High Affinity Ouabain Binding Sites Research Articles

Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87).

The activity of arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-controlling enzyme in melatonin synthesis is stimulated approximately equal to 100-fold by an adrenergic cyclic AMP mechanism in both neonatal and adult rat pineal glands. This stimulation is blocked in the adult gland by the depolarizing agents ouabain (1 microM) and K+ (80 mM) (Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H. (1975) Mol. Pharmacol. 11, 241-255). In the present study pineal glands obtained from prenatal to adult rats were used; it was found that K+ (80 microM) inhibited the adrenergic stimulation of N-acetyltransferase activity at all ages but that ouabain (1 nM to 1 mM) treatment was not inhibitory early in development. In contrast, in the neonate, ouabain (1-100 nM) enhanced adrenergic induction of N-acetyltransferase activity, and ouabain treatment alone (1-1000 nM) stimulated N-acetyltransferase activity. A small stimulation was also seen at one concentration (1 nM) in the adult. Analysis of the development of high affinity ouabain binding sites and Na+,K+-ATPase activity in the intact pineal gland indicated that the developmental pattern of both resemble the development of ouabain inhibition of the adrenergic stimulation of N-acetyltransferase activity. All are low for the first few days of life, gradually increase during the next 3 weeks of life, and then approach adult levels. Similarly, ouabain (1 nM to 1 mM) had no effect on 86Rb uptake in the 2-day-old gland but blocked (IC50 congruent to 20 nM) 86Rb uptake in the adult gland. These findings indicate ouabain probably has little inhibitory effect on the norepinephrine stimulation of N-acetyltransferase activity in the neonatal because a high affinity ouabain binding form of Na+,K+-ATPase activity, similar to the alpha + form identified in rat brain, is at very low levels in the pinealocyte. Accordingly, it appears that an ouabain-insensitive mechanism in the neonatal gland maintains membrane potential and that this mechanism plays a less important role in the adult. The explanation of why ouabain alone stimulates N-acetyltransferase activity and why it enhances the effects of norepinephrine in the neonatal pineal gland might be that ouabain acts on surviving neural elements present in the gland to cause the net release of a transmitter, perhaps norepinephrine, which then stimulates N-acetyltransferase activity.

Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk-cells with DNA encoding the beta-subunit of an avian brain sodium pump.

A cDNA encoding the beta-subunit of the (Na+ + K+)-ATPase was cloned from a chicken brain cDNA library, and its nucleotide sequence was determined. High cross-species sequence homologies were found both in coding and noncoding regions. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk-cells. The avian beta-subunit was expressed on the cell surface (1-8 X 10(5) molecules/cell) complexed with alpha-subunits of the murine (Na+ + K+)-ATPase. In the hybrid system there was rapid assembly of subunits, post-translational N-glycosylations of the beta-subunit at its three Asn-X-Ser (or Thr) positions, and modification of high mannose oligosaccharides to complex type. Avian beta-subunits expressed in the mouse cells had an apparent molecular weight of about 55,000 as compared with 47,000 in avian cells, due to post-translational modifications, presumably differences in complex oligosaccharides. Despite the high number of interspecies hybrid (Na+ + K+)-ATPase molecules, the cells had none of the high affinity ouabain binding sites (KD = 2 X 10(-7) M) characteristic of avian cells, consistent with the view that the ouabain binding site is located largely or exclusively on the alpha-subunit and is not greatly affected by alpha-beta interaction.

High affinity and low affinity ouabain binding sites in the rat heart

Ventricular muscle of rat heart has two classes of receptors which are responsible for the positive inotropic effect of ouabain. Low affinity receptors are apparently related to Na +, K +-ATPase. To determine if high affinity receptors are also sarcolemmal Na +, K +-ATPase of muscle cells, their characteristics were examined. Binding of [ 3H]ouabain to the high affinity binding site required ATP in the presence of Mg 2+ and Na +, was stimulated by Na + in the presence of Mg 2+ and ATP, and was inhibited by K +. Digoxin, digitoxin and cassaine all inhibited [ 3H]ouabain binding to the high affinity site. Cassaine was about an order of magnitude less potent than the glycosides. These results indicate similarities in high affinity ouabain binding sites in ventricular muscle of rat heart and Na +, K +-ATPase obtained from other sources. Destruction od sympathetic nerve terminals with 6-hydrodopamine failed to affect the high affinity ouabain binding sites indicating that high affinity sites do not represent the Na +, K +-ATPase in sympathetic nerve terminals. Labeling of Na +, K +-ATPase from [γ- 32P]ATP indicates that high affinity ouabain binding sites account for 25% of the total enzyme molecules present in ventricular muscle of rat heart.

The inotropic effect of ouabain and its antagonism by dihydroouabain in rat isolated atria and ventricles in relation to specific binding sites.

The inotropic effect of ouabain has been studied in rat ventricles and atria. The concentration-effect curve of ouabain may be fitted by a model assuming the existence of two saturable components. The component with the higher sensitivity to ouabain accounted for 30% of the maximal increase in systolic tension in ventricles and for only 5% in atria. Increase in diastolic tension was only apparent at ouabain concentrations required to observe the low sensitivity component. [3H]-ouabain binding has been examined in microsomes prepared from atria and ventricles. High and low affinity binding sites have been observed. The ratio of high and low affinity ouabain binding sites was 4 fold lower in microsomes from rat atria than from rat ventricles. This could account for the difference in the response of these two tissues to the inotropic action of ouabain. In ventricular strips the high sensitivity component was much less apparent in the presence of dihydroouabain than with ouabain. When ventricular strips were preincubated in the presence of dihydroouabain 3 microM, the increase in systolic tension evoked by ouabain 1 microM was significantly reduced. Cumulative concentration-effect curve studies showed dihydroouabain antagonism to the high sensitivity component.

Brain (Na +,K +)-ATPase: Biphasic interaction with erythrosin B

Brain (Na +,K +)-adenosine triphosphatase (EC 3.6.1.3) has both high and low affinity ouabain binding sites. It has been proposed that the high affinity ouabain binding sites characterize a nerve-specific form of the enzyme. Erythrosin B has been reported to inhibit high affinity ouabain binding selectively. The experiments in this paper were carried out in order to characterize the interactions of erythrosin B with (Na +,K +)-ATPase and to examine the specificity of erythrosin B for enzyme with high affinity for ouabain. Inhibition by erythrosin B was biphasic, with a rapid and a slow phase. The rapid phase appeared to be relatively specific for enzyme with high affinity for ouabain, while the slow phase was not. Inhibition by erythrosin B was accelerated by Mg 2+ and was retarded by ATP, K +, or Na + and ATP. Erythrosin B increased apparent affinity of the enzyme for K + and decreased apparent affinity for Na + and for ATP. These results indicate that erythrosin B interacts with an ATP site and has effects on cation affinities opposite to those of ATP. Erythrosin B inhibition is proportional to high affinity ouabain binding if brief incubation times and moderate concentrations are used.

High-affinity ouabain binding site and low-dose positive inotropic effect in rat myocardium.

The mechanism of the positive inotropic effect of digitalis glycosides remains unclear. One theory suggests a causal relationship between the binding to and consequent inhibition of (Na++K+) ATPase (the sodium pump) by digitalis and an increased myocardial contractile force1–6. By this mechanism, the increased force of contraction would occur secondary to an elevation of intracellular sodium concentration which then causes an increased intracellular concentration of calcium via a sodium–calcium exchange mechanism4–6. Another theory proposes that the binding of digitalis to (Na++K+) ATPase causes an increase in a sarcolemmal calcium pool6–9, suggesting that the inotropic effect could be due to a causal relationship between the formation of the digitalis–(Na++K+)ATPase receptor complex and increased myocardial calcium availability and utilization, exclusive of an inhibition of the sodium pump7–9. We now report that two distinct positive inotropic sites for ouabain exist in rat ventricular strips. The higher-affinity response (ED50 = 0.5 µM) correlates with an apparent high-affinity site which can be detected by 3H-ouabain binding to intact rat ventricular myocytes. These higher-affinity sites do not correlate with concentrations (IC50) of ouabain necessary to inhibit (Na++K+)ATPase activity of sarcolemma preparations prepared from rat ventricles, suggesting that in the rat ventricle the high-affinity site for the inotropic effect of ouabain may not be related to inhibition of (Na++K+)ATPase. The low-affinity site is, however, related to inhibition of this enzyme.