethylene catheter. At 5 rain intervals, tissues were transferred to succeeding vials containing buffer at either 2 or 37°C for the desired period of efflux after which the tissues were placed in plastic scintillation vials and dried overnight. The dry tissue weights were recorded, and the tissues dissolved in 2 ml of 0.1 M KOH at 90°C for 6 h (in capped vials). After cooling to room temperature 5 ml of water was added to the vials and, after shaking, 7.5 ml of a Triton X-100/toluene-based
scintillation cocktail. Scintillation fluid was also added to the 5 ml of buffer in each efflux vial and all samples were counted for their 3H-content. Efflux curves were constructed by successive additions of the amount of [3H]ouabain leaving the tissue to the amount present in the tissue at the end of the efflux interval.
2.4. Low temperature bulk wash As described for rabbit aorta (Deth and Lynch, 1980) estimates of bound [3H]ouabain could be made from the residual content of the labelled glycoside after a period of washout at 2°C in unlabelled K-free buffer. This procedure allowed more facile handling of a large number of tissues. Groups of tissues (i.e. 4-6) were washed for 20 min in 50 ml of K-free buffer and then transferred to fresh buffer for an additional 30 min. After this procedure tissues were processed as outlined above to determine [3H]ouabain content.
2.5. 86Rb uptake studies Aortic rings were incubated at 37°C in normal, K-contained buffer containing 5 # C i / m l of 86Rb with or without ouabain. After the desired interval, rings were washed in unlabelled buffer at 2°C for 1 min and then transferred to a test tube and dried in an oven at 90°C overnight. After being weighed, tissue radioactivity was measured in a gamma counter. Results were expressed as a percentage of the total uptake of 86Rb by control tissues, normalized to tissue dry weight. Significance of observed differences was evaluated using Student's t-test with P values of < 0.05 taken to be significant.
2.6. Drug sources Ouabain, digitoxin, digoxin, dihydro-ouabain, phenylephrine, iodoacetic acid and dinitrophenol were obtained from Sigma Chemical, terbutaline from Geigy Pharmaceutical and theophylline from J.T. Baker Chemical Co. [3H]Ouabain was obtained for New England Nuclear.
47
5oi 2.0'
2"C 1.0
rat aorta. Tissues were labelled for 30 min with 10 -7 M [3H]ouabain under K-free conditions and then washed out in K-free buffer at either 37 or 2°C. The tissue content during the 37°C wash could be described by two exponential decay processes with [3H]ouabain content and T1/2 values for fast and slow components of 2.46 × 10 -13 mol/mg, 4.5 min and 9.57 × 10 -14 mol/mg, 23.3 min respectively. At 2°C, loss from the slower component was greatly retarded (T]/2 = 238 min) while the fast component was little affected (T1/2 = 3.5 min).
3.2. Time course of [3H]ouabain binding
0.5-
The rate of entry of [3H]ouabain into the fast and slow compartments was measured by washing 0.2"
37"C
O0o:O i151
o.,
,b
Zb
3b
~0
minutes
5'0
do
Fig. 1. Washout of [3H]ouabain from rat aorta strips at 2 or 37°C. Tissues were incubated K+-free buffer containing 10 -7 M [3H]ouabain for 30 min then moved at 5 min intervals through a saries of vials containing K+-free buffer at either 2 or 37°C. The tissue content of [3H]ouabain was calculated from the amount remaining in the tissue after 1 h of washout and the [3H]ouabain content of each vial. Each value is the average ( +_S.E.M.) of eight tissues.
x
E
_= v
O
3.1. Effect of temperature on [3H]ouabain binding Our previous experience with rabbit aorta (Deth and Lynch, 1980) and the experience of other investigators (Landowne and Ritchie, 1970; Baker and Willis, 1972; Brading and Widdicombe, 1974; Liillmann et al., 1975) has indicated that [3H]ouabain equilibrates into two major tissue compartments which probably reflect extracellular and cellular compartments. These two can be differentiated on the basis of their rates of washout, and this difference can be further accentuated by reducing the temperature of the washout buffer. As shown in fig. 1, a similar pattern was found for
_
1.0
._=
3. Results
/ / ~
0.5-
~~ X~X~ X.-,.,.~ 9Omen.
~ " X~x~X~X~x--x--x--x ~X "%1~ ^'-'X~x----X--x--x ~' ~--'~""X~x~x~x-.-x x ~ X ~ ~x~x~x--x~x
si:
X~x..~
O.2
~ x " ' x ' ' x " - - x ~ x- - x
0.1
,b
zb
3'o 4b minutes
60 30 20 I0
min. rain. rain min.
5
min.
"
5'0
Fig. 2. Time course of [3H]ouabain uptake by rat aorta strips. Tissues were incubated for various times in K+-containing buffer with 10 -7 M [3H]ouabain then washed out at 2°C for 50 min as in fig. 1. Rapid and slow exchanging components of [3H]ouabain content were determined by a curve peeling technique and are summarized in the inset. Each value is the average of 4 tissues ( 5: S.E.M. in the inset).
48
out tissues at 2°C after 5, 10, 20, 30, 60 and 90 rain of [3H]ouabain labelling at 1 0 - 7 M (fig. 2). In this experiment normal K ÷ (4.5 mM) was present during labelling while K-free buffer was used during washout. As can be seen in fig. 2, the residual amount of [3H]ouabain after 50 min of washout gradually and significantly increased from 0.16 + 0.01 to 0.51 + 0.04 x 10 -13 m o l / m g over the 5 to 90 min interval. The slow component of washout, calculated from the Y-axis intercept of a line extrapolated from the last 5 efflux values, similarly increased as shown in the inset to fig. 2. The rapid component of [3H]ouabain washout, calculated by subtracting the slow component and fitting the residual to an exponential function, reached a value at 10 rain which was not significantly different from its value at 90 rain. This behavior is quite similar to the pattern observed for rabbit aorta.
3.3. Influence of K + and ATP depletion Based on the knowledge of the Na-K ATPase transport function, binding of cardiac glycosides to the enzyme should be sensitive to the concentration of K + at the extracellular surface of the cell, and be dependent on the availability of ATP within the cell (Akera and Brody, 1978). This reflects the ability of K ÷ to reduce the availability of the E2-P form of the enzyme which is the preferred configuration for glycoside binding, and the essential requirement of ATP for generation o f the E : P form. In rat aorta, the amount of slow component [3H]ouabain binding after 30 min of labelling at 10 -7 M was increased by K-free conditions (30 min prior to and during [3H]ouabain labelling) and suppressed by elevated K ÷ (18 mM prior to and during [3H]ouabain labelling) as compared to 4.5 mM K ÷. The y-intercepts were 7.3, 6.3 and 4.3 × 10 -14 m o l / m g for 0, 4.5 and 18 mM K + respectively. While significant, the effect of K + removal was less pronounced than previously observed for rabbit aorta in which case the ratio of binding at 0 vs. 4.5 mM was 2.1 as compared to 1.2 for rat aorta. When cellular ATP levels were depleted by a 30 min exposure to 10 .4 M dinitrophenol and 10 -3 M iodoacetic acid, slow component [3H]ouabain binding at 2 x 10 .7 M was reduced by approxi-
--ox 12 ""
o I0
Specific binding
8 c
;/
No°-.p.ci,,°
?, 4
binding
/
-,
2 I
0
20
40
I
60 80 Minutes
I
1
I00
120
Fig. 3. Determination of 'specific' binding of oual~ainto rat aorta strips. Tissues were incubated for various times in [3H}ouabain (2.5 × 10 - 7 M) with (e) or without (A) unlabelled ouabain (10 - 4 M). After the binding period tissues were processed via a bulk wash procedure as described in Methods. 'Specific' binding ( © ) is defined as ' t o t a l ' - ' n o n - s p e c i f i c ' binding. Each point is the mean _+S.E.M. of 5 values.
mately 90% (1.5 × 10 -14 m o l / m g vs. 1.8 x 10 -~3 mol/mg).
3.4. Definition of 'specific binding" The slow-exchanging, temperature-sensitive component of [3H]ouabain binding presumably includes a fraction of drug bound to Na-K ATPase sites, but also fractions which reflect non-saturable sites such as delayed extraceUular compartment exchange or transmembrane uptake. In order to distinguish among these fractions, the binding of [3H]ouabain was compared in the presence and absence of a high concentration of unlabelled ouabain (10 -4 M) which should occupy saturable sites with sub-micromolar affinity constants (K D values), while non-saturable fractions would be expected to exhibit quasi-linear binding behavior. By subtracting the latter "non-specific" binding from 'total binding', a 'specific binding' fraction can be defined. In these experiments tissues were processed in
49
groups using a bulk wash procedure at 2°C as described in Methods to mimic the efflux procedure and retain only slow-exchanging [3H]ouabain. When rat aorta segments were allowed to bind [3H]ouabain at 2.5 x 10 -7 M, specific binding followed a complex time course including an apparent decrease during the 30-90 min interval followed by an increase at 120 min (fig. 3). Nonspecific binding comprised approximately 20-35% of total binding and appeared to follow a rather linear time course (fig. 3) which might account for the somewhat upward trend of slow component binding evident in the inset to fig. 2. From these results an incubation time of 30 rain was chosen for subsequent binding studies.
f
'o_ x O~
E
¢n q)
.•.•
1612
--
o
~
"5 E v
~ Total binding o
8
-
.0
o
4
to nn
t,-I ~
I 2
,
I
I 5
I 4
I 5
3H -Ouoboin x 10-7 (M) ,4-
3.5. Saturability of 'specific binding'
12-
Tissues were allowed to bind [3H]ouabain at concentrations from 10 -8 to 5 × 10 -7 M in the presence or absence of 10 -4 M unlabelled ouabain in K÷-containing buffer and then processed via a bulk wash procedure. Specifically bound [3H]ouabain exhibited saturability with a plateau evident at concentrations above 2 × 10 -7 M (fig. 4 upper). A Scatchard analysis of specific binding (fig. 4 lower) yielded a K D value of 7.87 x 10 -8 M and a Bmax (density of binding sites) of 1.26 x 10-13 m o l / m g dry tissue weight. The fitted straight line had a correlation coefficient of 0.97, suggesting the presence of a single class of sites with the above binding characteristics. Non-specific binding was an approximately linear function of [3H]ouabain concentration which therefore represented a larger portion of total binding at higher [3H]ouabain levels (fig. 5). Specific binding approximated the portion of total binding which was earlier found to be ATP-dependent. A Scatchard plot of total binding yielded a somewhat higher K D of 1.08 x 10 -7 M and a Bm,x of 1.68 X 10 -13 m o l / m g which could be compared to previously obtained values for total binding to rabbit aorta (Deth and Lynch, 1980).
I0-
ekX "k
Kd = 7 . 8 7 x 1 0 - 8 M Bmox = 1.26x 10-15 moles/rag
3.6. Influence of phenylephrine or c-AMP levels on [ 3H]ouabain binding It has been proposed that a c-AMP-mediated increase in the rat of activity of the N a - p u m p may
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Fig. 4. Saturation binding of [3H]ouabain to rat aorta strips. (Upper) Tissues were incubated for 30 min in various concentrations of [3H]ouabain with (I) or without (A) unlabelled ouabain (10 -4 M). Tissues were then processedvia a bulk wash procedure. 'Specific' binding (O) is defined as [total binding][non-specific binding]. (Lower) Scatchard plot of specifically bound [3H]ouabain. Each point is the mean (__+S.E.M. where indicated) of 5 values.
leads to relaxation of smooth muscle (Schied et al., 1979) and we have shown that N a - K ATPasemediated ion transport is increased during aadrenergic receptor activation (Deth et al., 1983). To test whether the contractile state of the artery influenced the rate of [3H]ouabain binding, we pretreated tissues with 10 -5 M phenylephrine for 10 min and compared their ability to bind
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['Glycoside] Fig. 5, Competition for [3H]ouabain binding sites by cardiac glycosides. Tissues were incubated for 30 rain in buffer containing 5×10 -7 M [3H]ouabain and various concentrations of unlabelled glycosides (10-8-10 -4 M). After a 50 rain washout in K+-free buffer at 2°C, residual [3H]ouabain content was measured and expressed as a percent of binding without unlabelled glycoside. Each value is the mean of four determinations.
centrations of unlabelled glycosides (10-8-10 -4 M). Results were expressed as a percent of binding in the absence of any competing glycosides (fig. 5). At 10-4 M, unlabelled glycosides displaced from 61 to 81% of bound [3H]ouabain. IC50 concentrations were based on the fraction of [3H]ouabain displaced by each unlabelled glycoside and Kt~ values were computed by applying the ChengPrusoff relationship (1973) (KD -----IC~0/(1 + L / K 0 ) where L = 5 x 10 -7 M and K 0 = 7.8 x 10 -8 M). K D values were: digoxin 2.0 x 10 -7 M; ouabain 6.1 × 10 -8 M; digitoxin 6.8 x 10 -8 M; and dihydro-ouabain 2.0 x 10-6 M. This potency order parallels the reported ability of glycosides to compete for Na-K-ATPase sites in other tissues (Dutta et al., 1968; Haustein and Hauptmann, 1974; Godfraind and Ghysel-Burton, 1980).
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[3H]ouabain (10 -7 M) during continued PE exposure with binding by relaxed tissues. Although PE-treated tissues b o u n d significantly less [3H]ouabain after 5 min (3.5 + 0.4 vs. 5.9 + 0.5 m o l / m g × 10 -14) subsequently there was no difference between the two groups. In the same experiment other tissues were treated with 10 -5 M terbutaline and 10 -4 M theophylline to elevate c-AMP levels prior to and during a period of [3H]ouabain binding. N o difference in the amount of binding was found. Comparisons at different concentrations of [3H]ouabain similarly showed no marked difference in binding associated with PE or terbutaline and theophylline treatment.
3. 7. Competition for [3H]ouabain binding sites by other glycosides To determine whether [3H]ouabain binding sites in rat aorta exhibited molecular requirements similar to Na-K ATPase sites in other tissues, we examined the ability of several unlabelled glycosides to compete with [3H]ouabain for binding. Tissues were incubated for 30 min in buffer containing 5 x 10 -7 M [3H]ouabain and various con-
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Fig. 6. S6Rb uptake by rat aorta strips. Two groups of tissues were incubated in K + containing, S6Rb labelled buffer for 2, 5, 10 or 20 rain. One group was treated with 10 -3 M ouabain for 30 rain prior to and during 86Rb uptake and the residual 86Rb content after a I rain rinse is designated 'ouabain-insensitive 86Rb uptake'. Uptake by the other group (without ouabain) is designated 'total 86Rb uptake'. The difference between these two values represents 'ouabain-sensitive 86Rb uptake'. Each value is the mean of five tissues.
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Log [Ouobain ] Fig. 7. (a) Concentration dependence of ouabain-induced inhibition of 86Rb uptake in rat aorta strips. 86Rb uptake during 10 rain of exposure was compared in K+-containing buffer with various concentrations of ouabain (10-6-10-3 M) and expressed as a percentage of the maximum ouabain-sensitive uptake. Each value is the average of five determinations ( + S.E.M.). (b) Concentration dependence of ouabain-induced inhibition of S6Rb uptake in rabbit aorta strips. Same as in (a) except ouabain concentrations were 3 × 10-1°-10- 5 M. Results are the average (_.+S.E.M.) of five determinations.
3.8. Ouabain inhibition of 86Rb uptake Because of the ability of Rb + to substitute for K ÷ in the N a - K - A T P a s e mechanism, the
ouabain-sensitive component of 86Rb uptake has been used to assess the function of N a - K ATPase in isolated arteries (Haddy et al., 1978, 1980; Pamnani et al., 1979). 86Rb uptake by rat aortic
52 rings was linear over the first 10 min of exposure (fig. 6). Incubation with increasing concentrations of ouabain from 10 -5 to 2 x 10 -3 M caused a reduction in the amount of 86Rb accumulated (fig. 7a). Because of ouabain's limited solubility, it was not possible to clearly establish the maximum suppression by ouabain. However, similar studies using vanadate (10 #M) and ATP depletion (10 -4 M DNP and 10 -3 M IAA) as a means of completely inhibiting the Na-pump resulted in a similar suppression of 86Rb uptake (i.e. 55% of the total 86Rb uptake). Assuming this value to be 100% of the Na-pump-related uptake, half-maximal inhibition occurred at a ouabain concentration of 8.5 × 10 -5 M. Any underestimation of the 100% value would result in a higher IC50 value. 86Rb uptake by rabbit aorta was more sensitive to ouabain (fig. 7b). Prior incubation in low concentrations of ouabain (5 x 10-10 ___,10-8 M) caused an increase in the ouabain-sensitive component of 86Rb uptake while high concentrations reduced uptake. Using the value for zero ouabain as 100%, half-maximal inhibition occurred at a ouabain concentration of 3 x 10 -7 M. Increased 86Rb uptake at low ouabain levels most likely reflects the stimulatory influence of norepinephrine released from adrenergic nerve endings (Deth et al., 1983).
4. Discussion
Cardiac glycoside binding to Na-K-ATPase sites and the influence of glycosides on ion transport have both been extensively employed to study the role of the Na-pump in cellular function. The specificity of glycoside binding is well-documented (Schatzmann, 1953; Schwartz et al., 1969; Akera and Brody, 1978), although there is disagreement as to the relationship between binding and the functional effects that glycosides cause (Daniel et al., 1970; Murthy et al., 1974; Okita, 1977; Godfraind and Ghysel-Burton, 1980). The current studies were undertaken to investigate this relationship in the rat aorta. Our results have established the existence of relatively high affinity binding sites for [3H]ouabain which exhibit a K D of about 0.1 #M. T h i s
affinity is similar to values from studies on other intact smooth muscle preparations such as rabbit aorta (Deth and Lynch, 1980) or guinea-pig taenia coli (Brading and Widdecombe, 1974). However, this finding is ostensibly at odds with the often described resistance of Na-K ATPase in the rat to inhibition of ouabain (Allen and Schwartz, 1969; Friedman and Friedman, 1974; Ku et al., 1976; Urquilla et al., 1978; Fedan et al., 1978; Periyasamy et al., 1979). Allen and Schwartz (1969) observed that Na-K ATPase activity from rat heart and kidney was relatively insensitive to ouabain as compared to bovine or canine preparations. Urquilla et al. (1978) noted that ouabain (8 x 10 -5 M) or K-free buffer caused depolarization in the guinea pig vas deferens, but not in the rat vas deferens. In an extension of this observation, the total Na-K ATPase activity was found to be similar in membranes prepared from vas deferens of both species ruling out a scarcity of the enzyme as a basis for reduced glycoside sensitivity. Ouabain (8 x 10 -5 M) produced a 30% inhibition of the rat vas deferens derived Na-K ATPase. In the 86Rb-uptake studies shown in fig. 7, it is apparent that in rat aorta higher concentrations of ouabain are needed to produce full inhibition of the Napump-related transport activity, and the value of 30% inhibition is in reasonable agreement with the influence of 8 x 10 -5 M on 86Rb uptake. In fact 10 -3 M seems necessary to produce 100% inhibition, based on the observation that ATP-depletion or vanadate treatment produced essentially the same extent of inhibition of 86Rb uptake as 10 -3 M ouabain. Given that the Na-K ATPase of the rat has a low affinity for ouabain, the relevance of the high affinity binding sites for [3H]ouabain must be questioned. Several behaviors indicate that the site is needed a classical Na-K ATPase locus: (1) binding is saturable, yielding a Bmax similar to that reported for other tissues; (2) the affinity is similar to that reported for other tissues; (3) binding is greatly reduced in the absence of ATP; (4) binding is reduced in the presence of high K+; (5) structural requirements for binding are similar to other Na-K-ATPase sites. On the other hand several points are at variance with a Na-K ATPase loca-
53 tion: (1) binding to these sites does not result in inhibition of 86RB uptake; (2) K t free buffer increases [3H]ouabain binding above levels obtained at 4.5 mM K t to only a modest extent; (3) binding is not greatly altered during a-adrenergic agonist-induced contraction, which is known to increase the ouabain-sensitive 86Rb uptake (Deth et al., 1983). While the basis for the lower ouabain sensitivity of the rat Na-K ATPase has not been fully established, it has been shown to be retained in relatively purified enzyme preparations (Periyasamy et al., 1979), suggesting that differences in the functional environment of the intact cell are not involved (although see Mansier and Lelievre, 1982). The most straightforward explanation would be that the configuration of the glycoside binding site is different in the rat vs. more sensitive species. Within this context, a less favorable configuration could be a stable feature of the protein binding site itself or alternatively its presence could depend on the physical properties of the membrane environment, the state of aggregation of subunits, or other complex factors which might be preserved during enzyme purification. For instance, Wellsmith and Lindenmayer (1980) have provided kinetic evidence for the existence of two forms of the ouabain receptor with widely divergent affinities in canine cardiac membranes. The proportion of the two forms was shown to be dependent on the presence of Mg 2+, Na t and ATP, such that for 5 mM Mg 2+, 100 mM Na t and 5 mM ATP, the ratio of high affinity to low affinity sites was about 2 : 1 while in their absence, no significant high affinity binding was detected. The lower affinity form had a K o of 1.9 × 10 -7 M, and binding to this site did not result in inhibition of Na-K ATPase activity. In the same study, the addition of small quantities of detergent sodium dodecylsulfate (SDS) shifted the proportion of sites in favor of the higher affinity form. Since the active functional unit of the enzyme is thought to be a dimer of the ( a f t ) 2 form (a and fl being high and low molecular weight protein subunits, respectively) Craig and Kyte, 1980), the propensity for aggregation may play a role in determining ouabain sensitivity. Allen and coworkers (1981) also noted that SDS addition was necessary to unmask a latent
ouabain sensitive Na-K ATPase activity in membranes prepared from canine aorta. An additional mechanism by which alterations in ouabain sensitivity may be produced was reported by Charlemagne et al., (1980). Treatment of membranes prepared from a murine plasmacytoma with EDTA increased ouabain sensitivity by 300-1000 fold, while the readdition of Ca 2t along with either tropomyosin or proteins derived from the plasma membrane restored the lower ouabain sensitivity. Thus, it is implied that Ca 2t, acting in conjunction with membrane proteins is able to modulate the configuration of the ouabain binding site. A recent confirmation of this relationship is provided by Mansier and Lelievre (1982) who found that cardiac sarcolemmal membranes from the rat displayed only low ouabain affinity when normal calcium (2 mM) was present prior to homogenization, but a bimodal affinity (K D = 1.2 × 10 -8 M and 6 × 10 -5 M) when hearts were perfused with Ca-free buffer. Moreover, the higher affinity form is found to be present in isolated myocytes (Adams et al., 1982) and binding to this site results in an inotropic response. The high affinity [3H]ouabain binding sites we identify in intact rat aorta strips are most likely similar to these sites identified in rat myocardium. However, we have not observed alterations in arterial contractility at these concentrations, so a further comparison seems warranted. The work of Wellsmith and Lindenmayer (1980), discussed earlier, suggests that heterogeneity of cardiac glycoside binding sites may be a general feature of all species, and the discrepancy in affinity observed in the rat may reflect a rather extreme expression of a commonly available regulatory mechanism. Since the Ca 2t-dependent differences in rat cardiac sarcolemmal binding of ouabain were preserved in isolated membranes, one may tentatively hypothesize that the normal low sensitivity of rat arteries visa vis rabbit arteries is a result of relatively high Ca 2t levels at a regulatory site in the former species (i.e. the rat). This possibility may serve to explain the greater reliance of rat arteries on the activity of the Na-K ATPase for relaxation as compared to rabbit arteries (Webb and Bohr, 1978; Deth et al., 1983),
54 s i n c e N a - C a c h a n g e is c h a r a c t e r i z e d b y a l o w e r K D for C a 2+ a n d w o u l d t h e r e f o r e b e m o r e e v i d e n t a t h i g h e r C a 2 + levels. A l t e r n a t i v e to a s p e c i e s - d e p e n d e n t d i f f e r e n c e i n C a 2 + levels, t h e r a t p l a s m a m e m b r a n e m a y c o n t a i n a p r o t e i n e l e m e n t (cf. C h a r l e m a g n e et al., 1 9 8 0 ) w h i c h is e s s e n t i a l f o r t h e e x p r e s s i o n o f c a l c i u m ' s r e g u l a t o r y role. R a b b i t t i s s u e s m a y l a c k o r h a v e less o f t h i s p u t a t i v e p r o t e i n , r e s u l t i n g i n a p r e dominance of the higher affinity form. I n s u m m a r y , [3 H ] o u a b a i n b i n d s t o h i g h a f f i n i t y sites in rat aorta strips which exhibit characteristics e x p e c t e d o f a n N a - K A T P a s e locus, b u t d o n o t m e d i a t e i n h i b i t i o n o f i o n t r a n s p o r t . T h e s e sites may be analogous to Ca2+-dependent ouabain binding sites observed in the rat sarcolemma. Given a r e g u l a t o r y r o l e o f C a 2+ i n d e t e r m i n i n g o u a b a i n affinity, the lower affinity of rat arteries may be r e l a t e d t o a s p e c i e s - d e p e n d e n t d i f f e r e n c e i n C a 2÷ homeostatis.
Acknowledgement This work was supported by a grant-in-aid from the American Heart Association (81-1180) with funds contributed in part by the Massachusetts Heart Association to whom thanks are due.
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