The Na(+)/Ca(2+)-exchanger: an essential component in the mechanism governing cardiac steroid-induced slow Ca(2+) oscillations

Plasma membrane (PM) Na⁺, K⁺-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na⁺, K⁺-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca²⁺ oscillations in COS-7 cells by enhancing the interactions between Na⁺, K⁺-ATPase, inositol 1,4,5-trisphosphate receptor (IP₃R) and Ankyrin B (Ank-B) to form a Ca²⁺ signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca²⁺ oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca²⁺ oscillations. These oscillations depend on extracellular Ca²⁺ concentrations [Ca²⁺]_out and are inhibited by Ni²⁺. Furthermore, we found that these slow oscillations are Na⁺_out dependent, abolished by Na⁺/Ca²⁺ exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca²⁺ oscillations in COS-7 cells.     

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, aspecific inhibitor ofNa⁺-K⁺-ATPase,was coupled to epoxy agarose via a 13-atom spacer to make an affinitycolumn that specifically bindsNa⁺-K⁺-ATPase.Na⁺-K⁺-ATPasefrom rat and dog kidney was bound to the column and was eluted as afunction of enzyme conformation, altered by adding specificcombinations of ligands.Na⁺-K⁺-ATPasefrom both sources bound to the column in the presence of Na + ATP + Mgand in solutions containing 30 mM K. No binding was observed in thepresence of Na or Na + ATP. These experiments suggest thatNa⁺-K⁺-ATPasebinds to the column under the same conditions that it binds tountethered ouabain.Na⁺-K⁺-ATPasealready bound to the column was competitively eluted with excess freeNa + ouabain or with Na + ATP. The latter eluted active enzyme. Forcomparable amounts of boundNa⁺-K⁺-ATPase,Na + ouabain and Na + ATP eluted more rat than dogNa⁺-K⁺-ATPase,consistent with the lower affinity of the ratNa⁺-K⁺-ATPasefor ouabain. The ouabain-affinity column was used to purify activeNa⁺-K⁺-ATPasefrom rat kidney microsomes and rat adrenal glomerulosa cells. Thespecific activity of the kidney enzyme was increased from ~2 to 15 µmolP_i · mg¹ · min¹.Na⁺-K⁺-ATPasepurified from glomerulosa cells that were prelabeled with [³²P]orthophosphatewas phosphorylated on the -subunit, suggesting that these cellscontain a kinase that phosphorylatesNa⁺-K⁺-ATPase.

     

Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts

Insulin stimulates K⁺ transport by theNa⁺-K⁺-ATPase in human fibroblasts. In othercell systems, this action represents an automatic response to increasedintracellular [Na⁺] or results from translocation oftransporters from an intracellular site to the plasma membrane. Here weevaluate whether these mechanisms are operative in human fibroblasts.Human fibroblasts expressed the ₁ but not the₂ and ₃ isoforms ofNa⁺-K⁺-ATPase. Insulin increased the influx ofRb⁺, used to trace K⁺ entry, but did not modifythe total intracellular content of K⁺, Rb⁺, andNa⁺ over a 3-h incubation period. Ouabain increasedintracellular Na⁺ more rapidly in cells incubated withinsulin, but this increase followed insulin stimulation ofRb⁺ transport. Bumetanide did not prevent the increasedNa⁺ influx or stimulation ofNa⁺-K⁺-ATPase. Stimulation of theNa⁺-K⁺- ATPase by insulin did not produce anymeasurable change in membrane potential. Insulin did not affect theaffinity of the pump toward internal Na⁺ or the number ofmembrane-bound Na⁺-K⁺-ATPases, as assessed byouabain binding. By contrast, insulin slightly increased the affinityof Na⁺-K⁺-ATPase toward ouabain. Phorbol estersdid not mimic insulin action on Na⁺-K⁺-ATPaseand inhibited, rather than stimulated, Rb⁺ transport. Theseresults indicate that insulin increases the turnover rate ofNa⁺-K⁺-ATPases of human fibroblasts withoutaffecting their number on the plasma membrane or modifying theirdependence on intracellular [Na⁺].

     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa⁺-K⁺-ATPase, that partial inhibition ofNa⁺-K⁺-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa⁺-K⁺-ATPase, but not in cells expressingouabain-insensitive Na⁺-K⁺-ATPase, whereasinhibition of Na⁺-K⁺-ATPase by loweringextracellular K⁺ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa⁺-K⁺-ATPase significantly attenuatedattachment. Na⁺-K⁺-ATPase inhibition caused asustained increase in the intracellular Ca²⁺ concentrationthat obscured Ca²⁺ transients observed in untreated cellsduring attachment. Inhibitors of Ca²⁺ transporterssignificantly decreased attachment, but inhibition ofNa⁺/H⁺ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa⁺-K⁺-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca²⁺.

     

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na(+)-K(+)-ATPase activity

The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type. protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

Functional expression of putative H+-K+-ATPase from guinea pig distal colon

A guinea pig cDNAencoding the putative colonicH⁺-K⁺-ATPase-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonicH⁺-K⁺-ATPasewas cotransfected with cDNA for either rabbit gastric H⁺-K⁺-ATPaseor TorpedoNa⁺-K⁺-ATPase-subunit. In both expressions,Na⁺-independent,K⁺-dependent ATPase(K⁺-ATPase) activity was detectedin the membrane fraction of the cells, with a Michaelis-Menten constantfor K⁺ of 0.68 mM. The expressedK⁺-ATPase activity was inhibitedby ouabain, with its IC₅₀ value being 52 µM. However, the activity was resistant to Sch-28080, aninhibitor specific for gastricH⁺-K⁺-ATPase.The ATPase was not functionally expressed in the absence of the-subunits. Therefore, it is concluded that the cDNA encodes thecatalytic subunit (-subunit) of the colonicH⁺-K⁺-ATPase.Although the -subunit of the colonicH⁺-K⁺-ATPasehas not been identified yet, both gastricH⁺-K⁺-ATPaseandNa⁺-K⁺-ATPase-subunits were found to act as a surrogate for the colonic -subunit for the functional expression of the ATPase. The present colonicH⁺-K⁺-ATPasefirst expressed in mammalian cells showed the highest ouabainsensitivity in expressed colonicH⁺-K⁺-ATPasesso far reported (rat colonic inXenopus oocytes had an IC₅₀ = 0.4-1mM; rat colonic in Sf9 cells had no ouabain sensitivity).

     

Beta-subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Previous studies showed the presence of a significant fraction of Na⁺-K⁺-ATPase -subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na⁺-K⁺-ATPase with its signaling partners. Because both - and -subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na⁺-K⁺-ATPase, we have examined the relative distribution of two major subunit isoforms (₁ and ₁) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na₂CO₃ or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of ₁-subunits and 80–90% of ₁-subunits. Use of Na₂CO₃ was shown to inactivate Na⁺-K⁺-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained 75% of total myocyte Na⁺-K⁺-ATPase activity. Sealed isolated caveolae exhibited active Na⁺ transport. Confocal microscopy supported the presence of ,-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na⁺-K⁺ fluxes, and the sites where the pumping and signaling functions of Na⁺-K⁺-ATPase are integrated. Preferential concentration of ₁-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of ₁ and ₁ in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na⁺-K⁺-ATPase in cardiac caveolae. cardiac myocyte; caveolin; oligomer; ouabain; sodium pump     

九种常用杀虫剂对二化螟线粒体ATPase活力的抑制作用

研究了二化螟Chilo suppressalis线粒体Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的生物化学性质以及9种常用杀虫剂对这两种酶活性的影响。结果表明, 二化螟线粒体Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的最适反应条件为pH值7.4,温度37℃。 Na⁺-K⁺-ATPase的米氏常数（Km）为0.42 mmol/L,最大反应速度（Vmax）为302.47 nmol/(min·mg) 。Ca^2＋-Mg^2＋-ATPase的Km为0.40 mmol/L,Vmax为128.04 nmol/(min·mg)。药剂浓度为1×10^-4 mol/L时,5种菊酯类杀虫剂对离体ATPase活性抑制的顺序为：溴氰菊酯>联苯菊酯>百树菊酯>三氟氯氰菊酯和氟硅菊酯;对二化螟Na⁺-K⁺-ATPase的抑制率分别为40.12％、39.69％、27.27％、19.49％和18.71％;对Ca^2＋-Mg^2＋-ATPase的抑制率分别为29.27％、23.78％、19.88％、11.64%和14.34％。硫丹对二化螟Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的抑制率均为17.46％。甲胺磷和呋喃丹对Ca^2＋-Mg^2＋-ATPase的抑制率分别为27.16％和17.42％,对Na⁺-K⁺-ATPase则几乎没有抑制作用。实验结果还表明, 在1.6×10^-7～1×10^-4 mol/L的浓度范围内,上述9种杀虫剂对二化螟ATPase活性的抑制率存在明显的剂量-效应关系。     

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na⁺ and Cl^– across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na⁺-K⁺-Cl^– cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na⁺-K⁺-Cl^– cotransporter, as well as on Na⁺-K⁺-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na⁺-K⁺-Cl^– cotransporter and Na⁺-K⁺-ATPase activities as bumetanide-sensitive and ouabain-sensitive ⁸⁶Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [³H]-3-O-methyl-D-glucose and [¹⁴C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O₂ (vs. 19% normoxic O₂) significantly increased cotransporter activity as did 7.5% or 2% O₂ for up to 2 h. This was not associated with reduction in Na⁺-K⁺-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na⁺-K⁺-Cl^– cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume     

Alloxan-induced diabetes reduces sarcolemmal Na+-K+ pump function in rabbit ventricular myocytes

The effect of diabetes on sarcolemmal Na⁺-K⁺ pump function is important for our understanding of heart disease associated with diabetes and design of its treatment. We induced diabetes characterized by hyperglycemia but no other major metabolic disturbances in rabbits. Ventricular myocytes isolated from diabetic rabbits and controls were voltage clamped and internally perfused with the whole cell patch-clamp technique. Electrogenic Na⁺-K⁺ pump current (I_p, arising from the 3:2 Na⁺-to-K⁺ exchange ratio) was identified as the shift in holding current induced by Na⁺-K⁺ pump blockade with 100 µmol/l ouabain in most experiments. There was no effect of diabetes on I_p recorded when myocytes were perfused with pipette solutions containing 80 mmol/l Na⁺ to nearly saturate intracellular Na⁺-K⁺ pump sites. However, diabetes was associated with a significant decrease in I_p measured when pipette solutions contained 10 mmol/l Na⁺. The decrease was independent of membrane voltage but dependent on the intracellular concentration of K⁺. There was no effect of diabetes on the sensitivity of I_p to extracellular K⁺. Pump inhibition was abolished by restoration of euglycemia or by in vivo angiotensin II receptor blockade with losartan. We conclude that diabetes induces sarcolemmal Na⁺-K⁺ pump inhibition that can be reversed with pharmacological intervention. sodium transport; insulin; angiotensin II; cardiomyopathy; hyperglycemia     

Mechanism of depression in cardiac sarcolemmal Na+-K+-ATPase by hypochlorous acid

Oxidative stress during pathological conditionssuch as ischemia-reperfusion is known to promote the formationof hypochlorous acid (HOCl) in the heart and to result in depression ofcardiac sarcolemmal (SL)Na⁺-K⁺-ATPaseactivity. In this study, we examined the direct effects of HOCl on SLNa⁺-K⁺-ATPasefrom porcine heart. HOCl decreased SLNa⁺-K⁺-ATPaseactivity in a concentration- and time-dependent manner. Characterization ofNa⁺-K⁺-ATPaseactivity in the presence of different concentrations of MgATP revealeda decrease in the maximal velocity(V_max) value, without a change in affinity for MgATP on treatment of SL membranes with 0.1 mM HOCl. TheV_max value ofNa⁺-K⁺-ATPase,when determined in the presence of different concentrations ofNa⁺, was also decreased, butaffinity for Na⁺ was increasedwhen treated with HOCl. Formation of acylphosphate by SLNa⁺-K⁺-ATPasewas not affected by HOCl. Scatchard plot analysis of[³H]ouabain bindingdata indicated no significant change in the affinity or maximum bindingcapacity value for ouabain binding following treatment of SL membraneswith HOCl. Western blot analysis ofNa⁺-K⁺-ATPasesubunits in HOCl-treated SL membranes showed a decrease (34 ± 9%of control) in the ₁-subunitwithout any change in the ₁- or₂-subunits. These data suggestthat the HOCl-induced decrease in SLNa⁺-K⁺-ATPaseactivity may be due to a depression in the₁-subunit of the enzyme.

     

Differential Properties Between an Endogenous Brain Na+, K+-ATPase Inhibitor and Ouabain

By means of a Sephadex G-50 column and anionic exchange HPLC a cerebral cortex soluble fraction (II-E) which highly inhibits neuronal Na⁺-K⁺-ATPase activity has been previously obtained. Herein, II-E properties are compared with those of the cardenolide ouabain, the selective and specific Na⁺, K⁺-ATPase inhibitor. It was observed that alkali treatment destroyed II-E but not ouabain inhibitory activity. II-E presented a maximal absorbance at 265 nm both at pH 7 and pH 2 which diminished at pH 10. Ouabain showed a maximum at 220 nm which was not altered by alkalinization. II-E was not retained in a C-18 column, indicating its hydrophilic nature, whereas ouabain presented a 26-min retention time in reverse phase HPLC. Therefore, it is concluded that the inhibitory factor present in II-E is structurally different to ouabain.     

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells

Two H⁺-K⁺-ATPase isoforms are presentin kidney: the gastric, highly sensitive to Sch-28080, and the colonic,partially sensitive to ouabain. Upregulation of Sch-28080-sensitiveH⁺-K⁺-ATPase, or "gastric"H⁺-K⁺-ATPase, has been demonstrated inhypokalemic rat inner medullary collecting duct cells (IMCDs).Nevertheless, only colonic H⁺-K⁺-ATPase mRNAand protein abundance increase in this condition. This study wasdesigned to determine whether Sch-28080 inhibits transporters otherthan the gastric H⁺-K⁺-ATPase. In the presenceof bumetanide, Sch-28080 (200 µM) and ouabain (2 mM) inhibited⁸⁶Rb⁺ uptake (>90%). That⁸⁶Rb⁺ uptake was almost completely abolished bySch-28080 indicates an effect of this agent on theNa⁺-K⁺-ATPase. ATPase assays in membranes, orlysed cells, demonstrated sensitivity to ouabain but not Sch-28080.Thus the inhibitory effect of Sch-28080 was dependent on cellintegrity. ⁸⁶Rb⁺-uptake studies withoutbumetanide demonstrated that ouabain inhibited activity by only50%. Addition of Sch-28080 (200 µM) blocked all residualactivity. Intracellular ATP declined after Sch-28080 (200 µM) butrecovered after removal of this agent. In conclusion, highconcentrations of Sch-28080 inhibit K⁺-ATPase activity inmouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits

TheNa⁺-K⁺-ATPase is a heterodimeric plasmamembrane protein responsible for cellular ionic homeostasis in nearlyall animal cells. It has been shown that some insect cells (e.g., HighFive cells) have no (or extremely low)Na⁺-K⁺-ATPase activity. We expressed sheepkidney Na⁺-K⁺-ATPase - and -subunitsindividually and together in High Five cells via the baculovirusexpression system. We used quantitative slot-blot analyses to determinethat the expressed Na⁺-K⁺-ATPase comprisesbetween 0.5% and 2% of the total membrane protein in these cells.Using a five-step sucrose gradient (0.8-2.0 M) to separate theendoplasmic reticulum, Golgi apparatus, and plasma membrane fractions,we observed functional Na⁺ pump molecules in each membranepool and characterized their properties. Nearly all of the expressedprotein functions normally, similar to that found in purified dogkidney enzyme preparations. Consequently, the measurements describedhere were not complicated by an abundance of nonfunctionalheterologously expressed enzyme. Specifically, ouabain-sensitive ATPaseactivity, [³H]ouabain binding, and cation dependencieswere measured for each fraction. The functional properties of theNa⁺-K⁺-ATPase were essentially unaltered afterassembly in the endoplasmic reticulum. In addition, we measuredouabain-sensitive ⁸⁶Rb⁺ uptake in whole cellsas a means to specifically evaluateNa⁺-K⁺-ATPase molecules that were properlyfolded and delivered to the plasma membrane. We could not measure anyouabain-sensitive activities when either the -subunit or -subunitwere expressed individually. Immunostaining of the separate membranefractions indicates that the -subunit, when expressed alone, isdegraded early in the protein maturation pathway (i.e., the endoplasmicreticulum) but that the -subunit is processed normally and deliveredto the plasma membrane. Thus it appears that only the -subunit hasan oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the - heterodimer within theendoplasmic reticulum apparently does not require a Na⁺pump-specific chaperone.

     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na⁺,K⁺-ATPase activity, revealed the presenceof a ouabain-sensitive Na⁺,K⁺-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa⁺,K⁺-ATPase, which seems likely to be involved in the effluxof Na⁺ ions. (Received February 7, 1995; Accepted August 9, 1995)     

Modulation of embryonic NA+-K+-ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K⁺) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose-dependent negative effect of increasing K⁺ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two-cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-AT-Pase), we used two other conditions known to inhibit the Na⁺-K⁺-ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K⁺ concentrations (1.4 mM) or in medium supplemented with ouabain (50 μM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K⁺ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K⁺ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na⁺-K⁺-ATPase during the two-cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage. © 1993 Wiley-Liss, Inc.