Decreased Na/K ATPase ouabain binding sites in red blood cells of patients with insulin-dependent diabetes and healthy north African control subjects: relationship with diabetic neuropathy

Like other degenerative complications occurring during diabetes, development of neuropathy is determined mainly by the duration of disease and quality of control. However, there may be some predisposing factors. Activity of Na/K ATPase has been implicated in the pathophysiology of diabetic neuropathy. A decrease in the activity of this enzyme has been observed in red blood cells of poorly controlled diabetic patients and healthy North African subjects who are predisposed to diabetic neuropathy. This study was performed to characterize abnormalities of Na/K ATPase activity in these two populations. For this purpose we measured enzyme activity (hydrolysis of ATP) and the number of enzyme units (number of binding sites for ouabain) in the red blood cells of three groups of men, i.e., healthy Caucasian subjects, healthy North African subjects and Caucasian insulin-dependent diabetic patients. The level of Na/K ATPase activity and the number of enzyme units were about 30% lower in the red blood cells of diabetic patients and North African subjects, than in healthy Caucasian controls. In healthy North African subjects predisposed to neuropathy in case of development of diabetes, the decrease in enzymatic activity was correlated with a decrease in the number of enzyme units. This correlation was not observed in diabetic patients. We speculate that the constitutional decrease in Na/K ATPase activity in healthy North African subjects corresponds to a quantitative defect, whereas the acquired decrease in diabetic patients corresponds to a qualitative defect probably related to the structure of the lipid membrane.     

Functional communication between cardiac ATP-sensitive K+ channel and Na/K ATPase

Insecticide resistance often is blamed for failures of insecticides to control cat fleas, Ctenocephalides felis (Bouché). Yet the genetics and adaptive advantage of resistance traits remain unexamined. Lethal doses of insecticides that kill 50% of the population fluctuate 7-fold within a cat flea strain. Many reports of flea resistance may be attributable to variable mortality from effects of solvents, substrates, humidities, temperatures, colonization, and ages of fleas. Resistance ratios (ratios of lethal doses of a resistant to a susceptible strain) are < 690-fold in fleas; lower than many other arthropods. This, plus strain variability, hinders resistance detection. Relationships between resistance levels, control failures, and health threats are unclear. Insensitive acetylcholinesterase, knockdown recovery, glutathione transferase conjugation, and mixed function oxidase/cytochrome P450 are demonstrated resistance mechanisms in cat fleas. Ecological genetics of resistance in cat fleas probably involves flea transfer among hosts, host movements, refugia, founder effects, and mortality from abiotic factors. Understanding cat flea resistance requires population monitoring before, during, and after insecticide treatments using conventional and rapid molecular bioassays. Sustained insecticide release devices such as flea collars and long-lived insecticide residues for premises possibly contribute to the development of resistance. New systemic and topical insecticides, especially when given prophylactically, may act similarly. Eliminating insecticides prevents insecticide resistance but necessitates application of biorational tactics incorporating mechanical, environmental, and cultural controls. Using high temperatures, low humidities, host grooming and such tactics as decreasing doses, increasing action thresholds, rotating insecticides, and leaving spatial and temporal refugia may suppress cat flea resistance.     

Genetic factors, Na K ATPase activity and neuropathy in diabetics]

A genetic predisposition to develop a polyneuropathy in case of diabetes seems to exist. Some ethnic groups such as North Africans are prone to develop a diabetic polyneuropathy. To identify this predisposition could help in targeting a preventive treatment. We have observed that red cell Na/K ATPase activity was lower among diabetic patients than controls and even lower when diabetic neuropathy was present. Now an impaired NA/K ATPase activity has been implicated in the pathogenesis of diabetic neuropathy and ethnic differences in this enzyme activity have been demonstrated. For these reasons, we have compared red cell Na/K ATPase activity of European and North African individuals with or without diabetes and in case of diabetes with or without neuropathy. Among European subjects, Na/K ATPase activity was higher in 46 control subjects than in 84 insulin-dependent diabetic patients (405 +/- 16 nmol.mg Prot-1h-1 versus 282 +/- 10 p. < 0.05) and in the diabetic group Na/K ATPase activity was lower in the patients presenting with neuropathy (242 +/- 19 versus 323 +/- 12 p. < 0.05). The mean red cell Na/K ATPase activity was lower in 16 North African control subjects than in their European counterparts (296 +/- 26 p. < 0.05). The same observation was made when comparing 24 North Africans insulin dependent diabetic patients to the European diabetics (246 +/- 20 p. < 0.05). A low Na/K ATPase activity appears to be a risk marker of diabetic neuropathy. It could explain the propensity of North African patients to develop this diabetic complication. A restriction polymorphism exist on the first intron of the ATP1 A1 gene coding for the ATPase alpha 1 isoform. This isoform is preponderent in the nervous tissue and exclusive in red cells. Among European diabetic individuals, the presence of the restricted allele is strongly associated to diabetic neuropathy, confering a relative risk of 6.5 (95%, confidence interval 3.3-13). The restricted allele is associated to a lower Na/K ATPase activity but only among diabetic patients and not in control subjects. This fact suggests an interaction between genetic factors (the restriction polymorphism of ATP1 A1 gene) and environmental factors (diabetes) to induce a decrease in Na/K ATPase activity which in turn could favor the development of diabetic neuropathy. Among North African individuals the impairement of Na/K ATPase activity is not explained by the presence of this polymorphism. Other genetic factors remain to be identified.     

Platelet-activating factor inhibits (Na+,K+) ATPase activity in rat brain

In the present study, experiments were conducted to determine the effect of platelet-activating factor (PAF) on (Na+,K+)-ATPase in rat cerebral cortex. PAF, but not lysoPAF, inhibited (Na+,K+)ATPase activity, in a dose- and time-dependent manner, 10(-7) to 10(6) M being the most effective dose. These effects were abolished in the presence of PCA-4248, a PAF antagonist, indicating that the PAF effect may be mediated by its specific membrane receptors. Omission of external calcium caused an increase in the basal activity and abolished the PAF effect on (Na+,K+)ATPase. The present study demonstrates that PAF inhibits (Na+,K+)ATPase activity in the cerebral cortex and suggests that PAF released during certain pathological conditions, such as ischemia, may act on ATPase. This could be one possible mechanism of PAF action that needs further attention.     

Altered fatty acid, cholesterol and Na+/K+ ATPase activity in erythrocyte membrane of rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose cause remains obscure. Blood from 15 RA patients and controls was taken and their ghosts separated. The ghosts were analysed for cholesterol content, Na+/K+ ATPase activity and eicosapentaenoic acid. The cholesterol content in the ghosts of RA patients was significantly lower as compared with the set of controls. There was a major difference in the activity of Na+/K+ ATPase between the two groups with RA patients showing significantly elevated activity. The ghosts of the RA patients exhibited major abnormality in the polyunsaturated fatty acids of phospholipids with the level of eicosapentaenoic acid (omega-3, 20:5) being significantly reduced.     

Biological activity of C-peptide on the skin microcirculation in patients with insulin-dependent diabetes mellitus

19 insulin-dependent diabetes mellitus (IDDM) patients participated in a randomized double-blind crossover investigation to investigate the impact of human C-peptide on skin microvascular blood flow. The investigation was also carried out with 10 healthy volunteers. Blood pressure, heart rate, blood sugar, and C-peptide levels were monitored during a 60-min intravenous infusion period of C-peptide (8 pmol kg-1 min-1) or saline solution (154 mmol liter-1 NaCl), and 30 min after stopping the infusion. During the same time period, capillary blood cell velocity (CBV), laser Doppler flux (LDF), and skin temperature were assessed in the feet. In the verum arm, C-peptide levels increased after starting infusion to reach a maximum of 2.3+/-0.2 nmol liter-1 after 45 min, but remained below 0. 15 nmol liter-1 during the saline treatment. Baseline CBV was lower in diabetic patients compared with healthy subjects (147+/-3.6 vs. 162+/-4.2 micron s-1; P < 0.01). During C-peptide administration, CBV in IDDM patients increased progressively from 147+/-3.6 to 167+/-3.7 micron s-1; P < 0.001), whereas no significant change occurred during saline infusion or in healthy subjects. In contrast to the CBV measurements, the investigation of LDF, skin temperature, blood pressure, heart rate, or blood sugar did not demonstrate any significant change during the study. Replacement of human C-peptide in IDDM patients leads to a redistribution in skin microvascular blood flow levels comparable to levels in healthy subjects by increasing the nutritive CBV relative to subpapillary arteriovenous shunt flow.     

A non-ouabain Na/K ATPase inhibitor isolated from bovine hypothalamus. Its relation to hypothalamic ouabain

We have isolated from bovine hypothalamic and pituitary tissue the sodium pump inhibitor HHIF that is structurally different from ouabain. By mass spectrometric analysis this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from ouabain. It has been previously shown that HHIF inhibits the Ca2+-ATPase of the plasma membrane of synaptosomes. HHIF increases free calcium levels in cultured rat mesangial cells as well as mesangial cell contraction and proliferation. With the same purification procedure we have isolated in parallel HHIF and Ouabain from central nervous tissue. Ouabain elutes prior to HHIF in the final purification HPLC systems. This endogenous Ouabain has, in all the systems tested, the same chromatographic behavior that synthetic cold or [3H] Ouabain.     

Cellular Ca2+ ATPase activity in diabetes mellitus

Basal and maximal Ca2+ ATPase activity was studied in erythrocytes of 29 healthy controls, 15 patients with insulin-dependent diabetes mellitus (IDDM) and 22 patients with non-insulin-dependent diabetes mellitus (NIDDM). Basal and maximal Ca2+ ATPase activity was significantly decreased in insulin-dependent diabetes mellitus (8.4 +/- 0.5 and 22.5 +/- 1.1 pmol/10(6) RBC/min) and non-insulin-dependent diabetes mellitus (7.3 +/- 1.0 and 18.6 +/- 1.8 pmol/10(6) RBC/min) compared to healthy controls (9.3 +/- 1.0 and 24.6 +/- 1.1 pmol/10(6) RBC/min). Maximal Ca2+ ATPase activity showed a significant correlation to systolic blood pressure in both insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus. There was no significant correlation of maximal Ca2+ ATPase activity to fasting serum glucose concentration and to HbA1 levels. Maximal Ca2+ ATPase activity was significantly correlated to creatinine clearance in non-insulin-dependent diabetes mellitus, but not in insulin-dependent diabetes mellitus. It is concluded that a decreased cellular Ca2+ ATPase activity may predispose to the development of hypertension in diabetes mellitus.     

Inhibition of Na+/K+ ATPase under hypertonic conditions in rat mast cells

Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions.     

Angiotensin II AT1 receptors and Na+/K+ ATPase in human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells (HUVECs) at passage 4 specifically bound 70 +/- 12 fmol [3,5-3H]Tyr4-Ile5-angiotensin (Ang) II/mg protein, with a Kd of 0.9 +/- 0.36 nM. Binding was eliminated in cells preincubated with a monoclonal antibody (6313/G2) raised against the subtype AT1 of the Ang II receptor. Freshly seeded HUVECs were positive for 6313/G2 antibody by immunocytochemistry, and such immunoreactivity was still retained at passage 4. Incubation of HUVECs for 20 min with different concentrations of Ang II provoked a significant increment in Na+/K+ ATPase activity compared with controls, in a dose- and time-dependent manner. Maximal response was obtained with 1000 pM Ang II after 20 min stimulation and resulted in a 2.2-fold increment in Na+/K+ ATPase activity. This stimulation was abolished when cells were incubated with 1000 pM Ang II in the presence of 1 microM of the specific AT1 subtype inhibitor, DuP753. Moreover, preincubation of HUVECs with 6313/G2 or with 1 mM dithiothreitol also inhibited the stimulatory effect of Ang II. These results suggest that the AT1 receptor subtype mediates the Na+/K+ ATPase response to Ang II in these cells.     

Inactivation of rat brain Na+ K+ ATPase by sodium dodecylsulfate: effect of pH, magnesium ions and temperature

The relative stabilities to SDS inactivation of the rat brain Na(+)-ATPase catalytic subunit isoforms in the conditions of the surface charge modulation and temperature modification of the physical state of the membrane lipids were examined. The higher sensitivity of the Na+,K(+)-ATPase a1-isoform than a+ to SDS inactivation occurs under the conditions of the detergent treatment of microsomes at pH 7.5 and room temperature. The decrease in pH in ATP-free medium up to 6.2 or temperature elevation up to 37 degrees C eliminates the differences in SDS sensitivity of the Na+,K(+)-ATPase isoforms. The enhancement of the SDS binding with a subunit due to changes in membrane surface charge in the first case or increase of accessibility of the protein intramembrane regions for detergent due to the decrease of the packing density of the boundary lipids in the second case are supposed.     

Erythrocyte Na+/K(+)-ATPase and membrane and serum lipid profiles: as related to alcohol, body mass index and blood pressure

Erythrocyte Na+/K(+)-pump activities have been measured in hypertensives, alcohol consumers and obese persons, but the results have been variously reported as decreased, increased or unchanged. We analyzed the relationships between erythrocyte Na+/K(+)-ATPase activities and the membrane and serum lipid profiles in 83 middle-aged men, to clarify the reasons for these inconsistencies. Increases in erythrocyte Na+/K(+)-ATPase activity related closely to decreases in cholesterol to phospholipid (C/P) ratio of the erythrocyte membrane. Decreases in the C/P ratio in turn related closely to elevations of serum triglycerides (TG) with increasing body mass index, and weakly to the volume of alcohol consumed. Thus, erythrocyte Na+/K(+)-ATPase activities depend largely on the membrane and serum lipid profiles as related to body weight and alcohol consumption, and which may be a cause of the previous conflicting findings. Erythrocyte Na+/K(+)-ATPase showed a positive association with blood pressure, independently of age, body mass index and serum gamma-glutamyl transpeptidase levels. Although the biological link of elevated erythrocyte Na+/K(+)-ATPase with the rise in blood pressure remains unclear, it may be a reflection of hyperinsulinemia in the subjects with a higher blood pressure due to overweight or excessive alcohol consumption.     

Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans

We showed previously that the glycolipoprotein fraction prepared from Leptospira interrogans inhibited the Na+,K+ ATPase enzyme purified from brain or kidney and in isolated nephron segments (M. Younes-Ibrahim, P. Burth, M. V. Castro Faria, B. Buffin-Meyer, S. Marsy, C. Barlet-Bas, L. Cheval, and A. Doucet, C. R. Acad. Sci. Paris Ser. III 318:619-625, 1995). In the present communication, we have demonstrated that unsaturated fatty acids such as oleic and palmitoleic acids, which are adsorbed to this fraction, are effective inhibitors of the enzyme.     

Na/H exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis

We examined whether H(+)-ATPase, H(+)-K(+)-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H(+)-K(+)-ATPase (-6.4 +/- 0.9 vs. -1.4 +/- 0.5 pmol/mm.min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 +/- 1.7 vs. -6.6 +/- 0.9 pmol/mm.min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H(+)-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 +/- 0.7 vs. -4.4 +/- 0.7 pmol/mm.min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 +/- 23.7 vs. 76.6 +/- 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 +/- 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 +/- 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H(+)-K(+)-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins.     

Alpha 2 mRNA of Na+K+ ATPase is increased in astroctyes of rat hippocampus after treatment with kainic acid

The Na+K+ ATPase (Na+ pump) plays a central role in regulating cation homeostasis and is thought to have an important role in cell proliferation. The multitude of subunit isoforms comprising the functional Na+K+ ATPase has raised the possibility that specific subunit isoform combinations may be involved in different cellular processes. We have investigated the involvement of the specific isoforms in neurons and glia at the site of a CNS lesion. Intracerebroventricular injection of kainic acid was used to induce neuronal cell loss and reactive gliosis in rat hippocampus and levels of Na+K+ ATPase subunit isoform mRNA levels were determined in cells of rat hippocampus using in situ hybridization. alpha 2 mRNA levels increased 35-40% in CA1 and CA3 astrocytes between 1-3 weeks after KA injection with no significant change in other subunit isoform mRNA levels. In addition alpha 3 mRNA levels in CA1 pyramidal neurons were decreased by approx. 35%. Small neurons in the CA1 and CA3 region showed no changes in mRNA levels for any of the Na+K+ ATPase subunit isoforms. These results may indicate a possible role for alpha 2 subunit isoform in the conversion of glial cells from a normal phenotype to the reactive phenotype characteristic in this model of CNS injury.     

Functional activity of Na+,K(+)-pump in normal and pathological tissues

Na+,K(+)-ATPase, supporting the ionic homeostasis of the cell, is under control of Na+, K+, Mg2+, and ATP. The regulating effect of Mg2+ is rather unclear, whereas the Na+/K+ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na+,K(+)-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na+,K(+)-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na+,K(+)-ATPase--an integral protein of the outer cell membranes.     

Na/K pump current in guinea pig cardiac myocytes and the effect of Na leak

INTRODUCTION: Steady-state Na/K pump current (Ip) in adult guinea pig ventricular myocytes was studied to determine the effect on the Na/K pump of transmembrane Na leak, membrane potential, and pipette Na concentration. METHODS AND RESULTS: Using conventional whole cell, patch clamp techniques, Ip was identified as either Ko-sensitive or ouabain-sensitive current when most other membrane currents were inhibited. Control experiments showed that there were no Ko-sensitive currents other than Ip under the conditions of our experiments. Ip was found to be similar to that reported by others being voltage dependent between -130 and 0 mV and having a half maximal activation by Nai of 28 mM. Ouabain sensitivity was also measured, and it was found that there were two binding sites with the high affinity site comprising 5% to 10% of the total and having an apparent affinity 1000-fold higher than the low affinity site. Apparent affinity of both sites was shifted about 10-fold (higher affinity) by increasing Nai from 10 to 85 mM. When internally perfused with 0 Na solution, Na leak through the membrane was found to be linearly related to Na/K pump activity. In contrast to prior suggestions, Ip was not correlated with series resistance when there was a large transmembrane Na gradient. CONCLUSION: These data suggest that, under conditions of high transmembrane Na gradient, Na leak through the membrane plays a significant role in determining Na/K pump activity.