(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+) dependent ATPase in mouse submaxillary gland

Summary The influences of castration and of testosterone administration on (NaK)-ATPase in mouse submaxillary gland has been studied. Electron microscopical and histochemical data showing a profound change in the structure of the granular tubules after castration are also presented. Whereas testosterone administration is followed by a proliferation of the rough and smooth endoplasmic reticulum in the cells of the granular tubules, castration results in an opposite change. After castration, alkaline phosphatase, which is primarily localized in the basal membranes of the granular tubules, is drastically reduced.The tissue was fractionated, by the procedure of Katz and Epstein [15], and microsomal membranes were isolated by a modification of the procedure described by Schwartzet al. [29]. Plasma membranes were isolated by the method of Henninget al. [9]. As regards MgNaK-ATPase activity in plasma membranes, castration produced a slight decrease inV _max values. In the same membrane preparation, a completely opposite results was obtained for NaK-ATPase. In microsomal membranes a tremendous increase inV _max with a change inK _m occured when potassium chloride was varied. When sodium chloride was constant and KCl concentration varied, the same high increase inV _max was recorded, but inK _m the decrease was not so strongly pronounced. The conclusion was reached that the high specific activity of NaK-ATPase in castrated mouse submaxillary gland may be a consequence of a different amount of membrane protein per unit of tissue weight.     

Effect of insulin upon membrane-bound (Na+ + K+)-ATPase extracted from frog skeletal muscle.

1. Insulin stimulates the activity of membrane-bound ATPase isolated from frog skeletal muscle and from rat brain. The increase in activity of the membrane-bound ATPase system isolated from frog ranged from 9-8 to 53% at concentrations of Na+ (25 mM), K+ (10 mM), and ATP (2 mM) similar to those in in vivo experiments conducted previously (Moore, 1973). The increased activity of the membrane-bound ATPase is, therefore, at least as great as the insulin-induced increase in Na efflux (10-38%) from intact cells (Moore, 1973). If the concentration of Na+ is lowered to 4 mM and that of ATP lowered to 0-5 mM albumin, and 10(6) M, the increase in ouabain-inhibitable ATPase activity can reach as high as 400%. 2. Ouabain, at a concentration (10(-3) M) sufficient to inhibit stimulation of the frog ATPase by increasing Na from 4 to 25 mM, completely blocked the stimulation of ATPase activity due to insulin. 3. At 2 mM-ATP, 100 mM-Na+, and 20 mM-K+, conditions which maximally activate the (Na+ + K+)-ATPase, insulin did not increase the ATPase, activity. Stimulation was consistently seen at 10 mM-K+, 0-5 mM-ATP, and either 4 mM or 25 mM-Na+. 4. The finding that insulin does not stimulate the ATPase activity in conditions in which the (Na+ + K+)-ATPase component is maximally activated and especially the fact that ouabain can reproducibly inhibit insulin stimulation of the membrane-bound ATPase activity strongly suggest that interaction of insulin with its receptor upon the plasma membrane somehow stimulates the (Na+ + K+)-ATPase system (ouabain sensitive; ATP phosphohydrolase, EC (3.6.1.3). These results are consistent with previous studies of the effect of insulin upon Na efflux from intact cells (Moore, 1973) and support the previous conclusion that the component of Na efflux stimulated by insulin is active. The evidence suggests that insulin probably does not affect Vmax of the (Na+ + K+)-ATPase system, but may increase the affinity of the enzyme system to one or more effectors, most likely Na+, ATP, and perhaps K+. 5. Oxidized glutathione (2-7 X 10(-6) M), 10(-6) M, 10(-7) M, and 10(-8) M cyclic AMP did not affect the ATPase activity 10(-6)Malbumin, and . 6. The results are consistent with the view that the Na pump, (Na+ + K+)-ATPase, is intimately involved with the physiological action of insulin and may be transducer between the binding of insulin to its receptor on the plasma membrane and the cellular actions of insulin.     

Characterization of bumetanide-sensitive Na+ and K+ transport in rat skeletal muscle

In isolated rat soleus muscle an average of 23% of the total ²²Na influx was found to be suppressible by bumetanide (K_0.5=0.1 mm ) and furosemide (K_0.5=1 mm ), whereas the influx and efflux of ⁴²K were not affected. In extensor digitorum longus muscle, around 25% of the total ²²Na influx was suppressible by bumetanide (1 mm ). In the presence of ouabain, both diuretics decreased net intracellular accumulation of Na⁺, but caused no change in K⁺ content. In extensor digitorum longus (but not in soleus), bumetanide-suppressible ²²Na influx was stimulated by increasing extracellular osmolarity with the bumetanide having no effect on ⁴²K influx. Bumetanide-suppressible Na⁺ influx was almost abolished in Cl^--free buffer, but was unaffected by the omission of K⁺. In rat soleus, the inhibitory effects of bumetanide, amiloride and tetrodotoxin on ²²Na influx were found to be additive. The results indicate that a NaCl cotransport system is present in both fast- and slow-twitch skeletal muscles, and may participate in volume regulation. Due to the large pool of muscle cells, activation of NaKCl₂ cotransport is likely to entail the hazards of hypokalemia. The advantage of exerting volume control via NaCl cotransport is that this risk can be avoided.     

Characteristics of single Na+ channels of adult human skeletal muscle

The patch-clamp technique was used to study Na⁺ channels of human skeletal muscle. Preparations were from biopsies of quadriceps muscle from adults who were not suffering from neuromuscular diseases. Activity of Na⁺ channels was recorded from inside-out patches when the membrane potential was stepped from a holding potential of ±110mV to potential above a threshold of about ±65 mV. Single channel activity increased within minutes after hyperpolarizing the patch due to recovery from ultraslow inactivation. Up to ten Na⁺ channels were active in individual patches. Macroscopic currents were reconstructed by averaging single channel currents. The time-to-peak current declined from 1.6 ms at ±60 mV to 0.5 ms at +10 mV. The currents decayed mono-exponentially with time constants between 12.1 ms at ±60 mV and 0.4 ms at +10 mV (21°C). The conductance of single Na⁺ channels was 1.65 pS and the mean open time was voltage-dependent. At ±50 mV, the mean open time was 0.4 ms, while positive to ±10 mV it increased to values above 1 ms. In the threshold potential range, the number of openings per depolarizing pulse was larger than the number of channels under the patch-clamp pipette, indicating reopening of Na⁺ channels at this potential. Openings could be observed only rarely 10 ms after onset of depolarization and the macroscopic current produced by late openings was less than 0.1% of the peak current. Human skeletal muscle is thus suitable for investigation with the patch-clamp technique and the determination of properties of Na⁺ channels with this technique could be the basis for an assessment of possible defects of these channels in diseased muscle.     

Voltage-dependent K+ channels in the sarcolemma of mouse skeletal muscle

The voltage-dependent K⁺ channels of the mammalian sarcolemma were studied with the patch-clamp technique in intact, enzymatically dissociated fibres from the toe muscle of the mouse. With a physiological solution (containing 2.5 mM K⁺) in the pipette, depolarizing pulses imposed on a cell-attached membrane patch activated K⁺ channels with a conductance of about 17 pS. No channel activity was observed when the pipette solution contained 2mM tetraethylammonium (TEA), or 2 mM 4-aminopyridine (4-AP). Whole cell recordings from these very small muscle fibres showed the well-known delayed rectifier K⁺ outward current with a threshold of about -40mV. The whole-cell current was completely blocked by 2 mM TEA in the bath, suggesting that the TEA-sensitive channels in the patch were also delayed rectifier channels. The inactivation properties of the channels were studied in the cell-attached mode. Averaged single-channel traces showed at least two types of channels discernible by their inactivation time course at a test potential of 60 mV. The fast type inactivated with a time constant of about 150ms, the slow type with a time constant of about 400 ms. A little channel activity always remained during pulses lasting several minutes, indicating either the presence of a very slowly inactivating third type of K⁺ channel, or the tendency of the fast inactivating channels to re-open at constant voltage. No difference was seen in the single-channel amplitudes of the different types of K⁺ channels. The well characterized adenosine-5-triphosphate-(ATP)-sensitive and Ca²⁺-dependent K⁺ channels, although present, were not active under the conditions used. The results suggest that in mouse skeletal muscle the delayed rectifier channels to not only carry the outward current during excitation but are also responsible for the resting K⁺ conductance.     

(Na+, K+)-Activated ATPase in Microsomes from Mouse Pancreatic Islets

The intracellular concentration of Na⁺ in mouse pancreatic β-cells (95 meqv/l, Sehlin and Täljdedal 1974) is so high that the active pump mechanism to extrude Na⁺ must have a low activity compared with that of neurone and muscle cells (Bonting 1971).     

Inhibitory effect of insulin and cytoplasmic factor(s) on brain (Na(+) + K+) ATPase.

(Na+ + K+)ATPase activity in cerebral cortex was modulated by insulin action depending on the Mg2+ concentration. Thus, in homogenates in the presence of 1-3 mM Mg2+, insulin stimulated the enzyme, whereas in the presence of 4-6 mM Mg2+ inhibition was observed. Exposure of synaptosomal membranes to the soluble fraction resulted in inhibition of ATPase activity in a dose-dependent manner. The inhibitory effect of insulin was regulated by a cytoplasmic factor in a dose-dependent manner. Similar variations to those obtained with a crude synaptosomal fraction were obtained by using a partially purified ATPase. These results indicated the importance of soluble factors in the modulation of ATPase by insulin and add more evidence in support for a role of insulin as a neuromodulator.     

The regulation of the Na+,K+ pump in contracting skeletal muscle

Increased passive Na⁺,K⁺ fluxes necessitate an efficient activation of the Na⁺,K⁺ pump in working muscles to limit the rundown of the Na⁺,K⁺ chemical gradients and ensuing loss of excitability. Several studies have demonstrated an increase in Na⁺,K⁺-pump rate in working muscles, and in electrically stimulated muscles up to a 22-fold increase in active Na⁺,K⁺ transport has been observed. Excitation-induced increase in intracellular Na⁺ is believed to be the primary stimulus for Na⁺,K⁺ pumping in a contracting muscle. In muscles recovering from electrical stimulation, however, the activity of the pump may stay elevated even after intracellular Na⁺ has been reduced to below the resting level. Moreover, in rat soleus muscles 10-s stimulation at 60 Hz induced a 5-fold increase in the activity of the Na⁺,K⁺ pump although mean intracellular [Na⁺] was unchanged. These findings strongly suggest that a substantial part of the excitation-induced increase in Na⁺,K⁺-pump activity is caused by mechanisms other than increased intracellular [Na⁺]. The mechanism behind this activation is not clear, but may involve a change in the affinity of the Na⁺,K⁺ pump for intracellular Na⁺. In addition to intracellular [Na⁺], the Na⁺,K⁺ pump may be stimulated in contracting muscles by other factors such as catecholamines, calcitonin gene-related peptide (CGRP), free fatty acids and cytoskeletal links. Together, this activation may form a feed forward mechanism protecting muscles from loss of excitability during periods of contraction by increasing Na⁺,K⁺-pump activity prior to erosion of the Na⁺,K⁺ chemical gradients. During exercise of high intensity, however, intracellular [Na⁺] increases substantially constituting an additional stimulus for the pump.     

Immunocytochemical localization of (Na+ + K+)-ATPase in the rat hippocampus

The adult rat hippocampus was investigated by light microscopic immunocytochemistry for (Na+ + K+)-ATPase. In the CA1, CA2 and CA3 hippocampal regions, dense immunostaining for (Na+ + K+)-ATPase, exhibiting a punctate appearance, was demonstrated along the soma plasmalemma of hippocampal pyramidal cells in the stratum pyramidale, thus outlining these cells distinctly, and along dendrites extending into the stratum radiatum. (Na+ + K+)-ATPase immunostaining was dense in the neuropil of the strata oriens and radiatum of the rat hippocampus, but much lighter in the corpus callosum. Immunostaining at the periphery of pyramidal cell soma may be associated with the plexus formed by axon terminals of hippocampal basket cells.     

Inactivating ''ball'' peptide from Shaker B blocks Ca(2+)-activated but not ATP-dependent K+ channels of rat skeletal muscle.

1. Shaker B inactivating 'ball' peptide is shown to produce no detectable block of ATP-dependent K+ channels of rat skeletal muscle fibres at concentrations up to 300 microM or membrane potentials up to +30 mV. 2. The peptide does produce a voltage-dependent block of large-conductance Ca(2+)-activated K+ channels at lower concentrations (K1 = 55 microM at 0 mV). An appropriate point mutation (L7E) abolishes block. 3. Mean open times, corrected for missed closures, are little affected by the blocking peptide, but burst durations are substantially reduced. 4. The inactivating peptide increases occupancy of substates, whose amplitudes are 0.27 and 0.64 of fully open.     

Molecular stressors underlying exercise training‐induced improvements in K+ regulation during exercise and Na+,K+‐ATPase adaptation in human skeletal muscle

Despite substantial progress made towards a better understanding of the importance of skeletal muscle K⁺ regulation for human physical function and its association with several disease states (eg type‐II diabetes and hypertension), the molecular basis underpinning adaptations in K⁺ regulation to various stimuli, including exercise training, remains inadequately explored in humans. In this review, the molecular mechanisms essential for enhancing skeletal muscle K⁺ regulation and its key determinants, including Na⁺,K⁺‐ATPase function and expression, by exercise training are examined. Special attention is paid to the following molecular stressors and signaling proteins: oxygenation, redox balance, hypoxia, reactive oxygen species, antioxidant function, Na⁺,K⁺, and Ca²⁺ concentrations, anaerobic ATP turnover, AMPK, lactate, and mRNA expression. On this basis, an update on the effects of different types of exercise training on K⁺ regulation in humans is provided, focusing on recent discoveries about the muscle fibre‐type‐dependent regulation of Na⁺,K⁺‐ATPase‐isoform expression. Furthermore, with special emphasis on blood‐flow‐restricted exercise as an exemplary model to modulate the key molecular mechanisms identified, it is discussed how training interventions may be designed to maximize improvements in K⁺ regulation in humans. The novel insights gained from this review may help us to better understand how exercise training and other strategies, such as pharmacological interventions, may be best designed to enhance K⁺ regulation and thus the physical function in humans.     

Coexistence of two classes of glibenclamide-inhibitable ATP-regulated K+ channels in avian skeletal muscle

Avian skeletal muscle expresses two types of ATP-sensitive K⁺ channels which have a unitary conductance of 15 pS. These K⁺ channels can be distinguished pharmacologically by their high or low sensitivity to the antidiabetic sulphonylurea blocker glibenclamide. Both channels are activated by the K⁺ channel opener cromakalim. Chick skeletal muscle expresses high-affinity binding sites for [³H]glibenclamide (K _d=0.6 nM) which presumably correspond to the ATP-sensitive K⁺ channels with the greatest sensitivity to glibenclamide. The density of these high-affinity binding sites varies during muscle development. The maximum density (500 fmol/mg protein) appears at 16 days in ovo, i.e. at a period when myoblasts have di¤erentiated into myotubes and when innervation of myotubes has started. After this maximum, the level of [³H]glibenclamide-binding sites decreases to a plateau value of 100 fmol/mg protein at 2–5 days postnatal. When muscle cells are put in cultures, the high-affinity binding sites disappear rapidly. Neither glibenclamide nor cromakalim have any effect on normal physiological chick muscle contraction. They have no effect on contracture and/or ⁸⁶Rb⁺ efflux produced by metabolic poisoning.     

Changes in Na+, K+-adenosinetriphosphatase,citrate synthase and K+ in sheep skeletal muscle during immobilization and remobilization

The K⁺ balance and muscle activity seem to interact in a complex way with regard to regulating the muscle density of Na⁺-K⁺ pumps. The effect of immobilization was examined in ten sheep that had low muscle K⁺ content. Three additional sheep served as untreated controls. After being brought from pasture to sheep stalls one hindlimb was immobilized in a plaster splint for 9 weeks, and in five of the animals remobilization was carried out for a further 9 weeks. The weight bearing of the leg in plaster was recorded by a force plate. Open muscle biopsies from the vastus lateralis muscle were obtained before the study, after 9 weeks of immobilization, and after another 9 weeks of remobilization. The Na⁺-K⁺ pump density was measured as [³H]-ouabain binding to intact tissue, and citrate synthase activity was measured in tissue homogenate. The tissue content of K⁺ was measured in fat-free dried tissue. Muscle K⁺ content increased linearly by almost 70% through the 18-week period independent of intervention. Immobilization reduced thigh circumference by 8% (P < 0.05) . A slight decrease in the area of type I fibres at 9 weeks and a slight increase at 18-weeks was found. The [³H]-ouabain binding was reduced by 39% and 22% in the immobilized and control legs, respectively, whereas citrate synthase activity was reduced by about 30% in both legs after 9 weeks of immobilization. During remobilization both the [³H]-ouabain binding and the citrate synthase activity increased to the same level as in the control animals. The plaster cast significantly reduced mass bearing of the immobilized leg, and a corresponding reduction in muscle activity must be assumed to have occurred in both legs as judged from citrate synthase activity. We concluded from this study that the reduction in the [³H]-ouabain binding during immobilization independent of an increase in muscle K⁺ content points to muscle activity as a strong stimulus for control of Na⁺-K⁺ bump density.     

Isoprenaline-stimulated differential adrenergic response of K+ channels in skeletal muscle under hypokalaemic conditions

The mechanism underlying the hyperpolarization induced by isoprenaline in mouse lumbrical muscle fibres was studied using cell-attached patch and intracellular membrane potential ( V(m)) recordings. Sarcolemmal inwardly rectifying K(+) channels (K(IR): 45 pS) and Ca(2+)-activated K(+) channels (BK: 181 pS) were identified. Exposure to isoprenaline closed K(IR) channels and increased BK channel activity. This increase was observed as a shift from 50 to -40 mV in the voltage dependence of channel activation. Isoprenaline prevented hysteresis of V(m) when the extracellular [K(+)] fell below 3.8 mM. This hysteresis was due to the properties of the K(IR). The effects of chloride transport and isoprenaline on V(m) did not interact purely competitively, but isoprenaline could prevent the depolarization induced by hyperosmotic media equally as well as bumetanide, which inhibits the Na(+)/K(+)/2Cl(-) cotransporter. In lumbrical muscle this leads to hyperpolarization, but this might vary among muscles. The switch from K(IR) to BK as the component of total K(+) conductance was due to isoprenaline.     

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.     

Fuzzy space and control of Na+, K+-pump rate in heart and skeletal muscle

Since intracellular Na⁺ activity (aⁱ_Na) is one important determinant of Na⁺, K⁺-pump rate as well as excitability and the finely tuned contractility, it is surprising that the relation between aⁱ_Na and pump rate reported by different authors has k_0.5 varying between 10 and 40 mmol L^-1. Other data also point to a variable relation between pump rate and aⁱ_Na. During stimulation of isolated rat soleus muscles at 2 Hz, ouabain-sensitive ⁸⁶Rb uptake was increased in spite of the intracellular Na⁺ remaining unaltered. In isolated cardiomyocytes, a transient Na⁺, K⁺-pump current was observed upon activation by extracellular K⁺ in spite of good control of aⁱ_Na. Na⁺-loaded, isolated, sheep cardiac Purkinje fibres initially hyperpolarized over a period of up to 1 min upon activation of the Na⁺, K⁺ pump with no detectable change of aⁱ_Na. These examples are compatible with the existence of a micro-environment close to the membrane where diffusion is slower than in the rest of the cytosol, so that local aⁱ_Na may fluctuate or gradients may develop as visualized by Wendt-Gallitelli et al. (1993). We conclude that the reported relationships between Na⁺, K⁺-pump rate and aⁱ_Na in intact cells probably underestimate the true affinity of the Na⁺, K⁺ pump for Na⁺ due to a functional diffusion barrier beneath the sarcolemma, and also because of incomplete cell dialysis in whole-cell voltage clamp experiments. The Na⁺, K⁺ pump seems to be preferentially supplied with Na⁺ from the outside through neighbouring channels and transporters.     

Na+-K+ pump regulation and skeletal muscle contractility

In skeletal muscle, excitation may cause loss of K+, increased extracellular K+ ([K+]o), intracellular Na+ ([Na+]i), and depolarization. Since these events interfere with excitability, the processes of excitation can be self-limiting. During work, therefore, the impending loss of excitability has to be counterbalanced by prompt restoration of Na+-K+ gradients. Since this is the major function of the Na+-K+ pumps, it is crucial that their activity and capacity are adequate. This is achieved in two ways: 1) by acute activation of the Na+-K+ pumps and 2) by long-term regulation of Na+-K+ pump content or capacity. 1) Depending on frequency of stimulation, excitation may activate up to all of the Na+-K+ pumps available within 10 s, causing up to 22-fold increase in Na+ efflux. Activation of the Na+-K+ pumps by hormones is slower and less pronounced. When muscles are inhibited by high [K+]o or low [Na+]o, acute hormone- or excitation-induced activation of the Na+-K+ pumps can restore excitability and contractile force in 10-20 min. Conversely, inhibition of the Na+-K+ pumps by ouabain leads to progressive loss of contractility and endurance. 2) Na+-K+ pump content is upregulated by training, thyroid hormones, insulin, glucocorticoids, and K+ overload. Downregulation is seen during immobilization, K+ deficiency, hypoxia, heart failure, hypothyroidism, starvation, diabetes, alcoholism, myotonic dystrophy, and McArdle disease. Reduced Na+-K+ pump content leads to loss of contractility and endurance, possibly contributing to the fatigue associated with several of these conditions. Increasing excitation-induced Na+ influx by augmenting the open-time or the content of Na+ channels reduces contractile endurance. Excitability and contractility depend on the ratio between passive Na+-K+ leaks and Na+-K+ pump activity, the passive leaks often playing a dominant role. The Na+-K+ pump is a central target for regulation of Na+-K+ distribution and excitability, essential for second-to-second ongoing maintenance of excitability during work.