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.     

Das Problem des zellulären Herzglykosidrezeptors

Zusammenfassung Diese Übersicht beschreibt die Eigenschaften der Na⁺, K⁺-ATPase bzw. der Na⁺, K⁺-Pumpe in der intakten Membran und die hochspezifische Hemmung dieses Transportenzyms durch Herzglykoside.Es wird auf die Wechselwirkung zwischen Glykosid und Enzym eingegangen und da bei insbesondere auf die Steuerung der Glykosidbindung durch ATP, K⁺, Na⁺, Mg²⁺ und Ca²⁺.Die Bedeutung des Enzyms als wahrscheinlicher pharmakologischer Rezeptor für Herzglykoside wird herausgestellt. Zunächst erfolgt eine Beschreibung der Schwierigkeiten und Fortschritte bei der Herstellung eines Zusammenhanges zwischen positiv inotropem Effekt von Herzglykosiden und deren Bindung an die Na⁺, K⁺-Pumpen des Herzmuskels bzw. der Hemmung des Transportsystems durch Herzglykoside.Es werden neuere Erkenntnisse über die Verknüpfung von der Hemmung der Na⁺, K⁺-Pumpe, die zu einer Erhöhung der intrazellulären Na⁺-Konzentration führt, und der indirekten Erhöhung der intrazellulären Ca²⁺-Aktivität referiert.Mitteilungen über den Nachweis einer herzglykosidartigen endogenen Aktivität entsprechen der hohen Rezeptorspezifität für Herzglykoside.     

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.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

Age differences in hormonal regulation of Na+, K+-ATPase activity in the rat renal cortex

Laboratory of Physiological Genetics, Institute of Cytology and Genetics, Siberian Brach, Russian Academy of Sciences, Novosibirsk. (Presented by Academician of the Russian Academy of Medical SciencesV. P. Lozov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 8, pp. 150–153, August, 1992.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.     

Regulation of NaCl transport in the renal collecting duct: lessons from cultured cells

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na⁺, which is extruded by the basolateral Na⁺,K⁺-ATPase. Simian virus 40-transformed and “transimmortalized” collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na⁺,K⁺-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na⁺ currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na⁺ absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase_1/2. Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron.     

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (_mc = 12 mV) followed by a slower repolarization (_mc = –7 mV). The intracellular sodium activity, a_iNa⁺, rises from 13.2±2.4 meq/l by 6.7±0.5 meq/l within 5 min, a_iCl^– rises about 3 meq/l above the control value of 37.7±2.2 meq/l, while a_iK is constant (97.7 ±7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (a_iK⁺) is 5.2±0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1±0.1 meq/l).In chloride-free media a_sK⁺=4.2±0.1 meq/l and _mc hyperpolarizes by –13 mV. The depolarization due to glucose absorption increases (_mc = 14.1 ± 1.4 mV) and the repolarization ( _mc ^repol ) disappears. In addition, a_iNa⁺ rises from 16.3±2.4 meq/l by 9.9±1.5 meq/l within 5 min, a_iK⁺ remains constant and equal to the value in chloride containing solutions (88.5±2.8 meq/l); a_sK⁺ increases transiently (1.1±0.1 meq/l).Serosal Ba²⁺ (5 mM) depolarizes _mc (+14.2±1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes _mc and abolishes the repolarization.These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca²⁺, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.     

3H-Ouabain binding sites in porcine skeletal muscle as influenced by environmental temperature and energy intake

The influence of environmental temperature and energy intake on³H-ouabain binding sites in skeletal muscle has been investigated in young growing pigs at 8 weeks of age. Animals lived for several weeks at 35 or 10°C on a high (H) or low (L) level of energy intake. The four treatment groups were thus: 35H, 35L, 10H and 10L. The total number of³H-ouabain binding sites (B _max) in longissimus dorsi muscle (mean values ± SEM) were 221±66, 214±61, 350±76 and 486±114 pmol/g wet weight for the 35H, 35L, 10H and 10L groups respectively.B _max was significantly greater in those living in the cold than the warm (P<0.001). Moreover, at 10°C energy intake had a significant effect, withB _max being greater in the 10L than the 10H group (P<0.005). Level of energy intake had no influence onB _max at 35°C. The apparent dissociation constant was not affected by either temperature or intake. The elevatedB _max and hence the increase in number of Na⁺, K⁺-pumping sites in the cold is probably related to increased muscular activity associated with shivering. However, thyroid status also influences the number of Na⁺, K⁺-pumping sites and this may have been a contributory factor in the present study. In addition, the elevatedB _max suggests a greater potential for non-shivering thermogenesis associated with increased Na⁺, K⁺-ATPase concentration in the cold. Differences in relative stage of development between the four groups may help to explain the results forB _max in relation to level of energy intake.     

Inotropic effect of ouabain in hypertrophied rat heart

The contribution of the heterogeneous digitalis receptors to the inotropic effect of ouabain was studied in hypertrophied rat hearts (aortic stenosis) by using isolated Langendorff heart preparations. Development and washout of the biological effects as well as the dose/ response curves revealed two inotropic components of high and low drug sensitivity. Maximal inotropy was observed with 100 M ouabain in both control and hypertrophied rat hearts. The high-sensitivity component accounted for only one-third of the response in control hearts but for two-thirds in hypertrophied hearts. The respective apparent affinities (10–20 nM and 10–20 M) of the two inotropic components found in isolated hearts were similar to those of the high- and low-affinity Na⁺,K⁺-ATPase activities detected in isolated cardiac sarcolemma. Onset and reversion of the pharmacological effects of ouabain occurred at respective rates that were similar to those of the association and dissociation of ouabain to the Na⁺,K⁺-ATPase level. In hypertrophied heart, the high- and low-sensitivity components (or receptors) reacted seven- and threefold, respectively, more slowly than the corresponding sites in normal hearts. These alterations in inotropic responsiveness and propertries of the digitalis receptors in cardiac hypertrophy suggest that new regulations should be taken into account in the adaptation to pressure overload.     

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.     

Identification of Proteasome Components Required for Apical Localization of Chaoptin Using Functional Genomics

Abstract: the distinct localization of membrane proteins with regard to cell polarity is crucial for the structure and function of various organs in multicellular organisms. However, the molecules and mechanisms that regulate protein localization to particular subcellular domains are still largely unknown. To identify the genes involved in regulation of protein localization, the authors performed a large-scale screen using a Drosophila RNA interference (RNAi) library, by which Drosophila genes could be knocked down in a tissue- and stage-specific manner. Drosophila photoreceptor cells have a morphologically distinct apicobasal polarity, along which Chaoptin (Chp), a glycosylphosphatidylinositol (GPI)-anchored membrane protein, and the Na ⁺ , K⁺ -ATPase are localized to the apical and basolateral domains, respectively. By examining the subcellular localization of these proteins, the authors identified 106 genes whose knockdown resulted in mislocalization of Chp and Na⁺ , K⁺ -ATPase. Gene ontology analysis revealed that the knockdown of proteasome components resulted in mislocalization of Chp to the basolateral plasma membrane. These results suggest that the proteasome is involved, directly or indirectly, in selective localization of Chp to the apical plasma membrane of Drosophila photoreceptor cells.     

Erythrocyte cations and Na+,K+-ATPase pump activity in athletes and sedentary subjects

Summary The chronic effect of training on intraerythrocyte cationic concentrations and on red cell Na⁺,K⁺-ATPase pump activity was studied by comparing well-trained athletes with sedentary subjects at rest. Also the acute effect of a 50-min cross-country run on these erythrocyte measurements was studied in the athletes. At rest the intraerythrocyte potassium concentration was increased (P<0.01) in the athletes compared to that of the control subjects. The intraerythrocyte concentrations of sodium and magnesium and red cell Na⁺, K⁺-ATPase pump activity were, however, similar in the trained and the untrained subjects.As compared with the resting condition, the intraerythrocyte potassium concentration was decreased (P<0.05) after exercise in the athletes, and this was accompanied by a minor increase in the intraerythrocyte sodium concentration. Red cell Na⁺,K⁺-ATPase pump activity was slightly increased (P<0.05) after exercise.     

Training in inhibitory avoidance causes a reduction of Na+,K+-ATPase activity in rat hippocampus

Compelling evidence has indicated the involvement of Na(+),K(+)-ATPase in the mechanisms of synaptic plasticity. In the present study, we investigated the effect of inhibitory avoidance training on Na(+),K(+)-ATPase activity, at different times after training, in the rat hippocampus. Male adult Wistar rats were trained in a step-down inhibitory avoidance task and compared to those submitted to isolated footshock (0.4 mA) or placed directly onto the platform. Na(+),K(+)-ATPase activity decreased, by 60%, in hippocampus of rats sacrificed immediately after the isolated footshock, as well as immediately (0 min) and 6 h after training; this effect was not present 24 h after training. We also verified that enzyme activity was not altered in rats killed after just being on the platform. These findings suggest that Na(+),K(+)-ATPase activity may be involved in the memory consolidation of step-down inhibitory avoidance in the hippocampus.     

Water and solute secretion by the choroid plexus

The cerebrospinal fluid (CSF) provides mechanical and chemical protection of the brain and spinal cord. This review focusses on the contribution of the choroid plexus epithelium to the water and salt homeostasis of the CSF, i.e. the secretory processes involved in CSF formation. The choroid plexus epithelium is situated in the ventricular system and is believed to be the major site of CSF production. Numerous studies have identified transport processes involved in this secretion, and recently, the underlying molecular background for some of the mechanisms have emerged. The nascent CSF consists mainly of NaCl and NaHCO₃, and the production rate is strictly coupled to the rate of Na⁺ secretion. In contrast to other secreting epithelia, Na⁺ is actively pumped across the luminal surface by the Na⁺,K⁺-ATPase with possible contributions by other Na⁺ transporters, e.g. the luminal Na⁺,K⁺,2Cl⁻ cotransporter. The Cl⁻ and HCO₃ ⁻ ions are likely transported by a luminal cAMP activated inward rectified anion conductance, although the responsible proteins have not been identified. Whereas Cl⁻ most likely enters the cells through anion exchange, the functional as well as the molecular basis for the basolateral Na⁺ entry are not yet well-defined. Water molecules follow across the epithelium mainly through the water channel, AQP1, driven by the created ionic gradient. In this article, the implications of the recent findings for the current model of CSF secretion are discussed. Finally, the clinical implications and the prospects of future advances in understanding CSF production are briefly outlined.     

Studies on potassium induced coronary dilation in the isolated guinea pig heart

Summary Changes of coronary flow in the isolated perfused spontaneously beating guinea pig heart were induced by elevation of potassium concentration in the perfusion medium (4–16 meq/l). Potassium caused a dose-dependent transient increase of diastolic coronary inflow. The response was inhibited by ouabain (1.4×10^–7 M) or reduced temperature. Rubidium ions elicited almost identical vasodilator effects which were also inhibited by ouabain.Autoregulation of coronary flow, reactive hyperemia, and hypoxic coronary dilation were not significantly altered in the presence of ouabain.The results support the hypothesis that potassium as well as rubidium cause vasodilation by activating a Na⁺, K⁺-ATPase. On the other hand, they do not favour the view of an essential involvement of potassium ions in local regulation of coronary flow under the conditions studied.A preliminary report of this study was presented at the 42nd meeting of the German Physiologic Society in Hannover, Germany [Pflügers Arch.343, R20 (1973)].     

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.