Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.     

Effects of amphotericin B on ion transport proteins in airway epithelial cells

Topical intranasal application of the antifungal Amphotericin B (AmphoB) has been shown as an effective medical treatment of chronic rhinosinusitis. Because this antibiotic forms channels in lipid membranes, we considered the possibility that it affects the properties and/or cell surface expression of ion channels/pumps, and consequently transepithelial ion transport. Human nasal epithelial cells were exposed apically to AmphoB (50 microM) for 4 h, 5 days (4 h daily), and 4 weeks (4 h daily, 5 days weekly) and allowed to recover for 18-48 h. AmphoB significantly reduced transepithelial potential difference, short-circuit current, and the amiloride-sensitive current. This was not due to generalized cellular toxicity as judged from normal transepithelial resistance and mitochondrial activity, but was related to inhibitory effects of AmphoB on ion transport proteins. Thus, cells exposed to AmphoB for 4 h showed decreased apical epithelial sodium channels (ENaC) activity with no change in basolateral Na(+)K(+)-ATPase activity and K(+) conductance, and reduced amount of alphaENaC, alpha1-Na(+)K(+)-ATPase, and NKCC1 proteins at the cell membrane, but no change in mRNA levels. After a 5-day treatment, there was a significant decrease in Na(+)K(+)-ATPase activity. After a 4-week treatment, a decrease in basolateral K(+) conductance and in alphaENaC and alpha1-Na(+)K(+)-ATPase mRNA levels was also observed. These findings may reflect a feedback mechanism aimed to limit cellular Na(+) overload and K(+) depletion subsequently to formation of AmphoB pores in the cell membrane. Thus, the decreased Na(+) absorption induced by AmphoB resulted from reduced cell surface expression of the ENaC, Na(+)K(+)-ATPase pump and NKCC1 and not from direct inhibition of their activities.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis.

Sodium-potassium ATPase (Na+K(+)-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K+ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na+K(+)-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na+K(+)-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show 1) that rat testis and epididymal Na+K(+)-ATPase share some immunological determinants with the canine enzyme; 2) that the epididymal enzyme is located in the conventional basolateral position; and 3) that the distribution of Sertoli cell Na+K(+)-ATPase is probably apical and lateral rather than basal.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin stimulates the translocation of Na+/K(+)-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular 'light fraction' (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.     

State of the Na+, K+-ATPase system of the female rat brain cerebral cortex in the postnatal period during ethanol consumption in pregnancy and lactation

The ontogenetic development of the rat brain cortex Na+, K(+)-ATPase and Mg(2+)-ATPase activities under female ethanol (20% v/v) consumption in the third trimester of gestation or in postpartum period was studied. The weight characteristics (body, whole brain and cortex weight) of viable rats on the first day after birth were not affected critically by prenatal alcohol exposure. It is revealed that the delay of postnatal rat growth 10 days after birth under translactational ethanol consumption is accompanied by reliable decrease of plasma membrane Na+, K(+)-ATPase activity in comparison with control animals. The comparable decrease in activities was observed for the ouabain-sensitive and ouabain-resistant Na+, K(+)-ATPase components (isoform species). From the 20th day the differences in enzyme activity were not revealed. Mg(2+)-ATPase increases in postnatal period independent of Na+, K(+)-ATPase activity and it remains insensitive to postnatal maternal alcohol intake. It is suggested, the first ten day period of lactation is critical for ethanol effect on the developmental control of the brain Na+, K(+)-ATPase functional expression and the course of adaptive processes in the rat organism.     

Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.     

Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.     

Ethanol sensitivity of rat brain cortex Na(+), K(+)-ATPase in plasma membrane structural damage induced by sodium dodecyl sulfate

To investigate the role of rat brain cortex Na+, K(+)-ATPase plasma membrane microenvironment in ethanol effect in vitro on membrane the sensitivity of enzyme activity to alcohol was studied under membrane perturbation induced by sodium dodecyl sulfate. The increase of enzyme sensitivity to detergent inactivation in the presence of high ethanol concentrations and to alcohol inhibition after modification by Ds-Na was revealed. It is supposed that Na+, K(+)-ATPase sensitivity to ethanol is dependent on structural state of protein microenvironment in accordance with assumed differences in structural organization of the boundary lipids of the neuronal enzyme isoforms.     

The effect of antimembrane testicular antibodies on Na+, K(+)-ATPase activity in the testicles of rats of different ages]

The effect of large and small doses of rabbit antibodies specific to plasma membranes of the rat testicle cells has been studied in the experiments on Wistar rats of three age groups (preadolescent--aged 20 days, puberal--aged 5-7 months and old--aged 24-26 months). It is stated that incubation of plasma membranes by IgG fraction isolated from antimembrane testicular serum (IgG-ATCSm) in a large dose (43 g of protein G per 125 g of protein of membrane fraction) caused statistically reliable inhibition of Na+, K(+)-ATPase activity in the membranes of testicle cells of puberal and old rats. Preadolescent rats exhibit only a tendency to decrease the activity of this enzyme. Incubation of plasma membranes of testicle cells in rats of different age by small doses of IgG-ATCSm (0.43 g of protein G per 125 g of membrane protein) induced a statistically reliable increase of Na+, K(+)-ATPase activity in puberal and old animals and its slight increase in preadolescent rats. The IgG fraction isolated from normal rabbit serum (IgG-NRS) exerted a less pronounced effect on Na+, K(+)-ATPase activity parallel with retention of a tendency to a decrease of activity under the influence of large doses of the drug and to an increase with introduction of small doses.     

Chimeras of X+, K+-ATPases. The M1-M6 region of Na+, K+-ATPase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+, K+-ATPase is involved in K+ de-occlusion

In this study we reveal regions of Na(+),K(+)-ATPase and H(+),K(+)-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H(+),K(+)-ATPase was replaced by that of Na(+),K(+)-ATPase was phosphorylated in the absence of Na(+) and showed no K(+)-dependent reactions. Next, the part originating from Na(+),K(+)-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na(+),K(+)-ATPase, harbors the amino acids responsible for Na(+) specificity. Compared with Na(+),K(+)-ATPase, this chimera displayed a similar apparent Na(+) affinity, a lower apparent K(+) affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that the E(2)K form of this chimera is less stable than that of Na(+),K(+)-ATPase, suggesting that it, like H(+),K(+)-ATPase, de-occludes K(+) ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the beta-subunit are involved in K(+) occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na(+)-stimulated ATPase reaction of Na(+),K(+)-ATPase, while on the other hand it has the K(+) occlusion properties of H(+),K(+)-ATPase.     

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.     

Investigation of Na(+),K(+)-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism

Charge translocation by Na(+),K(+)-ATPase was investigated by adsorbing membrane fragments containing Na(+),K(+)-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na(+),K(+)-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na(+) and K(+) ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme alpha-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na(+),K(+)-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na(+) but not of K(+), the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na(+),K(+)-ATPase.     

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.