Erythrocyte Na+,K+ cotransport and Na+,K+ pump in black and caucasian hypertensive patients

Alterations in red blood cell (RBC) Na+,K+ pump and in Na+,K+ cotransport (CoT) have been described in essential hypertension (EH). We examined pump and CoT in 50 normotensive (NT) subjects and 58 EH subjects subdivided by race and family history of hypertension (+ FH). RBCs were preloaded with Na+ to obtain intracellular levels of 25 mM/liter cells by using the p-chloromercuribenzene sulfonic acid (pCMBS) method. Na+ and K+ efflux rates into a magnesium-sucrose medium were quantitated in the presence of ouabain and ouabain plus furosemide to define pump and CoT activity respectively. Mean intracellular Na+ content was higher (p less than 0.05) in black NT and HT subjects compared to Caucasians. Mean RBC CoT was lower in black EH compared to NT and compared to Caucasian NT and HT subjects. Conversely, Caucasian HT patients had higher mean CoT than NT subjects. Subdivision into + FH revealed very little effect of + FH on CoT in black NT and HT subjects. In Caucasian NT and HT subjects with + FH, mean CoT was significantly reduced (less than 0.3 mM/liter cells/hr) compared to those without + FH. A subgroup of Caucasian EH subjects displayed high CoT (greater than 0.6 mM/liter cells/hr); a + FH had little impact on the high CoT group. There was no correlation between RBC CoT activity and age, sex, severity of hypertension, urinary sodium excretion, and plasma aldosterone. There was a positive correlation (r = + 0.47; p less than 0.01) between CoT and upright plasma renin activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Ume?-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Ume?-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.     

Na and K dependence of the Na/K pump in cystic fibrosis fibroblasts.

The Na and K dependence of the Na/K pump was measured in skin fibroblasts from patients with cystic fibrosis and age/sex-matched controls. Under basal conditions, there was no difference between control and cystic fibrosis cells in protein per cell, intracellular Na and K content, or Na/K pump activity (measured as ouabain-sensitive 86Rb uptake). There was no difference in the Na dependence of the Na/K pump between cystic fibrosis cells and control cells. In cells from patients with cystic fibrosis, the Na/K pump had a significantly lower affinity for K (Km = 1.6 mM) when compared to normals (Km = 0.9 mM). This difference was demonstrated by using two independent experimental designs.     

Angiotensin increases Na+ entry and Na+/K+ pump activity in cultures of smooth muscle from rat aorta.

Angiotensin markedly altered the Na+ permeability of smooth muscle cells cultured from explants of rat aorta. The rate of net Na+ uptake was followed in the presence of ouabain in order to block Na+ efflux via the Na+/K+ pump. Angiotensin II (AII) or angiotensin III (AIII) increased net Na+ uptake by about 3-fold. Maximal stimulation of Na+ uptake was produced by about 10 nM AII. Bradykinin and the angiotensin antagonist [Sar1, Ileu5, Ala8]AII had no significant effect on net Na+ uptake. Angiotensin also enhanced the activity of the Na+/K+ pump, which was assayed by following the rate of ouabain-sensitive 86Rb+ uptake by the cells. AII and AIII nearly doubled ouabain-sensitive 86Rb+ uptake, but bradykinin, norepinephrine, and [Sar1, Ileu5, Ala8]AII had no effect. In the presence of ouabain, 86Rb+ uptake was not significantly affected by AII or AIII, indicating that angiotensin did not alter passive permeability to Rb+. Loading the cells with Na+, either by incubation in K+-free medium or exposure to the Na+-selective ionophore monensin, markedly increased ouabain-sensitive 86RB+ uptake. This result indicates that the activity of the Na+/K+ pump is limited by the low level of Na+ that is normally in the cells. AII had no effect on the activity of the Na+/K+ pump in Na+-loaded cells. These results suggest that AII or AIII stimulates the Na+/K+ pump in cultured aortic muscle cells by increasing its Na+ supply.     

C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y

Summary This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na⁺, K⁺-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na⁺, K⁺-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na⁺ affinity of the enzyme and all subsequent studies were performed at non-saturating Na⁺ concentrations. C-peptide stimulation of Na⁺, K⁺-ATPase activity occurred in a concentration-dependent manner in the dose range 10^–8–10^–6 mol/l. The presence of neuropeptide Y, 5×10^–9 mol/l, enhanced this effect and stimulation of Na⁺, K⁺-ATPase activity then occurred in the C-peptide dose range 10^–11–10^–8 mol/l. C-peptide stimulation of Na⁺, K⁺-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca²⁺-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na⁺, K⁺-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca²⁺-dependent intracellular signalling pathways.Abbreviations PTX Pertussis toxin - NPY neuropeptide Y - PCT proximal convoluted tubule - BSA bovine serum albumin - dB cAMP dibutyryl cyclic adenosine monophosphate - PP2B Ca²⁺/calmodulin-dependent protein phosphatase 2B - PKC protein kinase C - [Ca²⁺] intracellular calcium concentration     

Regulation of endocytic pH by the Na+,K+-ATPase in living cells.

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.     

Potassium translocation by the Na+/K+ pump is voltage insensitive.

The voltage dependence of steady and transient changes in Na+/K+ pump current, in response to step changes in membrane potential, was investigated in guinea pig ventricular myocytes voltage clamped and internally dialyzed under experimental conditions designed to support four separate modes of Na+/K+ pump activity. Voltage jumps elicited transient pump currents when the pump cycle was running forward or backward, or when pumps were limited to Na+ translocation, but not when they were made to carry out K+/K+ exchange. This result indicates that K+ translocation involves no net charge movement across the membrane field and is therefore voltage insensitive. The transient pump currents seen during Na+/K+ transport demonstrate that both forward and reverse pump cycles are rate limited not by the voltage-dependent step but by a voltage-independent step, probably K+ translocation. These findings severely constrain kinetic models of Na+/K+ pump activity.     

Altered erythrocyte Na+ + K+ pump in adolescent obesity

The number of $\text{Na}\text{K}$ pump units and the cation transport activity of the pump were measured in erythrocytes from two etiologically different groups of obese adolescents and a group of normal controls. There was a significant reduction in the number of pump units, as measured by saturation ouabain binding, in erythrocytes from adolescents with idiopathic, early onset obesity. Individuals whose obesity developed subsequent to the appearance of a variety of hypothalamic lesions showed no reduction in the red cell complement of $\text{Na}\text{K}$ pump when compared to controls and the cation transport activity of their cells was higher than both the controls and the subjects with idiopathic obesity. These results support data obtained in adults that reduced red cell $\text{Na}\text{K}$ pump levels are seen in a group of individuals with idiopathic obesity. They further suggest that such reductions are not likely to be secondary to the obese state per se.     

Serum rapidly stimulates ouabain-sensitive 86-RB+ influx in quiescent 3T3 cells.

Serum causes a 4-fold increase in 86Rb+ (a K+ tracer) influx in quiescent 3T3 mouse fibroblast cells. It is one of the earliest changes caused by serum, being seen in 2 min and reaching a maximum in 10 min. Removal of serum causes rapid reversal of this effect. Serum acts mainly by increasing the maximum velocity, Vmax, of entry. Ouabain inhibits entry of 86Rb+ (82-90%) both in the presence and absence of serum, but does not alter exit. The rapid increase in cation influx is unaffected by cycloheximide and by changes in cyclic AMP and GMP. Low concentrations of insulin, epidermal growth factor, and prostaglandins (E1 and F2alpha) produced a smaller (80%) activation of 86Rb+ entry. Ouabain, at a level that inhibits cation influx, also prevents the onset of DNA synthesis following serum addition; this is reversible effect dependent on the concentration of K+ in the medium. This suggests that cation pumping activity may be required for initiation of DNA synthesis.     

心肌梗死后心肌钾、钠和钙离子浓度变化及其意义

目的　探讨心肌梗死后心肌重塑过程中K+ 、Na+ 和Ca2 + 离子浓度变化及可能意义。方法　Wistar大鼠 18只 ,随机分为 3组 ,分别为假手术组、梗死后 3h组和梗死 2 1d组 ,结扎左冠状动脉前降支建立心肌梗死模型 ,其中假手术组不结扎冠状动脉。心肌总K+ 、Na+ 和Ca2 + 离子浓度的测定采用原子吸收光谱法。结果　①心肌梗死后 3h时心肌总K+ 离子浓度开始下降 ,但与假手术组比较无显著性差异 (P >0 0 5 ) ;在心肌梗死后 2 1d时 ,心肌总K+ 离子浓度明显下降 ,与假手术组比较有显著性差异 (P <0 0 5 ) ;②心肌梗死后 3h时心肌总Na+ 离子浓度开始显著增加 ,与假手术组比较有非常显著性差异 (P <0 0 1) ;梗死后 2 1d时心肌总Na+ 离子浓度仍保持较高水平 ,与假手术组比较仍有显著性差异 (P <0 0 5 ) ;③心肌梗死后 3h时心肌总Ca2 + 离子浓度就开始增加 ;梗死后 2 1d时心肌总Ca2 + 离子浓度仍保持较高水平 ,与假手术组比较有显著性差异 (P <0 0 5 )。结论　心肌梗死3h时 ,心肌总K+ 离子浓度开始下降 ,总Na+ 离子和Ca2 + 离子浓度开始显著增加 ;在梗死后 2 1d时 ,心肌总K+ 离子浓度继续下降 ,而总Na+ 和Ca2 + 离子浓度仍保持较高水平。这些变化可能导致心肌梗死后的心肌电激动的各向异性而易致室性心律失常     

Inhibition of Na+, K+-ATPase Activity by the Metabolites Accumulating in Homocystinuria

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na⁺, K⁺-ATPase, and Mg²⁺-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na⁺, K⁺-ATPase but not Mg²⁺-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na⁺, K⁺-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na⁺, K⁺-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.     

Multiple genes encode the human Na+,K+-ATPase catalytic subunit.

A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms.     

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.     

Erythrocyte Na+,K+-ATPase and nasal potential in pseudohypoaldosteronism

OBJECTIVES: Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disorder characterized by salt-wasting due to target organ unresponsiveness to mineralocorticoids. PHA1 comprises two clinically and genetically distinct entities; isolated renal and systemic forms. DESIGN: The aim of this study was to investigate red blood cell (RBC) Na+,K+-ATPase activity and nasal potential difference (PD) in two pairs of unrelated dyzygous twins; one with the systemic form of the disease (PHA1-S) and the second with the isolated renal form (PHA1-R). Total and ouabain-sensitive ATPase activities were measured spectrophotometrically by a method that couples ATP hydrolysis with NADH oxidation. Maximal PD and response to amiloride perfusion were evaluated by a standard technique. RESULTS: In the twins with PHA1-S, persistently low activity of RBC Na+,K+-ATPase was found during a 6-year follow-up. Normalization of plasma renin activity (PRA) and plasma aldosterone was observed at the end of the first year of life. Maximal nasal PD was low and there was no significant response to amiloride. In the twins with PHA1-R, RBC Na+,K+-ATPase activity was very low at the time of diagnosis and normalized at the age of 6-8 months. PRA reverted gradually to normal values, whereas aldosterone levels remained high during the 6 years of follow-up. Maximal nasal PD and response to amiloride were normal. CONCLUSIONS: The observed differences in RBC Na+,K+-ATPase activity and nasal PD response to amiloride between the two pairs of twins support the contention of different basic pathogenic mechanisms in the two forms of PHA1.     

Changes in intracellular Na+ during Na,K pump inhibition in sheep cardiac tissues.

The differences in inotropic and toxic sensitivities of cardiac ventricular tissue to cardiac glycosides were investigated in order to determine whether or not the reportedly greater sensitivity of Purkinje fibers compared to myocardium is the result of a fundamentally different response to Na,K pump inhibition. I measured the changes in intracellular Na+ activity (aiNa) in the two tissue types simultaneously during exposure to actodigin (4.0 microM) and ouabain (0.5 microM) under quiescent conditions. A sheep papillary muscle and a Purkinje fiber from the same heart were placed in an experimental chamber and measurements of aiNa from both were obtained with Na+ -sensitive microelectrodes. In five experiments in which all electrode impalements were successfully maintained, actodigin caused similar changes in aiNa in the two tissues (from 7.2 +/- 1.0 mM in control to 12.9 +/- 1.9 mM in the presence of drug in papillary muscles compared to 7.3 +/- 0.3 mM in control and 13.2 +/- 1.0 mM in Purkinje fibers; means +/- S.E.M.). After washout, exposure to ouabain increased aiNa in both papillary muscles and in Purkinje fibers (from 7.2 +/- 0.7 mM in control and 16.2 +/- 1.4 mM during exposure to drug in papillary muscles compared to 7.4 +/- 0.3 mM and 14.9 +/- 0.8 mM in Purkinje fibers). In fact, ouabain caused a greater increase of aiNa in papillary muscles than in Purkinje fibers (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte ghost Na+,K+-ATPase and blood pressure

To examine the relationship between body mass index, blood pressure, and the Na+,K+-adenosine triphosphatase (ATPase) system, we measured the erythrocyte ghost Na+,K+-ATPase and the erythrocyte Na+ concentration in 120 blacks and 127 whites (136 males and 111 females). Blacks showed a 13.9% higher erythrocyte Na+ (7.63 +/- 0.19 vs 6.70 +/- 0.11 [SEM] mEq/L; p = 0.0001) and a 16.1% lower erythrocyte ghost Na+,K+-ATPase activity (140.3 +/- 4.2 vs 167.3 +/- 4.7 nmol inorganic phosphate/mg protein/hr; p = 0.0002) than whites. Male subjects demonstrated a 6.4% higher erythrocyte Na+ (7.35 +/- 0.17 vs 6.91 +/- 0.14 mEq/L; p = 0.043) and an 11.5% lower Na+,K+-ATPase activity (145.7 +/- 3.7 vs 164.7 +/- 5.5 nmol inorganic phosphate/mg protein/hr; p = 0.0015) than female subjects. Significant (p less than 0.001) negative correlations were identified for the systolic, diastolic, and mean blood pressure levels and the erythrocyte ghost Na+,K+-ATPase. These findings were complemented by positive correlations for the blood pressure levels and erythrocyte Na+ concentrations. The body mass index was negatively correlated with erythrocyte ghost Na+,K+-ATPase and it accounted for 6.7%, 5.6%, and 6.1% of the variabilities in the systolic, diastolic, and mean blood pressure levels, respectively. Variabilities of 1.4% systolic, 12.3% diastolic, and 11.1% in mean arterial pressure were attributable to the erythrocyte ghost Na+,K+-ATPase activity. Provided that findings in erythrocytes also reflect the relative status of the vascular smooth muscle cell Na+,K+-ATPase, the predisposition of black, male, and obese persons to hypertension may relate, among other factors, to a lower activity of this enzyme system, which results in an increased vascular tone.     

Changes of extracellular Na+, K+, Ca2+ and H+ of the ischemic myocardium in pigs

Summary In open-chest pigs during severe myocardial ischemia [K⁺]e, [Ca²⁺]e and [H⁺]e increase, [Na⁺]e increases transiently reaching control values after 30 min. Extracellular osmolality of the ischemic area increases due to an H₂O-shift from the ECS to the ICS. The increase of [Na⁺]e and [Ca²⁺]e must be explained by the shrinkage of the ECS due to the H₂O-shift. The increase of [Ca²⁺]e is additionally caused by the decrease of pHe. The increase of [K⁺]e is mainly caused by the release of K⁺ from the ICS. The changes of [K⁺]e and [K⁺]i cause a decrease of the membrane potential to a range in which slow response potentials and re-entry excitations can occur. The increase of [K⁺e therefore seems to be a major factor to cause early post-ischemic arrhythmias.Supported by Deutsche Forschungsgemeinschaft, SFB 68, A 7     

Peroxynitrite induced decrease in Na^＋, K^＋-ATPase activity is restored by taurine

AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed to ONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured. RESULTS: Different concentrations of ONOO- (100, 200, 500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO- to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.     

Erythrocyte Na+, K+-ATPase activity in patients with congestive heart failure.

In order to determine if the Na+, K+-ATPase activity in erythrocyte membranes is altered in congestive heart failure, and to examine its clinical significance with respect to other clinical variables, erythrocyte Na+, K+-ATPase activity was measured in 51 patients with left ventricular ejection fractions <40% (coronary artery disease, n=26; dilated cardiomyopathy, n=25) and 24 control patients. Na+, K+-ATPase activity was lower in both coronary artery disease and dilated cardiomyopathy groups than control group even in the absence of digitalis use. There was a significant inverse correlation between Na+, K+-ATPase activity and plasma norepinephrine. The presence of non-sustained ventricular tachycardia was associated with a lower Na+, K+-ATPase activity in both groups with congestive heart failure without digitalis use than those without ventricular tachycardia. Plasma norepinephrine was higher in patients with non-sustained ventricular tachycardia than those without in the coronary artery disease group, but not in the dilated cardiomyopathy group. Na+, K+-ATPase activity may be helpful in predicting electrophysiologic instability in patients with heart failure.     

Cytochemically assayable Na+,K+-ATPase inhibition by Milan hypertensive rat plasma

The ability of plasma from 3- and 9-week-old Milan hypertensive rats and their normotensive controls to inhibit Na+,K+-adenosine triphosphatase (ATPase) was studied using cytochemical bioassay techniques in fresh tissue. With a validated cytochemical bioassay that measures the capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase activity in guinea pig proximal tubules as an indication of their capacity to inhibit Na+,K+-ATPase, the mean glucose-6-phosphate dehydrogenase-stimulating ability of the plasma of the 9-week-old Milan hypertensive rats and their normotensive controls was 586.0 +/- 88 and 23.4 +/- 8.3 U/ml (n = 7; p less than 0.001), while that of the 3-week-old Milan hypertensive rats (before the main rise in arterial pressure) and their normotensive controls was 99.9 +/- 27.4 and 7.8 +/- 1.8 U/ml (n = 7; p less than 0.001). With the use of a semiquantitative cytochemical assay that measures Na+,K+-ATPase activity directly, plasma from the adult hypertensive rats had a much greater capacity to inhibit Na+,K+-ATPase than the plasma of the control rats. The significantly raised levels found in the young hypertensive rats before the main rise in arterial pressure are consistent with the hypothesis that the rise in the ability of plasma to inhibit Na+,K+-ATPase is due to an inherited renal difficulty in excreting sodium.