Biochemical Phormacolog?. Vol. 33. NO. 1. pp. 47-53. 1984 Printed in Great Britain. 000&2952/84 $3.00 + O.Oll @ 1984 Perpamon Press Ltd. HETEROGENEITY OF OUABAIN SPECIFIC BINDING SITES AND (Na’ + K’)-ATPase INHIBITION IN MICROSOMES FROM RAT HEART F. NOEL and T. GODFRAIND Laboratoire de Pharmacodynamie G&kale et de Pharmacologic, Universite Catholique de Louvain, U.C.L. 7350, Avenue Emmanuel Mounier 73, B-1200 Bruxelles, Belgium (Received 14 March 1983; zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO accepted 12 July 1983) Abstract-Cardiac glycoside binding to microsomes prepared from rat heart ventricles and enriched in INa’ + K+kATPase was measured bv a rauid filtration techniaue. The relation between ouabain binding ;o microsodes and (Na’ + K’)-ATPke activity has also been kxamined. Data were statistically analyses by means of two different non linear regression methods. The experimental results were fitted the most closely by a model describing that ouabain specific binding occurred at two classes of independent sites. High affinity sites were characterized by a dissociation constant of 0.21 t 0.01 yM and a low capacity (9.4 2 1.4 pmoles/enzymatic unit). Low affinity sites were characterized by a dissociation constant equal to 13 + 3 $k4 and a capacity equal to 87 k 15 pmoles/enzymatic unit. Similar results were obtained with the more lipophilic glycoside digoxin. It was also observed that dihydroouabain, a ouabain derivative with a saturated lactone ring, competes with 3H-ouabain for the binding to the two classes of sites. Binding to these two classes of sites appeared to be associated with a corresponding inhibition of (Na+ + K’)-ATPase activity. The mechanism of the positive inotropic effect of cardiac glycosides is a matter of controversy. A cas- ual relationship between inhibition of Na-K pump and increase in contractile force of the heart has been proposed to be the only mechanism of action [l-3] but some authors do not support such an hypothesis [4-6]. Recently, it has been suggested that more than one mechanism could be responsible for ouabain inotropic effect because this glycoside evokes an increase in contractility much higher than the one observed when the Na-K pump is inhibited by lowering KO or by treatment with dihydroouabain [7]. Furthermore, there are several reports in the literature showing that ouabain binding to micro- somal preparations obtained from various tissues and species could occur at more than one specific binding site [8-171. The nature of such sites and their relation to (Na+ + K+)-ATPase has been the subject of several hypothesis [9,10,13,15,18]. The present experiments were designed to further study the characteristics of cardiac glycoside binding to microsomes prepared from rat heart ventricles and enriched in (Na+ + K+)-ATPase. We have also compared binding to inhibition parameters. The results confirm the existence of two classes of binding sites in the rat heart and indicate that both could be related to the inhibition of (Na’ + K+)-ATPase activity. A preliminary report on some of these results has already been published WI. MATERIALS AND METHODS (Na’ + K+)-A TPuse preparation. Atria and ven- tricles were dissected from hearts of female Wistar rats (2.5-3.5 months old). A fraction enriched in (Na’ + K’)-ATPase was prepared as described pre- viously for guinea-pig heart [19] with slight modifi- cations. A sulfhydryl group protecting agent (1,4- dithioerythritol 30mM) was added during hom- ogenization. After a prolonged NaI-treatment (45 min), the homogenate was diluted and centri- fuged at 50,000 g for 90 min. The resultant pellet was twice resuspended in a solution containing 0.25 M sucrose and 5 mMTris/HCl (pH 7.4) and then cen- trifuged for 1 hr at 100,OOOg. The final pellet was resuspended in 0.25 M sucrose containing 0.1% deoxycholate, 20mM maleateflris (pH 7.4) and stored overnight at -30”. After thawing. the super- natant from a 30 min spin at 20,000 g was centrifuged for 30 min at 100,OOOg to separate a microsomal fraction. The (Na’ + K+)-ATPase activities in this fraction were determined as previously described by measuring the inorganic phosphate (Pi) liberated [ 191. These activities were checked during each bind- ing experiment. The protein content was determined by the method of Lowry et al. [20]. (3H)-glycoside binding assays. Unless otherwise stated, the incubation medium contained 3 mM MgC12, 3 mM Pi-Tris, 1 mM EGTA abd 20mM maleatenris, pH 7.4 at 37°C (Mg-Pi medium). A rapid filtration technique was used to separate membrane-bound from free glycosides: samples of 200 ~1usually containing 150 ,ug protein (13@200 pg) were filtered at 10” on Whatman glass fibers filters (GF/‘F). The filters were washed three times with 20 ml of chilled solution (0.25 M sucrose, 5 mM Tris/HCl, pH 7.4 at Oo), then added to 10ml of a scintillation solution (Aqualuma-plus 25%-toluene 75%) and the radioactivity counted in a liquid scin- tillation counter (Kontron MR 300) with an effi- ciency determined by internal standardization (about