Quantitative correlation of cardiac glycoside binding to its receptor and inhibition of the sodium pump in chicken heart cells in culture

Abstract

The Machaniam of positive inotropic action of cardiac glycoaidea is still a matter of controvera1 (1). The disagreement aainly relatee to the queation whether increaaed force of contraction la cauaed by, and therefore neceasarily accompanied by, an inhibition of the sodium pump. At therapeutic levels of the drug . RodiuM pump has been deaeribed aa not inrlueneed, inhibited or even stimulated. depending on the apeciea invastigated, the drug applled and. the method used (1,2). The problem is further complicated by the preaenee of two different binding aites ror cardiae glycoaides. In rat heart, occupation of the high affinity site being linked to positive inotropic effect without inhibition of (Na++ K+)-ATPase aetivity or active (86Rb +)_uptake aa meaaure of sodium pump aetivity (3). In fev of the e~peri.ent6 deacribed, how ever, haa inhibition or atimulation of the aodium pump by cardiae glycoaides been correlated quantitatively with the pereentage of binding sites oeeupied b y cardiac glycoside molecu les . Methods: Huscle and non ~uscle cells from hearts of 9.12 day_old c hi ck embryos haye been prepared and separately cultured a8 described in detail for rat heart cells (4,5).Experiments have been ~arried out with museie cells after 2-~ days in c~!tu re, with non muscle cells after 1 aubcultivation (splitting rati o 1:2). At that time, muscle ce lla had forMed a synchronously beating monolayer, non musc le cells wer@ at conflue nc y. For measurement of {3H)_ouabain binding, (86Rb ++ K+)-uptake and cellular K+, cells (0.2-2.0 mg proteinjfla.sk) were incubated in Ilepes buffered (20 mM, pli 7.40)

Cardiac glycoside receptors in cultured heart cells—II

“…High affinity sites (K o :: 3.2 x 10-8 M) represent ca 7% and low affinity sites (K o = 7.1 x 10-6 M) ca 93% of total binding sites (Table 2); the lotal number (U x l()6sitesjcell being similar {o that found in chick heart muscle cells (860,000 sitesjcell ; Ko = 1.5 X 10-7 M; [25)) . Binding of ouabain to both sites ;s lowered by K" (Fig.…”

“…Additional evidence mayaiso come trom work with cultured heart cells trom chicken embryos: recently we have characterized cardiac glycoside receptors in cardiac muscle and non-muscle cells from chicken embryos [24,25]. Conceming ouabain binding, a single dass of binding sites has been identified in both celllypes ( K D "" 1.5 X 10-7 M and 1.9 x 10-7 M).…”

“…In cultured heart cells from chicken embryos, ouabain binds 10 a single d ass of binding sites (24,25). Applying the very same experimental procedures for the characterization of ouabain binding in rat heart muscle cells in culture, experimental evidence strongly suggests different types of saturable ouabain binding sites in these cells.…”

“…[24,25] . APP7.ing exactly the same experimental procedure 10 [ H]ouabain binding in rat heart muscle cells , a biphasic binding curve was obtained (Fig.…”

Cardiac glycoside binding sites in cultured heart muscle cells

“…2 a; for discussion see ref. 21,22), a linear correlation has been found in cardiac non muscle cells from this species (21,22).…”