Blood pressure in essential hypertension correlates with the concentration of a circulating inhibitor of the sodium pump

Summary The influence of serum from patients with essential hypertension on the sodium efflux rate constants of human lymphocytes and on the activity of isolated (Na⁺+K⁺)-ATPase was investigated. The ouabain-sensitive sodium efflux rate constant was significantly decreased (p<0.001) in the sera of 19 hypertensives (1.92±0.11 h^–1) compared with the sera of 30 normotensives (2.44±0.07 h^–1). The ouabain-insensitive sodium efflux was unaffected. These results corresponded with a significant difference (p<0.005) of (Na⁺+K⁺)-ATPase activity (1.03±0.04 mU/ml and 0.079±0.06 mU/ml), when an isolated (Na⁺+K⁺)-ATPase was incubated with the sera of 22 normotensives or 18 hypertensives. Both the rate constant of ouabain-sensitive sodium efflux and the (Na⁺+K⁺)-ATPase activity correlated significantly with the diastolic and systolic blood pressure (p<0.001). These data, therefore, demonstrated the close relationship between essential hypertension and the concentration of a circulating inhibitor of the sodium pump.Abbreviations ATP Adenosine triphosphate - EGTA Ethyleneglycol bis(2-aminoethyl)-N,N,N,N-tetraacetic acid This paper contains an essential part of the thesis of K.M. presented to the Fachbereich Veterinärmedizin, GiessenThis work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Scho 139/16-2) and by the Fonds der Chemischen Industrie, Frankfurt/Main     

Interspersed normoxia during live high, train low interventions reverses an early reduction in muscle Na+, K+ATPase activity in well-trained athletes

Hypoxia and exercise each modulate muscle Na⁺, K⁺ATPase activity. We investigated the effects on muscle Na⁺, K⁺ATPase activity of only 5 nights of live high, train low hypoxia (LHTL), 20 nights consecutive (LHTLc) versus intermittent LHTL (LHTLi), and acute sprint exercise. Thirty-three athletes were assigned to control (CON, n = 11), 20-nights LHTLc (n = 12) or 20-nights LHTLi (4 × 5-nights LHTL interspersed with 2-nights CON, n = 10) groups. LHTLc and LHTLi slept at a simulated altitude of 2,650 m (F_IO₂ 0.1627) and lived and trained by day under normoxic conditions; CON lived, trained, and slept in normoxia. A quadriceps muscle biopsy was taken at rest and immediately after standardised sprint exercise, before (Pre) and after 5-nights (d5) and 20-nights (Post) LHTL interventions and analysed for Na⁺, K⁺ATPase maximal activity (3-O-MFPase) and content ([³H]-ouabain binding). After only 5-nights LHTLc, muscle 3-O-MFPase activity declined by 2% (P < 0.05). In LHTLc, 3-O-MFPase activity remained below Pre after 20 nights. In contrast, in LHTLi, this small initial decrease was reversed after 20 nights, with restoration of 3-O-MFPase activity to Pre-intervention levels. Plasma [K⁺] was unaltered by any LHTL. After acute sprint exercise 3-O-MFPase activity was reduced (12.9 ± 4.0%, P < 0.05), but [³H]-ouabain binding was unchanged. In conclusion, maximal Na⁺, K⁺ATPase activity declined after only 5-nights LHTL, but the inclusion of additional interspersed normoxic nights reversed this effect, despite athletes receiving the same amount of hypoxic exposure. There were no effects of consecutive or intermittent nightly LHTL on the acute decrease in Na⁺, K⁺ATPase activity with sprint exercise effects or on plasma [K⁺] during exercise.     

Effect of extracellular volume expansion on renal cortical and medullary Na+−K+-ATPase

Summary The role of renal Na⁺–K⁺-ATPase in the acute changes in sodium reabsorption caused by isotonic volume expansion was evaluatedin vivo andin vitro in the rat and the dog. Duringin vivo volume expansion with isotonic saline in the rat, renal medullary Na⁺–K⁺-ATPase specific activity increased, while the simultaneously determined cortical Na⁺–K⁺-ATPase specific activity and kinetics remained unchanged. Furthermore, experimentsin vitro failed to demonstrate a circulating inhibitor of renal Na⁺–K⁺-ATPase both in plasma dialysates from volume-expanded rats and in plasma dialysates concentrated 20-fold by ultrafiltration from volume-expanded dogs. These results suggest that the decreased proximal tubular reabsorption of sodium during volume expansion is not mediated by inhibition of renal cortical Na⁺–K⁺-ATPase. The acute increment in medullary Na⁺–K⁺-ATPase observed could represent an adaptive response to increased sodium reabsorption by the loops of Henle, and raises the possibility that this enzyme may participate in relatively rapid adjustments in the transport of sodium by the renal tubule.     

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na⁺,K⁺-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na⁺,K⁺-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na⁺,K⁺-ATPase were used. Dose–response curves for ouabain, a specific Na⁺,K⁺-ATPase inhibitor, were obtained to ascertain which Na⁺,K⁺-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na⁺,K⁺-ATPase was studied in purified lamb kidney Na⁺,K⁺-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na⁺,K⁺-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na⁺,K⁺-ATPase. Possible interaction mechanisms are discussed.     

Erythrocyte sodium content and transport in borderline and mild hypertension

Summary Sodium content and transport of red blood cells were examined in 98 male blood donors. Regarding their blood pressure they were classified into the following groups: (a) 57 normotensives, (RR<140/90 mm Hg); (b) 24 borderline hypertensives (140/90RR<160/95 mm Hg); and (c) 17 hypertensives (RR>160/100 mm Hg). Compared with the normotensives the borderline hypertensives have significantly reduced red cell sodium content. The ouabain-resistant net Na⁺ uptake and the relative Na⁺ uptake, as a measure of the Na⁺/K⁺ pump, were significantly increased. With rising blood pressures the measured values turn to normal, so that no difference exists between the normotensive and hypertensive groups. It is supposed that in the initial or even prehypertensive state a considerable enhancement of the pump activity occurs, simultaneously accompanied by less marked increases in sodium influx, leading to a reduced intracellular sodium content. In the course of hypertension, possibly caused by the formation of a pump inhibitor, the sodium content of red cells turns to normal or supernormal values.Abbreviations BMI body mass index - BHT borderline hypertensive - Ca _ion ²⁺ ionized plasma calcium - HT hypertensive - k relative OR net Na⁺ uptake - [Na⁺]_i, [K⁺]_i intracellular sodium and potassium content in RBCs - NT normotensive - OR ouabain-resistant - RBCs red blood cells - Na OR net Na⁺ uptake     

[Na+] i /[K+] i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression

The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na⁺,K⁺-ATPase but is independent of the inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na⁺,K⁺-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na⁺,K⁺-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin–Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK. In these cells, 6-h exposure to 3 μM ouabain led to the internalization of ∼50% of plasmalemmal Na⁺,K⁺-ATPase. Prolonged incubation in a K⁺-free medium abolished ouabain-induced Na⁺,K⁺-ATPase internalization but did not affect the cytotoxic action of ouabain seen after 18-h incubation. Previously, it was shown that CTS-induced Na⁺,K⁺-ATPase internalization is mediated by its interaction with Src within caveolae. Neither caveolae damage by cholesterol depletion with methyl-β-cyclodextrin nor Src inhibition with 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyridine affected the death of ouabain-treated C7-MDCK cells. Actinomycin D at the 0.1-μg/ml concentration almost completely abolished ribonucleic acid synthesis but did not protect C7-MDCK cells from the cytotoxic action of ouabain. Our results show that neither Na⁺,K⁺-ATPase endocytosis nor de novo gene expression contributes to -independent cell death signaling evoked by prolonged exposure to CTS.     

Evidence for coupled sodium/hydrogen exchange in the rat superficial proximal convoluted tubule

Recent in vitro studies from the rat and rabbit have suggested a tightly coupled sodium/hydrogen ion exchanger on the luminal membrane of proximal tubules. The steep sodium gradient from the lumen to cell supplies indirect energy for hydrogen ions to be pumped from the cell to the lumen. However, a proton translocating pump has been demonstrated in other epithelia, which is independent of sodium transport and directly driven by ATP. To examine the role that sodium might play in the process of acidification, rat proximal convoluted tubules and their surrounding peritubular capillaries were perfused in vivo with artificial ultrafiltrate-like perfusion solutions. Total CO₂ absorption was measured by microcalorimetry during alterations in sodium transport by replacement of the sodium with an impermeant cation, choline, or by inhibition of the (Na⁺+K⁺)-ATPase by removing potassium from both perfusion solutions. Under control conditions the absolute rate of total CO₂ absorption was 140 pmol/mm·min. In the choline substitution and potassium removal experiments, absolute total CO₂ absorption fell to 23 and 28 pmol/mm·min, respectively. The data suggest that: 1) in the rat superficial proximal convoluted tubule approximately 80% of the bicarbonate absorption is tightly coupled to sodium transport; 2) this process is driven indirectly by the (Na⁺+K⁺)-ATPase system; and 3) the residual 20% of acidification appears to be mediated by another mechanism or may be a consequence of technical liminations.     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i

Recent studies have demonstrated the tissue-specific effect of Na⁺/K⁺ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na⁺/K⁺ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na⁺/K⁺ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na⁺/K⁺ pump inhibition in K⁺-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by ³H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K⁺ and Na⁺, whereas dissipation of the Na⁺ gradient abolished the antiapoptotic action of K⁺-free medium. Comparative analysis of these results and modulation of intracellular Na⁺ and K⁺ content by the above-listed stimuli showed that interaction of ouabain with Na⁺/K⁺-ATPase triggered necrosis independently of inhibition of Na⁺/K⁺ pump-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio, whereas protection against apoptosis under Na⁺/K⁺ pump inhibition in K⁺-depleted medium was mediated by [Na⁺]_i elevation. The role of Na⁺/K⁺ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Na+, K+-ATPase activity in neurons and glial cells of the olfactory cortex of the rat brain during the development of long-term potentiation

Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities were studied in neurons and glial cells of the olfactory cortex of the rat by quantitative cytophotometry in conditions of long-term potentiation (LTP), and significant changes in direction and extent were found. Na⁺, K⁺-ATPase activity decreased in neurons in the first 15 min after LTP, with subsequent elevation by 30 min. Mg²⁺-ATPase activity remained unchanged in these conditions. Glial cells showed significant increases in Na⁺, K⁺-ATPase activity in the initial period after LTP, with return to control by 30 min. Again, there were no significant changes in Mg²⁺-ATPase activity. The formation and persistence of LTP in neurons and glial cells was accompanied by significant changes in Na⁺, K⁺-ATPase activity, which were reciprocal in nature. Functional Neurochemistry Laboratory (Director N. A. Emel'yanov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 3, pp. 16–20, March, 1995.     

Dependence of the electrogenic pump current of Xenopus oocytes on external potassium

The membrane potential of Xenopus oocytes showed a variable response to an increase of the K⁺ concentration in the bathing solution, [K⁺]_e, from 2.5 mM to 20 mM. In 54% of the cases (n=52) the cells hyperpolarized (by max. 70 mV). In the presence of 10^–5 M ouabain, all cells depolarized suggesting that the hyperpolarization was caused by an electrogenic Na⁺/K⁺ pump. In cells stored overnight in a Na⁺-free solution the transition from 2.5 to 20 mM [K⁺]_e always caused depolarization indicating that the stimulation of the pump requires high internal sodium, [Na⁺]_i. Cells stored overnight in a Na⁺-rich solution had a [Na⁺]_i of 30.7±7 mM, i.e. the Na⁺/K⁺ pump was saturated with sodium (Lafaire and Schwarz 1986). With 9 such cells we determined the K⁺ activation of the Na⁺/K⁺ pump. The activation follows Hill kinetics with I_max=90.5 nA, K_s=2.3 mM, and n=1.68.     

Influence of bile acids on the (Na+−K+)-activated- and Mg2+-activated ATPase of rat colon

Summary The influence of various bile acids on the (Na⁺−K⁺)-ATPase and Mg²⁺-ATPase activity of rat colon is described. At a concentration of 0.6 mmol/l C and TC did not inhibit the (Na⁺−K⁺)-ATPase activity in contrast to GC. The taurine derivates TC, TCDC and TDC did not influence or even enhanced the (Na⁺−K⁺)-ATPase activity. All bile acids except C, TC and CDC depressed the Mg²⁺-ATPase activity. At higher concentrations only C and TC did not influence the (Na⁺−K⁺)-ATPase activity. C, GC and TC at 2.5 mmol/l decreased the (Na⁺−K⁺)-activated phosphatase with ATP as substrate. All other substrates tested did not influence the enzymic activity significantly. The results indicate that bile acids can inhibit the Na⁺-absorbing system in rat colon. Hence this inhibition can cause diarrhea.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

SIK1/SOS2 networks: decoding sodium signals via calcium-responsive protein kinase pathways

Changes in cellular ion levels can modulate distinct signaling networks aimed at correcting major disruptions in ion balances that might otherwise threaten cell growth and development. Salt-inducible kinase 1 (SIK1) and salt overly sensitive 2 (SOS2) are key protein kinases within such networks in mammalian and plant cells, respectively. In animals, SIK1 expression and activity are regulated in response to the salt content of the diet, and in plants SOS2 activity is controlled by the salinity of the soil. The specific ionic stress (elevated intracellular sodium) is followed by changes in intracellular calcium; the calcium signals are sensed by calcium-binding proteins and lead to activation of SIK1 or SOS2. These kinases target major plasma membrane transporters such as the Na⁺,K⁺-ATPase in mammalian cells, and Na⁺/H⁺ exchangers in the plasma membrane and membranes of intracellular vacuoles of plant cells. Activation of these networks prevents abnormal increases in intracellular sodium concentration.     

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct

The relation between transcellular Na⁺ absorption, intracellular Na⁺ concentration and Na⁺/K⁺-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na⁺ absorption was varied by perfusing the tubule with solutions containing different Na⁺ concentrations (nominally Na⁺-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K⁺ in the bathing solution was exchanged for Rb⁺. Intracellular element concentrations, including Rb⁺, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na⁺ concentration in the perfusion solution caused significant rises in intracellular Na⁺ concentration and Rb⁺ uptake of principal cells. Principal cell Na⁺ and Rb⁺ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na⁺-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na⁺ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na⁺ in the perfusion solution. In contrast, a comparable relationship between lumen Na⁺ concentration, intracellular Na⁺ concentration and basolateral Rb⁺ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na⁺ absorption. In addition, the data demonstrate that apical Na⁺ entry and basolateral Na⁺/K⁺-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na⁺ concentration leads to increased Na⁺ entry and augmented intracellular Na⁺ concentration, which then secondarily stimulates active basolateral Na⁺/K⁺(Rb⁺) exchange.     

Heterogenous Na+, K+-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissues

Na⁺, K⁺-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissue was measured using indirect immunofluorescence and confocal laser scanning microscopy. All four major sites of aggregated lymphoid tissue, i. e. Peyer's patch, sacculus rotundus, caecal patch and appendix, were studied. Na⁺, K⁺-ATPase expression was localized to the basolateral surface of cells of the follicle-associated epithelium (FAE) and adjacent villous or surface epithelia (non-FAE), where increased expression during enterocyte migration was evident. In the FAE, expression of Na⁺, K⁺-ATPase appeared to be lower in the specialized M cells than in enterocytic-type cells, although expression in both cell types was lower than in adjacent non-FAE. Quantification of immunofluorescent staining of Na⁺, K⁺-ATPase by confocal laser scanning imaging showed a reduction of expression in the FAE to approximately 20–60% relative to that in the adjacent non-FAE. These results are consistent with a primary role of the FAE in mucosal immunity with minimal involvement in active solute absorption.     

Na+-activated K+ current in cardiac cells: rectification,open probability,block and role in digitalis toxicity

The Na⁺-activated K⁺ current was studied in inside-out patches and in whole cells isolated from the guinea-pig cardiac ventricle. The single channel conductance showed inward rectification for K⁺ _i+ _e, but outward rectification for K⁺ _i>K⁺ _e The open probability was dependent on Na⁺ _i and Na⁺,K⁺-pump activity. In the presence of pump blockade the channel remained active at low Na⁺ _i Similar results were obtained in whole cells. These results suggest the existence of Na⁺ gradients depending on Na⁺,K⁺-pump activity and passive inward leak of Na⁺. The channel and whole cell current were blocked by R56865. The drug did not change the single channel conductance but markedly reduced open probability by shortening burst duration. The current may play an important role in action potential shortening during pump blockade.This work was supported by a grant of the National Fund for Scientific Research Belgium.3.0016.87.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.     

Sodium-activated potassium current in sensory neurons: a comparison of cell-attached and cell-free single-channel activities

Single-channel currents from Na⁺-dependent K⁺ channels (K_Na) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patchclamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of subconductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cellattached recordings at the cell resting potential the degree of K_Na activity, measured as the probability of the channel being open, P _o, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na⁺ around 30 mM and of K⁺ close to the physiological intracellular levels. However, in some cell-attached patches P _o was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na⁺ concentrations in the bath (100 mM). The excision of a highactivity patch in the presence of 30 mM Na⁺ resulted in a fall of P _o in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision all K_Na displayed a similar Na⁺ sensitivity, irrespective of the degree of activation observed in the cellattached mode. In inside-out patches the P _o values observed in the presence of either low or high concentrations of Na⁺ in bath solutions were not modified by internal Ca²⁺ (0.8–8.5 M). The variable degree of K_Na activation observed in cell-attached recordings suggests that either internal Na⁺ concentrations reach very high levels close to the membrane, or soluble factor(s) are involved in the modulation of K_Na activity: under such conditions, the Na⁺-activated K⁺ current may contribute to the maintenance of the resting membrane potential and to control neuronal membrane excitability.