Specific binding of cardiac glycoside drugs and endogenous digitalis-like substances to particulate membrane fractions from human placenta.

Abstract

We studied the characteristics of binding of cardiac glycosides to particulate membrane fractions from human placenta, to demonstrate that placental tissue is a suitable source of receptors for digitalis drugs. Moreover, we performed preliminary experiments with 125I-labeled digoxin and placental particulates to develop a radioreceptor assay for measurement of endogenous substances with activity similar to cardiac glycoside drugs (EDLS). Placental membrane fractions were incubated with [3H]ouabain (10 nmol/L) or 125I-labeled digoxin (50 pmol/L). With both ligands, binding followed a pseudo-first-order reaction kinetics and was saturable. Scatchard analysis revealed a single class of sites [for ouabain, KD = 20.2 +/- 5.8 nmol/L (mean +/- SEM), Bmax = 3.1 +/- 0.9 nmol per gram of protein; for digoxin, KD = 29.7 +/- 1.9 nmol/L, Bmax = 24.3 +/- 1.1 nmol per gram of protein]. As expected, digoxin was less potent than ouabain in displacing both tracers from digitalis drugs receptors; progesterone, cortisone, digitoxose, furosemide, bumetanide, and propranolol had no or little effect. Specific 125I-labeled digoxin binding was competitively inhibited by plasma and (or) urine extracts from newborns, adults, pregnant women, and patients with renal insufficiency. Inhibition of binding and volume of plasma and urine assayed were linearly related. These findings support the hypothesis that cardiac glycosides and EDLS can interact with the human placenta and suggest placental tissue to be a suitable source of receptors for cardiac glycosides.

Synthetic Candidates for EDLF

“…The dose dependencies of the inhibition of ( 125 I)digoxin binding by the three synthetic inhibiting compounds span two logarithmic units as observed with known cardic glycosides 10,11 . The concentration range, however, is well above (∼10,000‐fold) the typical range of the plant digitalis compounds 10,11 . These effects are completely different from those obtained by fatty acids 11 in which sharp dose‐response curves were obtained.…”

“…Working solutions were then freshly prepared in assay buffer (5 mM Na 2 HPO 4 , 5 mM MgCl 2 , 50 mM Tris‐HCl, pH 7.4 at 37°C, BSA 0.1%). For cross‐reactivity experiments at equilibrium, 0.3 mg/ml of human placental membranes prepared as previously described 10 were incubated for 5 hours at 37°C with increasing amounts of the synthetic compounds and/or 40 pM ( 125 I)digoxin (DuPont‐New England Nuclear, Boston, Massachusetts). Bound and free radioactivity was separated by filtration through Whatman GF/C glass fiber filters, and retained radioactivity was counted in a λ‐spectrometer.…”

“…Cross‐reactivity experiments by the sequential technique were performed as previously described 11 using identical dilutions of the synthetic compounds. ( 3 H)ouabain binding was measured as described previously 10 using filtration as above to separate bound from free ligand. To construct Lineweaver‐Burk plots, ( 3 H)ouabain (DuPont‐New England Nuclear) at concentrations of 70, 20, 10, and 5 nM was incubated with four concentrations (from 5 × 10 −7 to 10 −4 M) of the inhibiting synthetic compounds.…”

“…Using ( 3 H)ouabain and ( 125 I)digoxin as tracers, we demonstrated that human placenta contains large amounts of specific, high‐affinity digitalis receptors 10 . Human placenta membranes were then used to develop a radioreceptor assay (RRA) to detect EDLF in human plasma and urine 11 …”

Functional Characterization of an Endogenous Digitalis‐Like Factor in Human Newborn Plasma

“…The methods of purification of neonatal EDLF, 4 the preparation of membrane fractions enriched in (Na + /K + )‐ATPase from rat brains and kidneys 6 and from hearts, 8 the measurement of (Na + /K + )‐ATPase activities employing an enzymatic coupled assay, 9 the preparation of human placental membranes, 10 and binding experiments with 125 I‐digoxin 11 and 3 H‐ouabain 10 have previously been described. The sensitivity of (Na + /K + )‐ATPase to EDLF was measured after preincubation for 30 minutes at 37°C.…”