Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery

Madin-Darby canine kidney cells were used to study events in the postsynthetic processing and cell surface delivery of Na,K-ATPase. The photoactivable 2-nitro-5-azidobenzoyl (NAB) derivative of ouabain and an anti-ouabain antibody were employed in experiments designed to determine the time intervals required for newly synthesized Na,K-ATPase to achieve the capacity to bind ouabain and to arrive at the cell surface. Ouabain-binding capacity was assessed in Madin Darby canine kidney cells which were pulse-labeled with [35S]methionine. At various chase intervals cells were disrupted by probe sonication and the resultant vesicles were permeabilized. Vesicles were incubated with NAB-ouabain and, following UV photolysis, solubilized and subjected to immunoprecipitation with an anti-ouabain antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates revealed that newly synthesized Na,K-ATPase can carry out type II (Mg2+ and Pi supported) ouabain binding throughout the course of its postsynthetic processing. In contrast, the ability to carry out type I (Na+, Mg2+, and ATP-supported) ouabain binding is not attained until 10 min after the completion of the sodium pump's synthesis. Experiments in which intact pulse-labeled cells were incubated with NAB-ouabain revealed that the Na,K-ATPase arrives at the cell surface as soon as 50 min after its synthesis. These results suggest that postsynthetic processing is required before the newly synthesized Na,K-ATPase can display its full repertoire of catalytic functions. This processing seems to be complete prior to the newly synthesized sodium pump's arrival at the cell surface.     

Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes ofXenopus laevis

Full-grown prophase-arrested oocytes of Xenopus laevis were treated with 50 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) that does not activate protein kinase C. The effect on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed. (i) During application of PMA, current generated by the Na+/K+ pump decreases; in addition, Cl- and K+ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4 alpha PDD was ineffective. (ii) Ouabain binding experiments demonstrate that PMA (K1/2 = 7 nM), but not 4 alpha PPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The KD value for ouabain binding is not influenced. 4 alpha PDD was ineffective. (iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area. From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.     

Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.     

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.     

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation ofXenopus oocytes

Summary During progesterone-stimulated maturation of defolliculated full-grownXenopus oocytes, the activities of the transport systems forl-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms.A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8–12×10⁹ molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K⁺ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na–K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na–K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg⁺⁺ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na⁺-dependent incorporation of³²P from -[³²P]-ATP that can be reversed by the addition of K⁺. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.     

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.     

Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.     

Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.

The cardiac glycoside ouabain inhibits Na,K-ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild-type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K-pump in Xenopus oocytes injected with wild-type and mutated alpha 1 and wild-type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K-ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000-fold compared with wild-type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.     

Ouabain binding in rectal gland ofSqualus acanthias

In an attempt to examine the mechanisms of activation of (Na, K)-ATPase when epithelial transport is stimulated, the binding of ouabain to rectal gland tissue was measured before and after stimulation with dibutyryl cAMP and theophylline. Stimulation significantly altered the characteristics of ouabain binding to slices of Squalus acanthias rectal gland and to isolated rectal gland cells, accelerating the rate of binding and increasing the amount of ouabain bound at equilibrium when low concentrations of ouabain (10(-9) to 10(-7) M) were present in the medium. Scatchard plots of ouabain binding were nonlinear, suggesting at least two classes of binding sites, one of higher and one of lower affinity. Stimulation with cAMP and theophylline appeared to increase the affinity of the high-affinity site. Ouabain binding was increased by cAMP and theophylline even in the presence of furosemide (10(-4) M) or bumetanide (10(-5) M), and when Li+ was substituted for Na+, or NO3- for Cl- -maneuvers known to inhibit rectal gland secretion. The changes in ouabain binding induced by cAMP and theophylline do not appear, therefore, to be secondary to secretory activity but may reflect a change in the configuration, environment or location of existing enzyme so as to enhance its activity. Stimulation of ouabain binding cannot be demonstrated in whole homogenates of rectal gland, indicating that intact cells are necessary for the cyclic AMP-induced increase in ouabain binding to become manifest.     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

The static magnetic field effects on ouabain H3 binding by cancer tissue

Radioactive labeled ouabain was used for estimating the static magnetic field (SMF) induced cell volume changes. Ouabain is a specific inhibitor of Na+/K+ ATPase, and can be used for estimating its quantity--thus giving information about the cell volume changes. Ouabain binding by cancer and normal glandular tissues of breast cancer patients and normal glandular tissues of healthy women was measured after exposure of tissues to SMF 0.2T. SMF exposure led to a decrease of ouabain binding in both normal and cancer tissues when ouabain concentration in the external medium was 10(-9) M, while in the case of higher concentrations of ouabain (10(-7) M, 10(-6) M) an increase of ouabain binding was seen. The normal glandular tissues of healthy women were sensitive to SMF only at the highest concentrations of ouabain used in our experiments. The SMF-induced decrease of binding at low ouabain concentrations was considered as an evidence for the dehydration effect of SMF. It is suggested that the SMF could influence the cancer cell metabolism through cell hydration changes.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase.

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.     

Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques

Insulin (0.1-10 microM) reinitiates the meiotic divisions in Rana oocytes and produces a 14-20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na+ conductance of the membrane decreases by 40-50% with a concomitant increase in 22Na+ uptake from the medium. The increased uptake of Na+ during a period of decreasing Na+ conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pHi are Na+-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li+ for Na+. Insulin hyperpolarizes Li+ or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K+-sensitive components (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [125I]Insulin binding to the plasma membrane shows a class of high-affinity sites (Kd = 87 nM) with 40-50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na+-H+ exchange resulting in an increase in intracellular pH and Na+ uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na+ conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na+ pump which together with a decrease in Na+ conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.     

Inhibition of insulin receptors by vanadate and ouabain

Insulin binding studies were performed, using cells from 5 non-obese, non-diabetic subjects, on four separate days: 2 were paired control studies to demonstrate precision, and 2 other sets were binding studies in which one incubation solution was a control and the other contained either vanadate, (10(-4) M) or ouabain (10(-4) M). For both substances tracer binding of 125I insulin was reduced significantly, 27% by vanadate and 30% by ouabain. Furthermore, at all points on the binding curve these substances inhibited binding by 18-98%, in a pattern consistent with reduced receptor number. The concentrations of vanadate or ouabain which we used did not change cell volume or inhibit trypan blue dye exclusion, as an index of cell viability. Because vanadate and ouabain inhibit Na+K+ATPase and have largely dissimilar effects on a variety of cell systems, our observations may reflect specific involvement of Na+K+ATPase in binding or closely related processes.     

Ouabain binding site in a functioning Na+/K+ ATPase

The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.     

Ouabain binding to renal tubules of the rabbit

It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)-ATPase

The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na+ + K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37-110 micrograms) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 microM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 64.7 +/- 2.0 nM and a binding capacity (Bmax) of 10.1 +/- 1.5 pmol/mg protein. In the presence of 100 microM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 +/- 2.7 nM (p less than 0.01), whereas the Bmax remained virtually unchanged (12.5 +/- 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na+ + K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.