Lindsay Brown

Multiplicity of cardiac glycoside receptors in the heart

Abstract The recent discovery of different types of cardiac glycoside binding site may help to interpret seemingly contradictory experimental findings. Both high and low affinity binding sites have been demonstrated using contracting heart muscle, cardiac cell membrane preparations and heart muscle cells in culture. Erland Erdmann and colleagues discuss recent results and consider whether these different receptors account for the very low glycoside concentrations (∼10−9m) that are effective therapeutically compared with the higher concentrations (∼10−7m) that are effective in vitro .

DIGITALIS STRUCTURE-ACTIVITY RELATIONSHIP ANALYSES CONCLUSIONS FROM INDIRECT BINDING STUDIES WITH CARDIAC (Na+ + K+)-ATPase

We have performed direct and indirect binding studies with [3H]ouabain or [3H]digitoxin on beef or guinea pig cardiac (Na+ + K+)-ATPase to measure the potencies of a broad range of cardiotonic steroids for structure-activity relationship (SAR) studies for comparison with previously determined positive inotropic potencies. The positive inotropic potencies of twelve compounds on contracting guinea pig left atria correlated well with the equilibrium dissociation constants (KD values) from the inhibition of [3H]ouabain binding to guinea pig cardiac (Na+ + K+)-ATPase (r = 0.98 for seven 5 beta-compounds, r = 0.95 for five 5 alpha-compounds). Further we calculated KD values from the inhibition of [3H]ouabain binding data for a total of 33 digitalis derivatives on the digitalis-sensitive beef cardiac (Na+ + K+)-ATPase. For the 27 compounds tested on both beef cardiac (Na+ + K+)-ATPase and guinea pig left atria, the potencies showed a significant correlation (r = 0.92 for 22 5 beta-compounds, r = 0.96 for five 5 alpha-compounds. For seven compounds, KD values were measured on beef cardiac (Na+ + K+)-ATPase using inhibition of binding of [3H]digitoxin. These values correlated well (r = 0.99) with the KD values from the [3H]ouabain studies. These results show that: (1) The significant correlation observed between KD values on guinea pig cardiac (Na+ + K+)-ATPase and positive inotropic potency in guinea pig left atria is further evidence that the pharmacological receptor for inotropy is part of the enzyme, (2) Inhibition of the binding of [3H]ouabain or [3H]digitoxin can be used to determine the relative potencies of unlabelled digitalis derivatives. The similar relative potencies on beef and guinea pig cardiac (Na+ + K+)-ATPase of a broad range of digitalis derivatives indicate that the binding site is similar for both species; and (3) SAR studies indicate that functional groups on these steroids have the same influence on potency on either the positive inotropy or cardiac (Na+ + K+)-ATPase studies.

Cardiac glycoside receptors in cultured heart cells II

The binding of [3H]ouabain has been studied in (Na+ + K+)-ATPase enriched cardiac cell membranes, as well as in cardiac muscle and non-muscle cells in culture--all obtained from hearts of neonatal rats. The binding has been correlated with ouabain-induced inhibition of (Na+ + K+)-ATPase (cardiac cell membranes) and the inhibition of active (86Rb+ + K+)-influx (cardiac muscle and non-muscle cells in culture). Furthermore, the effect of ouabain on the amplitude of cell-wall motion and contraction velocity has been studied in electrically driven cardiac muscle cells. In muscle and non-muscle cells, two classes of ouabain binding sites have been identified. In rat heart muscle cells, the high affinity binding site has a dissociation constant (KD) of 3.2 X 10(-8) M and a binding capacity (B) of 0.2 pmole/mg protein (80,000 sites/cell); the values for the low affinity binding site are: KD = 7.1 X 10(-6) M; B = 2.6 pmole/mg protein (10(6) sites/cell). The binding to both types of binding sites is depressed by K+ and abolished after heat denaturation of the cells. The kinetics of [3H]ouabain binding to rat heart muscle cells (association and dissociation rate constants, K+- and temperature-dependence of association and dissociation processes) have been characterized. In rat heart muscle and non-muscle cells, the binding of [3H]ouabain to the low affinity site results in inhibition of the (86Rb+ + K+)-influx (EC50 = 1.3 and 1.5 X 10(-5) M ouabain), a decrease in cell-K+ (EC50 = 1.9 and 1.4 X 10(-5) M) and an increase in cell-Na+ (10(-5)-10(-4) M). The ouabain-induced positive inotropic effect (increase in amplitude of cell-wall motion, increase in contraction velocity) in cardiac muscle cells is observed only at ouabain concentrations greater than or equal to 5 X 10(-6) M, and it is therefore probably attributed to occupation of the low affinity binding site. Coupling of occupation of the low affinity site by ouabain with drug-induced inhibition of the sodium pump and with drug-induced positive inotropic action is further substantiated by kinetic measurements. In contrast, occupation of the high affinity binding site does not produce any measurable inhibition of the sodium pump activity or positive inotropy.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiac glycoside receptors in cultured heart cells. I: Characterization of one single class of high affinity receptors in heart muscle cells from chick embryos

Binding of (3H)-ouabain and ouabain-induced inhibition of the sodium pump and of the (Na+ + K+)-ATPase have been characterized in cultured cardiac muscle and non muscle cells, as well as in cardiac cell membranes--all obtained from chick embryos. In both cell types, ouabain binds to a single type of binding sites in a temperature-dependent manner. The association rate but not the dissociation rate, is lowered by K+; specific binding is lost after heat-denaturation of the cells. Binding parameters (association and dissociation rate constants, activation energies for association and dissociation) are similar in muscle and non muscle cells. The dissociation constant of specific ouabain binding is 1.5 X 10(-7)M in cardiac muscle cells, and 1.9 X 10(-7)M in cardiac non muscle cells, the binding capacity being 2.6 and 2.1 pmoles/mg protein respectively. Specific binding of ouabain to the cells is coupled to inhibition of the sodium pump, as can be seen from ouabain-induced inhibition of active (86Rb+ + K+)-uptake, decrease in cellular K+, and increase in cellular Na+ (EC50 = 10(-7)-10(-6)M). The data obtained with cardiac cells are in good agreement with results found for ouabain binding (dissociation constant 4.3 X 10(-7)M) and (Na+ + K+)-ATPase inhibition (EC50 = 1.4 X 10(-6)M) in cardiac cell membranes prepared from the same tissue. Due to the experimental evidence it is concluded that the binding site for ouabain is identical with the cardiac glycoside receptor of these cells. In cardiac non muscle cells, binding of ouabain to its receptor is strictly coupled to inhibition of active K+-transport in a stoichiometric manner. In cardiac muscle cells, however, active K+-transport is inhibited by less than 10% when up to 40% of cardiac glycoside receptors have bound ouabain. It is assumed that this non-stoichiometric coupling of receptor occupancy and sodium pump inhibition in cardiac muscle cells may prevent substantial changes of Na+- and K+-contents in the heart in the presence of therapeutic levels of cardiac glycosides.

The positive inotropic response to milrinone in isolated human and guinea pig myocardium

SummaryThe bipyridine derivative, milrinone, produced positive inotropic effects in isolated, contracting right ventricular papillary muscles and left atria from guinea pigs as well as in human papillary muscle strips. The inotropic effect was biphasic in guinea pig papillary muscles (EC50, high affinity, 1.5×10−6 mol/l, about 35% of maximal effect; apparent EC50, 3×10−5 mol/l with a maximal effect at 2×10−4 mol/l) but monophasic in guinea pig left atria (EC50, 6×10−5 mol/l) and in human papillary muscle strips (EC50, 5.8×10−5 mol/l). In guinea pig papillary muscles, reserpine pretreatment or l-practolol preincubation reduced the low concentration effect only. In the presence of l-practolol, carbachol reduced the low concentration effect only. In the presence of l-practolol, carbachol reduced but not abolished the inotropic effects of milrinone (3×10−6 mol/l, 1×10−4 mol/l) in both guinea pig and human myocardium. This antagonism was prevented by atropine preincubation. The maximum inotropic effect of milrinone was similar to that of ouabain and calcium in guinea pig myocardium but markedly less than either calcium or ouabain in human myocardium. Milrinone inhibited crude guinea pig and human cardiac phosphodiesterase activity in vitro but did not inhibit 3H-ouabain binding to partially purified human cardiac (Na++K+)-ATPase-containing membranes.We conclude that the primary mode of action of milrinone in both guinea pig and human myocardium is through inhibition of phosphodiesterase. The reduced maximal inotropic effect of milrinone compared with calcium in human but not in guinea pig myocardium indicates basic differences between healthy animal and diseased human cardiac muscle.

Positive inotropic effects of the calcium channel activator Bay K 8644 on guinea-pig and human isolated myocardium

Summary1. The positive inotropic effects of the dihydropyridine calcium activator Bay K 8644 were studied in guinea-pig isolated contracting myocardium and human papillary muscle strips obtained from patients undergoing mitral valve replacement or cardiac transplantation. 2. Bay K 8644 produced a slowly developing, concentration-dependent positive inotropic response in all cardiac tissues studied. In guinea-pig papillary muscle, the increase in force of contraction was half-maximal at 3.9 × 10−8 mol/l and the maximal inotropic effect was comparable to that obtained with ouabain, dobutamine or calcium. The guinea-pig left atrium (EC50, 2.1 × 10−7 mol/l) was fivefold less sensitive than the papillary muscle. 3. The maximal inotropic response to dihydroouabain was significantly increased after preincubation with Bay K 8644 (1 × 10−6 mol/l) in papillary muscles from both guinea-pig and human. In guinea-pig papillary muscles, the maximal inotropic response to dobutamine was not changed by preincubation with Bay K 8644 whereas in human papillary muscle strips, Bay K 8644 increased the inotropic response to dobutamine. 4. Bay K 8644 increased force of contraction (EC50, 4 × 10−8 mol/l) in human papillary muscle strips from patients undergoing mitral valve replacement. However, the maximal inotropic response to Bay K 8644 was reduced to 32 ± 4.4% that of calcium (15 mmol/l) measured in the same muscle strips. 5. A further reduction in maximal inotropic response to Bay K 8644 to 13 ± 1.2% that of calcium (15 mmol/l) with no change in potency was measured in human papillary muscle strips taken from terminally failing hearts of cardiac transplant recipients. 6. There was a significant correlation between the preoperative left ventricular ejection fraction and the maximal inotropic response to Bay K 8644 in isolated human papillary muscle strips. 7. These results suggest that Bay K 8644 affects excitation-contraction coupling of cardiac muscle so as to increase the maximal inotropic effect of the digitalis glycosides. Further, the inotropic response of human myocardial tissue to calcium channel activator Bay K 8644 may be reduced in states of pathological heart function.

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

SummaryThe sodium pump, (Na++K+)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured 3H-ouabain binding and (Na++K+)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. 3H-ouabain binds to one site of high affinity (KD1.9±0.2×10−9 mol/l) with a maximal binding capacity of 7.5±0.8×1014 binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na++K+)-ATPase activity (IC50 arachidonic acid: 7.5×10−5 mol/l, linoleic acid: 6.5×10−5 mol/l) and Mg2+-ATPase activity (IC50 arachidonic acid: 9×10−5 mol/l, linoleic acid: 4×10−5 mol/l). Arachidonic acid inhibited 3H-ouabain binding, (IC50 3.2×10−5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10−3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na++K+)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na++K+)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.

The inotropic effects of dopamine and its precursor levodopa on isolated human ventricular myocardium

SummaryThe direct positive inotropic effects of dopamine and its precursor, levodopa, were measured using isolated, contracting human papillary muscle strips taken from patients during mitral valve replacement. Levodopa did not produce any positive inotropic effect at concentrations up to 3×10−3 M. The positive inotropic effects of dopamine were observed at concentrations above 1×10−5 M with the maximal effect at 3×10−3 M — concentrations higher than those observed in therapy. This inotropic effect was reduced by the β1 antagonist, 1-practolol (1×10−6 M); the β2 antagonist, ICI 118,551 HCl (1×10−6 M); the dopamine antagonist, haloperidol (3×10−6 M); the neuronal uptake inhibitor, cocaine (3×10−5 M), but not by the α1, prazosin (1×10−7 M). This indicates that dopamine exerts its positive inotropic effects on human heart muscle mainly through release of noradrenaline, together with possible interactions at β-and dopamine-receptors. The maximal inotropic effect of dopamine was about 50% that of calcium (15 mM, 6.2±0.7 mN) or ouabain (1×10−7 M, 5.0±0.8 mN) when measured in the same muscle strips, possibly due to the reduced cardiac noradrenaline content together with the reduced β-receptor number in congestive heart failure. This concentration of ouabain (1×10−7 M) gave almost maximal inotropy without marked toxicity; when dopamine was then added, only toxicity developed without any further increases in force of contraction. Any haemodynamic benefits of dopamine therapy in optimally digitalis-treated patients are probably due to other cardiovascular effects such as vasodilatation.

Binding of dihydrodigitoxin to beef and human cardiac (Na+ + K+)-Atpase: Evidence for two binding sites in cell membranes

The specific binding of three cardiac glycosides, 3H-ouabain, 3H-digitoxin and 3H-dihydrodigitoxin, to beef cardiac (Na+ + K+)-ATPase was compared. Non-specific binding was defined as that in the presence of 0.1 mM unlabelled compound, or in the absence of ligands. The dissociation constants (KD-values) calculated from the inhibition of 3H-ouabain binding were: ouabain, 2.9 X 10(-9)M; digitoxin, 1.1 X 10(-9)M; and dihydrodigitoxin 2.7 X 10(-8)M. The concentrations which inhibited beef cardiac (Na+ + K+)-ATPase by 50% were: ouabain, 5.9 X 10(-9)M; digitoxin, 1.6 X 10(-9)M; and dihydrodigitoxin, 2.5 X 10(-8)M. Ouabain and digitoxin showed straight Scatchard plots for one site of high affinity (ouabain, KD = 2.6 X 10(-9)M; digitoxin, KD = 1.7 X 10(-9)M). However, dihydrodigitoxin gave a curved Scatchard plot. Analysis of this binding by the methods of M. J. Weidemann, H. Erdelt and M. Klingenberger (Eur. J. Biochem. 16, 313 (1970) for two binding sites gave the following results: for Mg2+,Pi-supported binding, the KD of the high affinity site was 1.6 X 10(-8)M with a capacity similar to that for ouabain of about 30 pmole/mg protein. For binding supported by Na+,ATP,Mg2+, the KD-value of the high affinity site was 5.3 X 10(-8)M of similar capacity. The low affinity binding site (KD = 4.0 X 10(-6)M for Mg2+,Pi; KD = 5.5 X 10(-6)M for Na+,ATP,Mg2+) bound about 350 pmole/mg protein. The low affinity site but not the high affinity site was also present in heat-denatured enzyme. Binding supported by Mg2+,Pi showed one low affinity site only for ouabain and dihydrodigitoxin in the presence of 200 mM Na+. The high affinity sites for these three cardiac glycosides were further characterized by measurement of the association and dissociation rate constants. The specific binding of 3H-ouabain and 3H-dihydrodigitoxin to human cardiac (Na+ + K+)-ATPase was measured. 3H-Ouabain showed a straight Scatchard plot for one high affinity site only (KD = 4.5 X 10(-9) M, capacity about 15 pmole/mg protein). 3H-Dihydrodigitoxin gave two binding sites: a high affinity site (KD = 1.8 X 10(-8) M) of similar capacity to ouabain, and a low affinity site (KD = 2.0 X 10(-6) M) of about 10-fold greater capacity.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-additive positive inotropic effects of amrinone and ouabain on cat papillary muscles.

SummaryAmrinone has been shown to produce haemodynamic benefits in digitalis-treated patients. Since amrinone is a positive inotropic agent on isolated heart muscle, these benefits may mean that amrinone increases the maximal ouabain-induced increase in force of contraction, without causing toxicity. We have therefore measured, in cat right ventricular papillary muscles, the inotropic effects of ouabain, amrinone alone and amrinone with a maximally effective, non-toxic ouabain concentration (2×10−7 M). Ouabain is much more potent than amrinone (EC50-values: ouabain, 8×10−8 M, amrinone, 1–2.8×10−3 M). The highest amrinone concentration used (6×10−3 M) produced a significantly lower increase in force of contraction than ouabain (2×10−7 M) in the same muscles. After ouabain (2×10−7 M) produced a stable effect, no further increase in force of contraction was observed with any amrinone concentration. Sustained arrhythmias were observed in five of six muscles at 3×10−3 M amrinone with ouabain (2×10−7 M), but in only one of these muscles with amrinone 3×10−3 M alone. Since the positive inotropic effects of amrinone are not additive with those from a maximally effective ouabain concentration, the haemodynamic benefits seen in patients are probably due to non-cardiac effects of amrinone such as vasodilatation.