Sodium flux and lipid spectrum in the erythrocyte membrane in essential hypertension.

In the erythrocyte membranes of normotensive individuals and patients with essential hypertension, sodium flux behavior, cholesterol content, phospholipid spectrum, and the fatty acid composition of the phospholipids were measured. There was no difference between normotensives and hypertensives in sodium influx in the erythrocyte. In both groups, 1 microM norepinephrine resulted in a significantly equal increase of sodium influx after an incubation period of 15 min. Compared with normotensives, erythrocytes in essential hypertensive patients showed a decreased sodium efflux. The cholesterol/phospholipid quotient of the erythrocyte membrane of hypertensive subjects increased in comparison with that of normotensive individuals. No differences between the two groups were found in the phospholipid pattern and in the fatty acid composition of phosphatidylserine/-inositol, -choline, and sphingomyelin. However, in phosphatidylethanolamine of the erythrocyte membrane in essential hypertensives in contrast to normals, a decreased content of the fatty acid fraction 22:0/20:3 and in increased content of the fatty acid fraction of 20:5/22:2/22:3 was found. The dependence of the Na turnover on the lipid spectrum of the erythrocyte membrane is discussed. The altered lipid composition of the erythrocyte membrane in essential hypertensives does not seem to cause the altered Na efflux behavior.

Na,K-ATPase in excitation-contraction coupling of vascular smooth muscle from cattle.

Lowering the extracellular K+ content from 6 to 0.6 mM causes a rise, and elevation from 6 to 8.5 mM a fall of 45Ca++ efflux from the vascular smooth muscle cells of the arteria carotis communis of cattle. In contrast, a level of 17 mM K+ has no influence. Removal of extracellular calcium does not block these effects. 10(-4) M ouabain also induces a rise in Ca++ efflux, additional potassium reduction then being without effect; 10(-9) M ouabain is of no influence. The 45Ca++ efflux kinetics correlates with the activity of the isolated Na,K-ATPase. Tonus increases of the vascular strips by 10(-4) M ouabain and potassium deficiency cannot be blocked by 4 mM lanthanum or removal of extracellular calcium. Unlike sodium, potassium stimulates the active Ca++ binding and the activity of the Ca-ATPase of the microsomal fraction. The ative Ca++ binding of the mitochondria is stimulated by both ions. It is postulated that the activity of the plasma membrane Na,K-pump is able to regulate the tonus of big arteries through alteration of Ca++ storage processes.

An ouabain-insensitive Na-ATPase of the arterial vascular muscle cell and its relation to ouabain-sensitive Na,K-ATPase.

A Na+-stimulated, Mg++-requiring ATPase (Na-ATPase), which is insensitive to ouabain, has been demonstrated in the carotis and coronary arteries of different species. In dependence on the sodium concentration half-maximal activities of Na-ATPase are found in the range from 16 to 24 mM Na+. A replacement of Mg++ by Ca++ leads to a partial loss of activity. It does not, however, change its sensitivity to sodium. Compared to Na,K-ATPase, the Na-ATPase shows a considerably lower sensitivity to calcium. p-Chloro-mercuribenzoate, N-ethylmaleinimide, chloropromazine, sodium fluoride, ethanol and sodium azide influence the activity of the Na-ATPase in a characteristic way corresponding to the reactivity of Na,K-ATPase. Noradrenaline and isoprenaline do not lead to any significant change of its activity. The possible separate existence of a Na-ATPase independent of Na,K-ATPase, as well as its potential importance for cellular metabolism are discussed.

Uptake of noradrenaline by human erythrocytes.

If human red cells are incubated with either noradrenaline or isoprenaline, these catecholamines are adsorbed to the cells surface first in a fast reaction. In a second, slower reaction they enter the cell membrane and are accumulated against the concentration gradient by the cells. It is supposed that the catecholamines enter the cells by simple diffusion and that they are reversibly bound to hemoglobin within the cells. It is possible that this binding plays an important role in the transport of catecholamines in the blood stream where, it is assumed, they are protected from oxidation.

[Demonstration of protein kinase activities in the coronary artery of cattle]. / Nachweis von Proteinkinase-Aktivitäten in der Arteria coronaria des Rindes

Protein kinase activities were identified in a soluble and a particulate fraction from the A. coronaria of cattle. For both protein kinase activities Mg++ is essential. Protamine was used as a substrate of the protein kinase activity of the soluble fraction. The pH optimum of the protein kinase activity of the soluble fraction is around 6.5. The Km-value of the protein kinase for ATP is 1.9 +/- 0.4 - 10(-5) M. cAMP stimulates the protein kinase activity more effectively than cGMP. Ca++ cannot replace Mg++; monovalent cations (Na+ and K+) show no influence. The protein kinase activity of the fraction was determined via endogenous phosphorylation. By means of the cAMP-dependent particulate protein kinase 72 to 80 percent of the serine residues are phosphorylated. The pH optimum of the protein kinase activity of the particulate fraction lies around 7.0. The Km-value of the enzyme for ATP is 6.6 +/- 0.8 - 10(-5) M. cGMP stimulates the protein kinase of the particulate fraction better than cAMP. For the protein kinase activity of this fraction Ca++ replaces Mg++ in the endogenous phosphorylation but not in the exogenous phosphorylation (protamine). In the presence of Mg++ and in the additional presence of Na+ or K+, the protein kinase activity is suppressed in the endogenous phosphorylation whereas it is stimulated in the exogenous phosphorylation.

The binding of noradrenaline to human erythrocytes.

When human erythrocytes are incubated in a suspension medium containing noradrenaline, they take up noradrenaline in a reaction proceeding in two phases. In a rapid first reaction noradrenaline is bound by the cells by adsorption at pH-values above 6.0; this reaction follows Freundlich's isotherms. Subsequently, noradrenaline is taken up much more slowly by transmembranous diffusion into the cells. Neither reaction can be influenced by inhibitors such as N-ethyl maleinimide, ouabain, alpha- or beta-receptor blockers.

[Proof of adenylate cyclase activity in the coronary artery of cattle]. / Nachweis von Adenylatzyklase-Aktivität in der Arteria coronaria des Rindes

In two fractions obtained from the bovine A. coronaria adenylate cyclase activity was identified and characterized. The adenylate cyclase activity of the 75,000 X g sediment shows a pH optimum at 7.4. The temperature dependence of this adenylate cyclase activity is linear when represented in the Arrhenius plot, and an Arrhenius activation energy of 13.2 kcal Mol-1 can be calculated for the enzyme reaction. The Km-value of the enzyme to ATP is 6 +/- 0.6 - 10(-4) M. The adenylate cyclase activity of the 75,000 X g sediment can be stimulated by NaF. 5'AMP and adenosine inhibit the adenylate cyclase activity of the 75,000 X g sediment. With regard to the enzyme activity, Mn++ and Co++ replace Mg++, but not Ca++. The monovalentcations Na+ and K+ do not influence the adenylate cyclase activity. In a particulate fraction containing plasma membranes, adenylate cyclase activity was also identified. This adenylate cyclase activity can be stimulated by catecholamines, noradrenaline, and isoproterenol. This stimulation can, however, only be proved for the enzyme in the coronaries of 9-week-old and 2-year-old animals. The adenylate cyclase activity from the coronaries of adult animals is not affected by catecholamines. These findings are discussed with regard to hypertension frequently found in adult animals.

Characterization of Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in smooth muscular cells of the sheep's common carotid artery.

The Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in the mitochondrial and microsomal fraction of smooth muscular cells of the sheep's common carotid artery have been characterized in more detail. Optimal enzyme activities were found for all ATPases to be at pH 7.5-8.0 and 45 degrees C-50 degrees C. The energies of activation were found to be at 5-9 kcal/mole for both ATPases. Two-thirds of the (Na+ + K+)-stimulated Mg-ATPase were found to be ouabain-sensitive and thus attributed to the coupled (Na, K)-transport system. The pI 50 values of ouabain for microsomal and mitochondrial fractions are 6.3 and 6.0, respectively. The highest activity of (Na+ + K+)-stimulated Mg-ATPase is at 5-10 mM K+ and more than 50 mM Na+. One-third of the (Na+ + K+)-stimulated Mg-ATPase activity was found to be due to a stimulation of Mg-ATPase by Na+ alone, which is not inhibited by ouabain. The relationship of this activity to the ouabain-sensitive part of the (Na+ + K+)-stimulated Mg-ATPase and to Na+-transport is discussed. For the Mg-ATPases apparent KM(ATP) values were determined to be 1.4 and 1.0 mM, resp., and for the (Na+ + K+)-stimulated Mg-ATPases 0.15 and 0.14 mM, resp.

Demonstration of a Mg-ATPase and a Na,K-ATPase from the arteria carotis communis of the sheep.

Besides the Mg-ATPase, a Na,K-ATPase can be demonstrated in different fractions of smooth muscles of the A. carotis communis of the sheep. The highest activity of Mg-ATPase is observed in the heavy microsomal fraction. The Ca-ion may act as a complete substitute for the Mg-ion in the Mg-ATPase. The proportion of Na,K-ATPase is between 10 and 40%, depending on the preparative conditions used in the individual fractions. Fractionated salt treatment (LiBr, KC1, KBr) improved the assay of Na,K-ATPase but increased strength of the Tris-HC1-buffer considerably reduced its activity.