Activation of sodium transport in human erythrocytes by beta-adrenoceptor stimulation in vivo.

Beta-adrenoceptor stimulation in vivo shifts potassium into the cells. To examine whether human erythrocytes participate in this process, we measured, along with serum or plasma potassium, the concentrations of potassium and sodium in erythrocytes. Beta-adrenoceptor stimulation was obtained by infusion of either fenoterol or hexoprenaline into 6 volunteers at rest or by endogenous amines provoked in 14 volunteers during ergometric exercise. Metabolic effects were followed at rest on serum insulin, C-peptide, and growth hormone levels, and during exercise on pH on lactate concentration in blood. The potassium concentration (mean +/- S.E.M.) dropped (p less than 0.01) in serum from 4.64 +/- 0.37 to 3.19 +/- 0.43 mmol x l-1 in the first hour at rest and in plasma from 5.70 +/- 0.93 to 4.63 +/- 0.45 in 90 sec directly after exercise. The concentration of erythrocyte sodium dropped (p less than 0.001) from 9.68 +/- 0.73 to 8.81 +/- 0.62 mmol x l-1 in cells and from 9.62 +/- 1.16 to 8.55 +/- 1.24 during exercise for 90 s, respectively. Changes in the concentration ratio of cellular sodium to potassium confirmed this sodium shift. An increased sodium transport in erythrocytes due to beta-adrenoceptor stimulation in vivo appears to complement a shift of serum potassium into the cells and may be mediated by the membrane-bound sodium, potassium ATPase.

Ferritin in erythrocytes and plasma of patients with iron overload.

Erythrocyte and plasma ferritin was followed in 13 patients with iron overload undergoing phlebotomies for at least 6 months in comparison with untreated patients and normal males. Plasma ferritin was widely scattered with an average of only twice the normal, whereas erythrocyte ferritin was highly elevated to about twelve times the normal (p less than 0.0001). - The time course of plasma and erythrocyte ferritin during phlebotomy therapy was analyzed in 3 patients with idiopathic hemochromatosis. Three stages were established: 1. plasma ferritin dropped gradually into the normal range while erythrocyte ferritin remained high, 2. appropriate phlebotomies maintained normal plasma ferritin and high erythrocyte ferritin, and indicated a monthly uptake of dietary iron of 150-200 mg at a steady state, 3. at low plasma ferritin levels, erythrocyte ferritin was rapidly decreased by further intensive phlebotomy therapy. Based on the presumed net removal of iron, 1 microgram/l plasma ferritin was equivalent to 3-6 mg of body iron and 1 microgram/l erythrocyte ferritin to somewhat less than 1 mg of body iron. - An elevated erythrocyte ferritin during phlebotomy therapy in iron overload not only depends on body iron stores like plasma ferritin but may also be regulated by the activity of erythropoiesis.

Transfer of Tris buffer and effects on K+ loss in human red blood cells and reconstituted ghosts.

The rate of volume changes of human red blood cells in the presence of Tris-HCl is pH-dependent. At 37 degrees C, t 1/2 is 25-30 min at pH 7.4 and 10-20 min at pH 8.4. Hemolysis in Tris-HCl is delayed by H2DIDS but is promoted by low concentrations of bicarbonate. This bicarbonate effect has been reversed by inhibiting carbonic anhydrase with acetazolamide. K+ loss of red blood cells is increased at 37 degrees C in isotonic NaCl solutions containing in addition Tris-HCl. This Tris effect is enhanced from pH 6.4 to 8.4. At pH 8.4 K+ loss is stimulated about 3-fold by addition of 160 mM Tris-HCl. The onset of the Tris effect is delayed at pH 7.4 and below, but not at pH 8.4. Such a delay is absent after preincubation of the cells with Tris-HCl. After binding H2DIDS to red cells, no Tris-dependent increase of K+ loss has been observed. K+ loss of reconstituted red cell ghosts with equal internal and external chloride concentrations remained unaffected by Tris-HCl added to the external solution. In ghosts containing sucrose for isotonicity instead of choline chloride K+ loss is smaller but is stimulated by Tris-HCl approaching the rate in those ghosts with equal internal and external chloride concentrations. The transfer of Tris-HCl into red blood cells depends on the pH and on the chloride shift. As there is evidence that Tris-HCl raises the intracellular pH and reduces the Donnan potential at the membrane, K+ loss of red cells may be increased following an intracellular buffer interaction of hemoglobin and Tris-HCl.

Erythrocyte and plasma ferritin in normal subjects, blood donors and iron deficiency anemia patients.

Ferritin concentration has been determined with an immunoradiometric assay in plasma and washed sedimented erythrocytes after hypotonic lysis. There was a gradual decrease of plasma ferritin in the sequence normal males, normal females, blood donors and patients with iron deficiency anemia. Erythrocyte ferritin remained unchanged in normal males and females and in blood donors, but dropped significantly in the anemic patients. Correspondingly, the ratio of erythrocyte to plasma ferritin rose from less than 2 in healthy males up to 8 in persons with iron deficiency. Little, if any effect on plasma and erythrocyte ferritin was observed in 12 male and female volunteers when taking iron for 4 weeks. In 2 patients with iron deficiency anemia the blood counts were normalized within 2-3 months during oral iron substitution, accompanied by a drastic increase of the erythrocyte ferritin concentration to values far above normal. In contrast, the plasma ferritin concentration remained below normal. Thus, in iron deficiency erythrocyte ferritin is synthesized with priority in the presence of iron and, in addition to plasma ferritin, appears to be a useful parameter of the iron status.

[The problem of the cellular receptor for cardiac glycosides (author's transl)]. / Das Problem des zellulären Herzglykosidrezeptors.

This review concerns the Na+, K+ -ATPase as well as the Na+, K+ -pump in the intact membrane and the highly specific inhibition of this transport system by cardiac glycosides. The interaction between glycoside and enzyme and the regulation of the kinetics of glycoside binding by ATP, K+, Na+, Mg2+ and Ca2+ are described. Emphasis is placed on the significance of the Na+, K+ -pump as the pharmacological receptor for cardiac glycosides. The problem encountered and progress made in attempting to correlate the inotropic action of cardiac glycosides with the binding of these drugs to the heart muscle and with the inhibition of the Na+, K+ -pump are reported. Recent results concerning increases of the intracellular Na+ concentration which are obtained by a partial inhibition of the Na+, K+ -pump and which are followed by an elevation of the intracellular Ca2+ -activity are reviewed. The discovery of a digitalis-like endogenous activity corresponds to the high specificity of the receptor for cardiac glycosides.

The current concept for the cardiac glycoside receptor.

This brief review emphasizes the significance of the Na+,K+-ATPase or the Na+,K+ pump of the intact membrane as the pharmacological receptor for cardiac glycosides. The properties of transport enzyme and the regulation of glycoside binding are described. An outline is given of the problems encountered and of the progress made in attempting to correlate the inotropic action of cardiac glycosides with the binding of these drugs to the heart muscle and with the inhibition of the Na+,K+ pump. Furthermore, the correlation of intracellular Ca2+ activity an Na+ concentration with the inhibition of the Na+,K+ pump is discussed. The existence of a digitalis-like endogenous activity may also indicate an important role of the Na+,K+ pump as a receptor for a physiological regulatory control of cardiac contractility.

Comparison of the side-dependent effects of Na and K on orthophosphate-, UTP-, and ATP-promoted ouabain binding to reconstituted human red blood cell ghosts.

This paper is concerned with analyzing the sidedness of action of various determinants which alter the rate of ouabain binding to human red blood cell ghosts. Thus, ouabain binding promoted by orthophosphate (Pi) and its inhibition by Na are shown to be due to inside Pi and inside Na. External K inhibits Pi-promoted ouabain binding and Nao acts to decrease the effectiveness of Ko. Similarly, inside uridine triphosphate (UTPi) stimulates the rate of ouabain binding which can be antagonized by either Nai or Ko acting alone. The actions of Nai and Ko are different when ouabain binding is promoted by Pi and UTPi compared to inside adenosine triphosphate (ATPi). With ATPi, the ouabain binding rate is only affected when Nai and Ko are both present. Possible differences in the mechanism of action of K and Na on Pi-and UTP-promoted binding are discussed in the light of their sidedness of action.

Effects of Mg and Ca on the side dependencies of Na and K on ouabain binding to red blood cell ghosts and the control of Na transport by internal Mg.

The effect of alteration in the concentration of internal Mg on the rate of ouabain binding to reconstituted human red blood cell ghosts has been evaluated as well as the effect of Mgi on Na:Na compared to Na:K exchange. It was found that the dependence of the rate of ATP-promoted ouabain binding on the combined presence of Nai and Ko which occurs at high [Mg]i is lost when the concentration of Mgi is lowered. The sensitivity of the external surface for Ko is also changed since Ko can now inhibit the ouabain binding rate in the absence of Nai; on the other hand Nao at low [Mg]i can stimulate ouabain binding indicating that the relative affinity of the outside surface for Nao has either increased or that for Ko has decreased or both. Thus the effects of changes in [Mg]i result in a change in the side-dependent actions of Na and K and emphasize the possible difficulties of interpreting results obtained on systems lacking sidedness. Mgi was found to be required for Pi-promoted ouabain binding and that the inhibitory action of Nai increased as [Mg]i was increased. In addition, Ca was found to be most effective in inhibiting the rate of ATP-promoted ouabain binding when Na and K were present together than when either was present alone. Na:K exchange was found to be more sensitive to the concentration of Mgi than Na:Na exchange; at low [Mg]i Na:K exchange could be stimulated without changing the extent of Na:Na exchange. These results are consistent with the idea that conformational states of the pump complex are directly influenced by [Mg]i.

Side-dependent effects of internal versus external Na and K on ouabain binding to reconstituted human red blood cell ghosts.

The side-dependent effects of internal and external Na and K on the ouabain binding rate, as promoted by inside MgATP, has been evaluated utilizing reconstituted human red blood cell ghosts. Such ghost systems provide the situation where [Na]i, [K]i, [Na]o, and [K]o can each be varied under conditions in which the others are either absent or fixed at constant concentrations. It was found that, in the presence of Ko, increasing either [Na]i or [K]i resulted in decreasing the rate at which ouabain was bound. Changes in [Na]i or [K]i in the absence of Ko were without effect on the ouabain binding rate. Thus, the ouabain binding rate was found to vary inversely with the rate of Na:K and K:K exchange but was independent of the rate of Na:Na exchange. The effect of Ko in antagonizing ouabain binding, as well as the influence of Nao on this interaction, were found to require the presence of either Nai or Ki. The results are interpreted in terms of a model relating the availability of the ouabain binding site to different conformational states of the pump complex. Differences were observed in the ouabain binding properties of red cell ghosts compared to microsomal preparations but it is not known whether the basis for the differences resides in the different preparations studied or in the lack of control of sidedness in the microsomal systems.