Evaluation of sodium and potassium pump activity and number in diabetic erythrocytes

The Na+,K+-ATPase (= Na pump), which produces the concentration gradient of Na+ and K+ across the cell membrane, was studied in diabetic erythrocytes. The activity and number of Na pumps, which are functional and quantitative expression of the Na+,K+-ATPase in erythrocytes, were measured by ouabain-sensitive 42K or 43K influx and by [3H]ouabain binding, respectively. The turnover rate of the pumps was calculated from the two measurements to evaluate in situ activity of the pump. The Na pump activity was found to be higher in the diabetic (193 +/- 12 nmol K+/h . 10(9) cells) than in the normal group (164 +/- 6) (P less than 0.05), though the affinities of the pump for extracellular K+ were not different. The number of Na pumps and the Kd of the pumps for ouabain in the diabetic group were not significantly different from those in the normal group (354 +/- 12 site/cell and 4.33 +/- 0.20 nM). The turnover rate of the diabetic group tended to be higher than that of the normal group. The rate was about one third of the molecular activity reported for Na+,K+-ATPase under optimum conditions. These results indicate that the enzymatic properties as well as the number of Na pumps were not altered in diabetic erythrocytes despite the increased Na pump activity. Therefore the increased activity of the erythrocyte Na pump in diabetes mellitus suggests an increase of cation permeability associated with a possible disorder in the diabetic membrane.     

Univalent cation transport in human and rat erythrocytes: its regulation by protein kinase activators and compression

The effect of the activators of protein kinase A (dibutyryl-cAMP) and protein kinase C (beta-phorbolic ether), as well as cell compression, on the rate of 22Na and 86Rb, a radioactive potassium analogue, incorporation by human and rat erythrocytes was investigated. Protein kinase A and protein kinase C activation was accompanied by the activation of Na+, K+-ATP-ase in human and rat erythrocytes as well as increased Na+, K+ cotransport rate in rat erythrocytes. Human erythrocytes responded to protein kinase C activation by a 2 or 3-fold increase in Na+/Na+-antitransport rate, and both human and rat erythrocytes exhibited a manifold increase in the Na+/H+ metabolism rate. Cell compression depressed Na+, K+-ATP-ase activity and increased the rates of Na+/H+ metabolism and the frusemide-inhibited component of potassium transport, the latter two effects being particularly obvious in rat erythrocytes. It is suggested that protein kinase C activation and/or erythrocyte compression may be a direct cause of increased plasmatic membrane permeability for univalent cations in primary hypertension.     

Erythrocyte thermogenesis in hyperthyroid patients: microcalorimetric investigation of sodium/potassium pump and cell metabolism

Erythrocyte thermogenesis was studied by microcalorimetry in 11 patients before and after treatment for hyperthyroidism. Cell heat production rate and intracellular Na+ and K+ levels were measured in plasma suspensions of erythrocytes with and without specific inhibition of Na/K ATPase by ouabain. The ouabain induced change in the heat production rate (the Na/K pump thermal power); the erythrocyte intracellular Na+ content and the ouabain sensitive Na+ transport were used to estimate the Na/K pump function. The mean value for heat production rate was 131 +/- 4 mW/L erythrocytes before treatment, which is significantly higher than in euthyroid subjects. A significant decrease (P less than 0.01) to normal levels was recorded following therapy. This decrease, as determined in samples with ouabain, correlated to changes in serum levels of triiodothyronine, T3, (r = .74, P less than 0.01). The Na/K pump thermal power was 11 +/- 2 mW/L erythrocytes (8 +/- 2% of total heat production rate) before and 9 +/- 2 mW/L erythrocytes (8 +/- 2%) after treatment. These two values were not different from those obtained in euthyroid subjects. The erythrocyte Na+ content decreased from 9.9 +/- 2.1 to 4.9 +/- 0.5 mmol/L erythrocytes (P less than 0.001) following normalization of thyroid function. The decrease in intracellular Na+ concentration correlated to the decrease in serum T3 levels, but only when calculated from the data obtained in samples with ouabain (r = .60, P less than 0.05). The relative increase in intracellular Na+ concentrations following addition of ouabain was significantly lower (P less than 0.05) before than after treatment for hyperthyroidism, 37 +/- 10% and 61 +/- 5%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between erythrocyte volume and sodium transport in the Milan hypertensive rat and age-dependent changes

The relationship between differences in red blood cell (RBC) volume and ion transport across the erythrocyte cell membrane were investigated in the Milan Hypertensive (MHS) and Milan Normotensive (MNS) rat strains, under different experimental conditions and during ageing. The results obtained indicate that: the difference in Na+/K+ cotransport between MHS and MNS disappear when the RBC volume of the two strains becomes equal under hypotonic swelling; MHS RBCs are osmotically more fragile than those of MNS, probably because of a different membrane structure rather than a different amount of membrane surface, and the smaller volume and the lower Na+ content of MHS RBCs are maintained throughout the life span, while Na+/K+ pump activity and Na+/K+ cotransport undergo age-dependent changes, related to the development of hypertension. All these findings suggest that a primary abnormality of the cell membrane structure of MHS, probably located in the cytoskeleton, is responsible for the cell functional alterations that we previously demonstrated to be genetically associated with MHS hypertension.     

Effects of C-peptide on microvascular blood flow and blood hemorheology

Beside functional and structural changes in vascular biology, alterations in the rheologic properties of blood cells mainly determines to an impaired microvascular blood flow in patients suffering from diabetes mellitus. Recent investigations provide increasing evidence that impaired C-peptide secretion in type 1 diabetic patients might contribute to the development of microvascular complications. C-peptide has been shown to stimulate endothelial NO secretion by activation of the Ca2+ calmodolin regulated enzyme eNOS. NO himself has the potency to increase cGMP levels in smooth muscle cells and to activate Na+K+ATPase activity and therefore evolves numerous effects in microvascular regulation. In type 1 diabetic patients, supplementation of C-peptide was shown to improve endothelium dependent vasodilatation in an NO-dependent pathway in different vascular compartments. In addition, it could be shown that C-peptide administration in type 1 diabetic patients, results in a redistribution of skin blood flow by increasing nutritive capillary blood flow in favour to subpapillary blood flow. Impaired Na+K+ATPase in another feature of diabetes mellitus in many cell types and is believed to be a pivotal regulator of various cell functions. C-peptide supplementation has been shown to restore Na+K+ATPase activity in different cell types during in vitro and in vivo investigations. In type 1 diabetic patients, C-peptide supplementation was shown to increase erythrocyte Na+K+ATPase activity by about 100%. There was found a linear relationship between plasma C-peptide levels and erythrocyte Na+K+ATPase activity. In small capillaries, microvascular blood flow is increasingly determined by the rheologic properties of erythrocytes. Using laser-diffractoscopie a huge improvement in erythrocyte deformability could be observed after C-peptide administration in erythrocytes of type 1 diabetic patients. Inhibition of the Na+K+ATPase by Obain completely abolished the effect of C-peptide on erythrocyte deformability. In conclusion, C-peptide improves microvascular function and blood flow in type 1 diabetic patients by interfering with vascular and rheological components of microvascular blood flow.     

Activation of the sodium pump blocks the growth hormone-induced increase in cytosolic free calcium in rat adipocytes

GH promptly increases cytosolic free calcium ([Ca2+]i) in freshly isolated rat adipocytes. Adipocytes deprived of GH for 3 h or longer are incapable of increasing [Ca2+]i in response to GH, but instead respond in an insulin-like manner. Insulin blocks the GH-induced increase in [Ca2+]i in GH-replete cells and stimulates the sodium pump (i.e. Na+/K+-ATPase), thereby hyperpolarizing the cell membrane. Blockade of the Na+/K+-ATPase with 100 microM ouabain reversed these effects of insulin and enabled GH to increase [Ca2+]i in GH-deprived adipocytes. Both insulin and GH activated the sodium pump in GH-deprived adipocytes, as indicated by increased uptake of 86Rb+. Decreasing availability of intracellular Na+ by blockade of Na+/K+/ 2Cl- symporters or Na+/H+ antiporters abolished the effects of both hormones on 86Rb+ uptake and enabled both GH and insulin to increase [Ca2+]i in GH-deprived adipocytes. The data suggest that hormonal stimulation of Na+/K+-ATPase activity interferes with activation of voltage-sensitive calcium channels by either membrane hyperpolarization or some unknown interaction between the sodium pump and calcium channels.     

Therapeutic response to canrenone of patients with essential hypertension as a function of sodium transport anomalies and ouabain sensitivity of erythrocytes

The presence of Na+ transport abnormalities (decreased affinity of the Na+/K+ pump or the Na+, K+ cotransport for internal Na+, increased Na+:Li+ countertransport, increased Na+ leak), Na+ content, Na+/K+ pump activity and sensitivity to ouabain were investigated in erythrocytes from 13 patients with essential hypertension. According to the presence or absence of Na+ transport abnormalities, the patients were divided into two groups: TrNa(+) (n = 9) and TrNa(-) (n = 4) respectively. Compared with TrNa(-) patients, TrNa(+) patients were characterized by: (i) a higher arterial pressure (131.4 +/- 11.8 vs 110.0 +/- 13.2 mmHg, p less than 0.05), (ii) an increased erythrocyte Na+ content (8.9 +/- 1.0 vs 6.3 +/- 0.8 mmol/l.cells, p less than 0.01) associated with (iii) a decreased rate constant of Na+/K+ pump activity (235 +/- 26 vs 309 +/- 45 h-1, p less than 0.05) and (iv) a higher sensitivity to ouabain (0.76 +/- 0.23 vs 1.12 +/- 0.26 microM, p less than 0.05). Oral administration of canrenone 50 mg per day during 7 weeks decreased mean arterial pressure by 10-30 mmHg in 6 out of the 9 TrNa(+) patients. Conversely, it decreased mean arterial pressure in only one out of the 4 TrNa(-) patients. The hypotensive effect of canrenone in TrNa(+) patients was not associated with normalization of their Na+/K+ pump activity. Canrenone did not modify the sensitivity to ouabain of either the TrNa(+) or the TrNa(-) patients. Before treatment, acute injection of ouabain provoked an inhibition of the erythrocyte Na+/K+ pump, without any change in Na+ content.(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.     

Sodium and potassium ion transport accelerations in erythrocytes of DOC, DOC-salt, two-kidney, one clip, and spontaneously hypertensive rats. Role of hypokalemia and cell volume

Sodium (Na+) and potassium (K+) transport by the furosemide-sensitive Na+-K+ transport system, the Na+-K+ pump, and the cation leak(s) were studied in erythrocytes from DOC-water, DOC-salt, two-kidney, one clip (Sprague-Dawley), and spontaneously hypertensive rats (Wistar-Kyoto). Rubidium (Rb+) was used as a tracer for K+. After 4 weeks of DOC-salt hypertension, inward K+ (Rb+) transport by the furosemide-sensitive system was increased threefold, and the inward Na+ leak and the red cell Na+ content were elevated by about 50%. The rise in cell Na+ accelerated K+ inward and Na+ outward transport by the Na+-K4 pump, DOC-water hypertension caused similar but less pronounced changes. In two-kidney, one clip hypertension, the Na+ leak and the Na+-K+ pump rates were slightly elevated, and furosemide-sensitive Rb+ uptake tended to be increased. In spontaneously hypertensive rats, furosemide-sensitive Rb+ uptake was accelerated by 50%. The marked hypokalemia in DOC-water and DOC-salt hypertension was associated with a slight loss of red cell K+ and an increase in mean cellular hemoglobin content (MCHC), indicative of cell shrinkage. Hypokalemia induced by dietary K+ deficiency caused alterations in red cell cation transport, content, and cell volume which were qualitatively similar but more pronounced than those seen in DOC-salt hypertension. Osmotic shrinkage in vitro induced a severalfold acceleration of furosemide-sensitive Rb+ uptake, similar to that observed in rat erythrocytes shrunken in vivo in K+-deficient states. It is concluded that the acceleration of furosemide-sensitive K+ (Rb+) transport in erythrocytes of mineralocorticoid hypertensive rats is largely caused by the hypokalemia and consecutive red cell K+ loss and shrinkage, respectively. Mean cellular hemoglobin content (MCHC) is thus a parameter that must be considered in studies on Na+ and K+ transport across the membrane of rat erythrocytes.     

Effect of type I (insulin-dependent) diabetes mellitus on key glycolytic enzymes of red blood cells

Summary The effect of type I (insulin-dependent) diabetes mellitus on the key glycolytic enzymes of red cells was studied. The activities of hexokinase, phosphofructokinase and pyruvate kinase were found to be significantly (p<0.01) increased in diabetic patients. Treatment with insulin restored the enzyme activities to normal. The increased activities of the key enzymes may help to regulate red cell ATP level in response to the elevated Na:K pump rate in diabetes. The increased activities of these enzymes may also be due to a greater proportion of young erythrocytes in diabetic patients because of a shortened red cell life span as compared to normal.     

Role of membrane-bound calcium in the changes of ion permeability and Na+, 5+ and ATPase activity of the erythrocytes in hypertension]

The content of calcium bound to the erythrocyte membrane and the effect of intracellular calcium concentration on the activity of Na+, K+-ATPase in the reconstituted erythrocytes were studied in 20 patients with essential hypertension and in 20 individuals with normal pressure. In incubation of the erythrocytes in a solution containing EDTA much more calcium is removed from the outer surface of their membrane in patients with essential hypertension than in the control group (60 +/- 5 mEq/l and 41 +/- 3 mEq/l, respectively). When the intracellular calcium concentration varies from 0 to 500 mumol/l, which corresponds to a rise in the free calcium (Ca2f+-3) concentration to 41 mumol/l, a difference in the changes of Na+, K+-ATPase activity of the reconstituted erythrocytes is noted. When intracellular calcium concentration is 50 mumol/l (Ca2f+-3 mumol/l), ATP-ase activity in patients with essential hypertension is 21% less than that in individuals with normal pressure (P less than 0.005). The authors explain the difference in the kinetics of Na+, K+-ATPase changes by the different degree of calcium depletion of the inner surface of the erythrocyte membrane in relatively low Ca2f+ values in the internal medium. The data obtained are evidence of the altered calcium-binding capacity of the erythrocyte membrane, which may cause the increased permeability of the erythrocyte membrane to sodium and potassium ions in patients with essential hypertension, which the authors had revealed earlier. The authors consider the revealed changes to be a fragment of a more extensive membrane defect which may be the principal cause of activation of the servomechanisms which maintain arterial pressure.     

The acute effects of glycemic control on axonal excitability in human diabetic nerves

OBJECTIVE: To investigate whether glycemic control is associated with reversible changes in axonal excitability in human diabetic nerves. It is known that voluntary contraction or compression ischemia alters nerve Na+/K+ pump activity, and axonal excitability changes due to the pump activity can be estimated by threshold tracking. METHODS: Threshold, the current required to produce a compound muscle action potential 50% of maximum, was determined from the stimulus-response curve, and threshold changes produced by maximal voluntary contraction or ischemia were measured before and after insulin treatment in 10 diabetic patients. RESULTS: Within 3 weeks of the start of treatment, the threshold changes became greater following voluntary contractions (+13+/-4% versus +23+/-5%; mean+/-SEM; p=0.04) and during ischemia (-5+/-2% versus -11+/-2%; p=0.04). CONCLUSIONS: The extent of threshold fluctuation depends on multiple metabolic factors associated with diabetes such as decreased Na+/K+ ATPase activity, increased anaerobic glycolysis, and tissue acidosis, and nerve excitability can respond quickly to glycemic control in diabetic patients.     

High resolution 23Na-nuclear magnetic resonance study of stroke-prone spontaneously hypertensive rat erythrocytes

The intracellular Na+ content of washed erythrocytes from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) was measured by a high resolution 23Na-nuclear magnetic resonance (NMR) technique using a non-permeant aqueous shift reagent, dysprosium triethylenetetramine hexaacetic acid, Dy(TTHA)3-. The initial intracellular Na+ of freshly isolated and washed erythrocytes was very low (approximately 5 mmol/l) and increased progressively with prolonged incubation in isotonic salt solution at 37 degrees C. There was no significant difference in the erythrocyte Na+ concentration between SHRSP and WKY over the entire period of measurement, nor was any difference detected in their osmotic fragility or total cellular volume, although the osmotic fragility decreased with incubation time. The high energy phosphate metabolites were also studied in the same erythrocytes by 31P-NMR. The level of intracellular ATP decreased with incubation at 37 degrees C but showed no difference between the SHRSP and WKY samples. Inclusion of 1 mmol/l ouabain in the incubation medium substantially retarded the breakdown of intracellular ATP and resulted in a concomitant increase in intracellular Na+. However, neither the ouabain-sensitive nor the ouabain-insensitive component of Na+ influx altered in SHRSP erythrocytes compared with WKY erythrocytes in paired experiments. Our results do not support the hypothesis that altered Na+ transport, resulting in an increase in erythrocyte Na+ concentration, is associated with spontaneous hypertension.     

Effect of creatine on erythrocyte rheology in vitro

To evaluate how creatine influences erythrocyte deformability, we determined its effect on erythrocyte filterability in 9 subjects with insulin dependent diabetes (IDDM) without complications, 14 diabetics with uremia and 10 non-diabetic controls. The short-term incubation (15 min at 37 degrees C) of diabetic erythrocytes with 3 mM creatine improved cell filterability (assessed according to the Reid method) from IDDM subjects without complications by 28.4% and that from diabetics with uremia by 18.9%. No rheological effect of creatine was found in erythrocytes from non-diabetic controls. However, a significant protective effect against erythrocyte filterability impairment induced by treatment of red blood cells from non-diabetic controls with hydrogen peroxide was observed with 3 mM (p < 0.04) and 5 mM (p < 0.01) creatine, respectively. Measurement of the thiobarbituric acid (TBA) reactivity was used to assess hydrogen peroxide induced formation of malondialdehyde (MDA). We found that creatine inhibits hydrogen peroxide-induced erythrocyte MDA-formation in a dose dependent manner by 20.4%, 22.3% and 41.4% for 1, 3 and 5 mM creatine, respectively. These results suggest that creatine by its ability to inhibit erythrocyte lipid peroxidation may contribute to the maintenance of normal cell deformability.     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Effect of protein kinase C activation on cytoskeleton and cation transport in human erythrocytes. Reproduction of some membrane abnormalities revealed in essential hypertension

Certain manifestations of alterations of membrane cytoskeleton, protein kinase C activity, and ion transport were revealed in erythrocytes of patients with essential hypertension: 1) the average volume of erythrocytes is reduced by 4%; 2) about 7% of the total number of erythrocytes is represented by cup-shaped forms compared with 1.5 to 3.0% in the control group; 3) basal phosphorylation of Band 4.9 protein is increased 1.6-fold to 1.8-fold; 4) activity of protein kinase C is increased by 60 to 70%; 5) the rate of proton electrochemical gradient (delta mu H+)-induced Na+-H+ exchange is increased twofold. Treatment of erythrocytes of healthy donors with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar but more marked changes in cell shape (17% of cup-shaped forms), volume reduction (by 7%), an increase of Band 4.9 protein phosphorylation (threefold), and an increase in the rate of Na+-H+ exchange (fourfold). Protein kinase activation does not modify Na+-Li+ exchange and slightly increases (by 20-50%) Na+-K+ pump activity, Na+-K+ cotransport, and the rate of 45Ca influx. It may be assumed that the increase of protein kinase C activity is one of the most probable molecular mechanisms conditioning abnormalities of the membrane skeleton and Na+-H+ exchange in primary hypertension.     

23Na and 39K NMR studies of ion transport in human erythrocytes.

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4).     

Evidence for coordinate genetic control of Na,K pump density in erythrocytes and lymphocytes

The erythrocyte is widely used as a model cell for studies of the Na,K pump in health and disease. However, little is known about the factors that control the number of Na,K pumps expressed on the erythrocytes of a given individual, nor about the extent to which erythrocytes can be used to validly assess the pump density on other cell types. In this report, we have compared the interindividual variance of Na,K pump density in erythrocytes of unrelated individuals to that seen with identical twins. Unlike unrelated individuals, in whom pump parameters, ie, ouabain binding sites. 86Rb uptake, cell Na concentration vary widely, identical twin pairs showed no significant intrapair variation for these values. Thus, a role for genetic factors is suggested. In addition, we established and validated a method for determining Na,K pump density and pump-mediated 86Rb uptake in human peripheral lymphocytes. Using this method, we show that whereas Na,K pump density differs markedly between erythrocytes (mean of 285 sites per cell) and lymphocytes (mean 40,600 sites per cell), there is a strong and highly significant correlation (r = 0.79, P less than 0.001) between the pump density in these cell types in any given individual. Taken together, these studies suggest that genetic factors are important determinants of Na,K pump expression, and that pump density appears to be coordinately regulated in two cell types in healthy individuals.     

Blood pressure and erythrocyte Na+ transport systems in a French urban male population

This paper reports an investigation of blood pressure (taken as a continuous variable) as a function of: erythrocyte Na+ content; Na+,K+ pump; Na+,K+ cotransport and Na+,Li+ countertransport fluxes, and passive cation permeabilities in fresh erythrocytes from 129 French males who were living in an urban area and were not under treatment for any medical condition (after allowing for the effects of age, body mass index, alcohol and tobacco consumption). In contrast with previous findings in a North American population, we were unable to confirm that blood pressure was correlated with erythrocyte Na+ content and Na+,K+-AT-Pase activity. Conversely, the only transport parameter correlated (negatively) with blood pressure was outward Na+,K+ cotransport [r = -0.20, P less than 0.05 and r = -0.19, P less than 0.05, for systolic (SAP) and diastolic arterial pressure (DAP), respectively; n = 114]. When allowing for age, body mass index and alcohol consumption, the correlation coefficient between the Na+,K+ cotransport system and blood pressure increased from -0.20 to -0.28 (P less than 0.01) for SAP and from -0.19 to -0.28 (P less than 0.01) for DAP (n = 105). We conclude that the correlations between blood pressure and erythrocyte Na+ transport function could differ between North American and French (or Mediterranean) populations. In any case, a decreased pump or outward Na+,K+ cotransport activity may lead hypertensive subjects to a similar increase in cell Na+ (and Ca2+) content in the vascular wall.     

Kinetic study of the Ca2+ pump in erythrocytes from essential hypertensive patients

Ca2+ pump kinetics were investigated in erythrocytes from 22 essential hypertensive patients and 20 normotensive controls (under initial-rate and steady-state conditions, using Sr2+ as a Ca2+ analogue). The mean value of the apparent dissociation constant for total internal Ca2+ (KCa) was slightly but significantly increased in the hypertensive population (73 +/- 7 versus 55 +/- 3 mumol/l cells, mean +/- s.e.m., P = 0.042 Mann-Whitney U-test). The statistical analysis showed that this was due to six essential hypertensives who exhibited a dissociation constant for Ca2+ that was higher than the upper 95% normal confidence limit (KCa = 116 +/- 7 mumol/l cells), and abnormally high maximal pump rates (7.7 +/- 0.6 versus 5.0 +/- 0.2 mmol/l cells per h in normotensives, P less than 0.001). In addition, the apparent dissociation constant for Ca2+ was inversely correlated with plasma renin activity, although the correlation was only borderline (P = 0.076). In the remaining 16 hypertensive patients, all kinetic parameters of the Ca2+ pump were within the normal range. Finally, a simultaneous study of Na+ transport kinetics suggested that erythrocyte Ca2+ and Na+ transport abnormalities were independent phenomena. Our results do not support the concept that primary hypertension (as a whole entity) is associated with a ubiquitous defect in the plasma membrane Ca2+ pump. However, in some essential hypertensive patients (about 25%) the erythrocyte Ca2+ pump exhibited an apparent decreased affinity for internal Ca2+. A similar defect in vascular smooth muscle may induce a delayed Ca2+ extrusion after the opening of Ca2+ channels, a disturbance likely to be translated into increased vascular reactivity.