Regulation of the Na+/K+-ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na⁺/K⁺-ATPase or Na⁺/K⁺-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na⁺ from cells in exchange for K⁺ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits ( and ) each expressed in several isoforms. Many hormones regulate Na⁺/K⁺ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na⁺/K⁺-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na⁺ concentration, altered Na⁺ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na⁺/K⁺-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na⁺/K⁺-ATPase control by insulin in diabetes and related disorders is addressed.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Na+, K+-ATPase Isozyme Diversity; Comparative Biochemistry and Physiological Implications of Novel Functional Interactions

Na⁺, K⁺-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na⁺, K⁺-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na⁺, K⁺-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na⁺, K⁺-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the subunit isoformspossess considerably different kinetic properties and modes of regulationand the subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na⁺, K⁺-ATPase isozymes. This review focuseson recent developments in Na⁺, K⁺-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa⁺, K⁺-ATPase function.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: Dependence on medium osmolality and regulation by signalling systems

Summary To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and ⁸⁶Rb and ²²Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 l per 10⁶ cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P< 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by 7%, P<0.05) and by ethylisopropyl amiloride (by 13%, P< 0.005). Ouabain-sensitive ⁸⁶Rb influx was decreased by 30–40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an eightfold increase in bumetanide-inhibited ⁸⁶Rb influx and ⁸⁶Rb efflux. The (ouabain and bumetanide)-insensitive component of ⁸⁶Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive ⁸⁶Rb efflux was increased by 1.5–2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited ²²Na influx was increased by sixfold following osmotic-shrinkage of VSMC. The data show that both Na⁺/H⁺ exchange and Na⁺/K⁺/2Cl^– cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na⁺/K⁺/2Cl^– cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na⁺/K⁺/2Cl^– cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na⁺/H⁺ exchange activity. NaF increased Na⁺/H⁺ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na⁺/K⁺/2Cl^– cotransporter and the Na⁺/H⁺ exchanger.The authors gratefully acknowledge the financial support of the Swiss National Foundation, grant No. 3.817.087. Bernadette Weber is thanked for preparing the figures.     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Fluorescent labelling of Na+,K+-ATPase in intact cells by use of a fluorescent derivative of ouabain: Salinity and teleost chloride cells

Summary Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na⁺,K⁺-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 M anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 M anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na⁺,K⁺-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na⁺,K⁺-ATPase in other tissues and species.     

Effect of Extracellular pH on Presteady-State and Steady-State Current Mediated bythe Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> Pump

A ouabain sensitive inward current occurs in Xenopus oocytes in Na⁺ and K⁺ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pH_o) produces a conducting pathway through the Na⁺/K⁺ pump that is permeable to H⁺ and blocked by [Na⁺]_o. An alternative suggestion is that the current is mediated by an electrogenic H⁺-ATPase. Here we investigate the effect of pH_o and [Na⁺]_o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pH_o the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pH_o, as predicted by a model in which protonation of the Na⁺/K⁺ pump reduces the energy barrier between the internal solution and the Na⁺ occluded state. The model also predicts that acidic pH_o increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. ²²Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pH_o suggest that both Na⁺ and H⁺ are permeant. The acid-induced inward current is reduced by high [Na⁺]_o, consistent with block by Na⁺. A least squares analysis predicts that H⁺ is four orders of magnitude more permeant than Na⁺, and that block occurs when 3 Na⁺ ions occupy a low affinity binding site (K _0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na⁺ and H⁺ through the Na⁺/K⁺ pump.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane