Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland

Summary Scatchard analysis of³H ouabain bound to isolated rectal gland cells as a function of increasing ouabain concentrations produced a concave curvilinear plot that was resolved into two specific sites with either a high (I) or low (II) affinity for ouabain. Cyclic cAMP/theophylline (±furosemide, 10^–4 m) increased the amount of³H ouabain bound to the high-affinity site I. Vanadate, a phosphate congener which promotes formation of the ouabain-binding state of the enzyme, mimicked the effects of cAMP/theophylline at low concentrations of ouabain, suggesting that cAMP/theophylline increases binding to site I by enhancing the rate of turnover of resident enzyme. Enhanced⁸⁶Rb uptake seen following cAMP/theophylline administration was primarily associated with increased flux through the high-affinity ouabain site, and this stimulation was not obliterated by the co-administration of furosemide. A model was presented which suggested the presence of two noninteracting pools of enzyme or isozymes which exhibit either a high or low affinity for ouabain. Cyclic AMP both stimulated turnover via site I, and modified the kinetics of binding of³H ouabain to site II. The (ave)K _d of³H ouabain for site II was increased from 3.6 m (controls) to 0.5 m (cAMP/theophylline) and the Hill coefficient was modified from 0.45 (controls) to 1.12 (caMP/theophylline), suggesting a transition from a negative- to a noncooperative binding state. While furosemide reversed the effects of cAMP/theophylline on site II kinetics, it did not obliterate cAMP/theophylline effects on site I. This suggests that cAMP may alter the intrinsic turnover rate of this particular pool of Na,K-ATPase in shark rectal gland.     

Oxygen consumption in the rectal gland of the dogfishScyliorhinus canicula and the effects of cyclic AMP

Summary Oxygen consumption was measured in slices of dogfish (Scyliorhinus canicula) rectal gland tissue and compared with spleen and kidney. Concentrations of dibutyryl cAMP and theophylline that have previously been shown to stimulate secretion from the perfused gland and to produce a large increase in ouabain binding in this tissue, greatly increased oxygen consumption in the rectal gland but were without effect on oxygen consumption in the spleen and the kidney. Addition of theophylline alone increased rectal gland oxygen consumption and no further increase was detected on addition of cAMP at the concentration used (0.05 mmol l^–1).Ouabain (10^–4 M) significantly decreased oxygen consumption in all three tissues studied and completely abolished the increase in oxygen consumption of the rectal gland produced as a result of the addition of cAMP and theophylline.These findings are discussed in relation to the apparent specific effect of cAMP on the rectal gland and its possible mode of action in this tissue.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.     

Cyclic AMP stimulates ouabain-insensitive ion movement in shark rectal gland

Summary When the active sodium-potassium pump (Na–K-ATPase) of shark rectal glands is blocked by ouabain, the concentration of intracellular ions changes in the direction of equilibrium with extracellular fluid. These changes were examined when isolated perfused glands were in the basal state and also when they were stimulated to secrete with cAMP and theophylline, to see whether stimulation affected the passive movement of sodium, potassium and chloride across cell membranes. In basal glands 10^–4M ouabain induced an increase of 30 meq/l in intracellular [Na⁺] and a decrease in intracellular [K⁺] of about 50 meq/l after 30 min, while intracellular [Cl^–] was unchanged. In stimulated glands, these movements were exaggerated. The increase in intracellular [Na⁺] averaged 112 meq/l, and the decrease in intracellular [K⁺], 96 meq/l (P<0.01), while mean intracellular [Cl^–] rose by 80 meq/l. Furosemide, 10^–4M, partially reversed the accelerated changes in intracellular electrolytes seen after ouabain was added to stimulated glands. These results are consistent with an action of cAMP upon a ouabaininsensitive cotransport of sodium, potassium and chloride in the rectal gland, analogous to that described in avian erythrocytes.Some of these results have been previously reported in abstract form in Bull Mt Desert Isl Biol Lab (Silva et al. 1979a).     

The alpha-2 isomer of the sodium pump is inhibited by calcium at physiological levels

The inhibition of the (Na,K)ATPase by calcium was investigated in plasma membrane preparations of rat axolemma, skeletal muscle and kidney outer medulla. Ouabain titration curves demonstrated that physiological calcium (0.08-5 microM) inhibited mainly the high affinity alpha 2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40-50% of the activity with a Ki of 1.9 +/- 0.9 x 10(-7) M. In skeletal muscle high and low ouabain affinity components were present in equal amounts and calcium inhibited only the high affinity component with a Ki of 1.3 +/- 0.3 x 10(-7) M. Kidney enzyme had a low affinity for ouabain and showed very little sensitivity to calcium in the physiological range. It was demonstrated that high calcium levels inhibit the enzyme in a general sense, irrespective of the isomer, with a Ki of 6.5 +/- 6 x 10(-4) M for the kidney and 5.9 +/- 4 x 10(-4) M for the axolemma enzymes. In axolemma, enzyme activity was studied as a function of sodium concentration. Physiological calcium reduced Vmax while not significantly changing K 0.5 for sodium binding.     

Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias

Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane are closest to the mitochondria. These results establish that most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Increases in Na/K pump numbers in isolated human lymphocytes exposed to lithium in vitro. Reversal by myo-inositol and by inhibitors of protein kinase C and the Na/H antiport

Lithium (1-8 mM) caused a dose-dependent increase in the number of [3H]ouabain binding sites and in sodium/potassium (Na/K) pump activity in normal lymphocytes after incubation for 72 h. The increase in Na/K pump activity was due to an increase in the Vmax of the pump, with no change in the apparent affinity (Km) for potassium (rubidium). There was no change in the turnover number of the pump and the intracellular sodium concentration fell. The increase in [3H]ouabain binding sites was prevented by the addition of myo-inositol (10 mM), by inhibition of the protein kinase C with staurosporine (100 nM) and by inhibition of the Na/H antiport with dimethylamiloride (50 microM). These results suggest that the increase in Na/K pump activity caused by lithium is due to an increase in pump numbers and not due to increased activity of individual pumps or to an alteration in the affinity of the pumps for potassium. The increase in Na/K pump numbers and activity in lymphocytes exposed to lithium for 72 h may be related to altered Na/H antiport activity secondary to inhibition of phosphoinositol breakdown by lithium.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Mode of action of theophylline on sodium efflux in barnacle muscle fibers

Summary The response of the Na efflux in unpoisoned barnacle fibers to 10mm theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10mm theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500mm EGTA completely abolishes the biphasic action of 10mm theophylline. External application of 10mm theophylline following removal of external Ca²⁺ fails to bring about a biphasic effect. Ca²⁺ restoration, however, results in a moderate rise in the Na efflux. External application of 10mm theophylline stimulates the Na efflux into Ca²⁺-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10mm theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10mm theophylline. Injection of cAMP into ouabainpoisoned fibers, following internal application of Corbin's inhibitor and external application of 10mm theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10mm theophylline, fails to reduce the Na efflux. Fibers injected with 1mm and 100mm EGTA and exposed to 10mm theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10mm. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10mm theophylline. Theophylline (10mm) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3mm-HEPES ASW or 10mm-Ca²⁺-3mm-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa (pCa-log₁₀[Ca²⁺]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.     

Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.     

Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87)

The activity of arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-controlling enzyme in melatonin synthesis is stimulated approximately equal to 100-fold by an adrenergic cyclic AMP mechanism in both neonatal and adult rat pineal glands. This stimulation is blocked in the adult gland by the depolarizing agents ouabain (1 microM) and K+ (80 mM) (Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H. (1975) Mol. Pharmacol. 11, 241-255). In the present study pineal glands obtained from prenatal to adult rats were used; it was found that K+ (80 microM) inhibited the adrenergic stimulation of N-acetyltransferase activity at all ages but that ouabain (1 nM to 1 mM) treatment was not inhibitory early in development. In contrast, in the neonate, ouabain (1-100 nM) enhanced adrenergic induction of N-acetyltransferase activity, and ouabain treatment alone (1-1000 nM) stimulated N-acetyltransferase activity. A small stimulation was also seen at one concentration (1 nM) in the adult. Analysis of the development of high affinity ouabain binding sites and Na+,K+-ATPase activity in the intact pineal gland indicated that the developmental pattern of both resemble the development of ouabain inhibition of the adrenergic stimulation of N-acetyltransferase activity. All are low for the first few days of life, gradually increase during the next 3 weeks of life, and then approach adult levels. Similarly, ouabain (1 nM to 1 mM) had no effect on 86Rb uptake in the 2-day-old gland but blocked (IC50 congruent to 20 nM) 86Rb uptake in the adult gland. These findings indicate ouabain probably has little inhibitory effect on the norepinephrine stimulation of N-acetyltransferase activity in the neonatal because a high affinity ouabain binding form of Na+,K+-ATPase activity, similar to the alpha + form identified in rat brain, is at very low levels in the pinealocyte. Accordingly, it appears that an ouabain-insensitive mechanism in the neonatal gland maintains membrane potential and that this mechanism plays a less important role in the adult. The explanation of why ouabain alone stimulates N-acetyltransferase activity and why it enhances the effects of norepinephrine in the neonatal pineal gland might be that ouabain acts on surviving neural elements present in the gland to cause the net release of a transmitter, perhaps norepinephrine, which then stimulates N-acetyltransferase activity.     

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.     

Hydrolytic properties of the (Na+ + K+)-ATPase isozymes for beta-(2-furyl)acryloyl phosphate, a pseudosubstrate for the sodium pump

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), a synthetic substrate for the (Na+ + K+)-ATPase by the partially purified enzyme from rat brain and rat kidney, has been assessed. Using previously determined FAPase reaction conditions, it was discovered that the KI for ouabain of the alpha 2/3 isozyme of the (Na+ + K+)-ATPase was approximately 10(-5) M, while for the alpha 1 isozyme the KI was approximately 10(-3) M. These values were an order of magnitude higher (lower affinity) than the KI's for ouabain as determined when using ATP in a coupled assay for (Na+ + K+)-ATPase activity: approximately 10(-6) M and approximately 10(-4) M for the alpha 2/3 and alpha 1 isozymes, respectively. This discrepancy was alleviated by altering established reaction conditions. Previously published FAPase studies have overlooked this fact, since either the properties of the isozymes of the (Na+ + K+)-ATPase were unknown at that time, or ouabain titration profiles were never performed.     

(Na, K)ATPase activity in membrane preparations of ouabain-resistant HeLa cells.

Membrane preparations from two independent ouabain-resistant HeLa cell clones, HI-B1 and HI-C1, each appear to contain two species of (Na,K)ATPase. Two-thirds of the total (Na,K)ATPase in each mutant is indistinguishable from the enzyme in preparations of wild type cells with respect to ouabain binding, ouabain inhibition of (Na,K)ATPase activity, and dependence of ATP hydrolysis on Na, Mg, K, and ATP concentration. The remaining (Na,K)ATPase activity in the mutants is up to 1000 and 10 000 times, respectively, more resistant to ouabain than wild type enzyme. Resistance results from a lower affinity of the mutant enzymes for the inhibitor. The presence of Na, K, or Mg has little or no effect on the degree of resistance expressed by the mutant enzymes, although the resistance of the wild type enzyme varies 400-fold in the presence of different ligands. Incubation with 5 X 10(-8) M ouabain abolishes the activity of the wild type enzyme without affecting the activity of the resistant enzymes. Using this procedure we compared the parameters of ATP hydrolysis via the resistant and wild type enzymes. Ouabain-resistant (Na,K)ATPase of HI-C1 has an apparent K0.5 for potassium 3-4 times higher than that of either wild type enzyme or the resistant enzyme of HI-B1.     

Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.