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.

     

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).

     

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+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its alpha - and beta -subunits

The mechanisms involved in receptor-mediated inhibition ofNa⁺-K⁺-ATPaseremain poorly understood. In this study, we evaluate whether inhibitionof proximal tubuleNa⁺-K⁺-ATPaseactivity by dopamine is linked to its removal from the plasma membraneand internalization into defined intracellular compartments.Clathrin-coated vesicles were isolated by sucrose gradientcentrifugation and negative lectin selection, and early and lateendosomes were separated on a flotation gradient. Inhibition ofNa⁺-K⁺-ATPaseactivity by dopamine, in contrast to its inhibition by ouabain, wasaccompanied by a sequential increase in the abundance of the-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocationbetween these organelles. A similar pattern was found for the-subunit. The increased incorporation of both subunits in allcompartments was blocked by calphostin C. The results demonstrate thatthe dopamine-induced decrease inNa⁺-K⁺-ATPaseactivity in proximal tubules is associated with internalization of its- and -subunits into early and late endosomes via aclathrin-dependent pathway and that this process is protein kinase Cdependent. The presence ofNa⁺-K⁺-ATPasesubunits in endosomes suggests that these compartments may constitutenormal traffic reservoirs during pump degradation and/orsynthesis.

     

Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport: role of Cl- and F-actin

Previous data indicate that adenosine 3',5'-cyclicmonophosphate activates the epithelial basolateralNa⁺-K⁺-Clcotransporter in microfilament-dependent fashion in part by direct action but also in response to apicalCl loss (due to cellshrinkage or decreased intracellularCl). To further addressthe actin dependence ofNa⁺-K⁺-Clcotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed.Na⁺-K⁺-Clcotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellularCl concentration and alsoto activation of K⁺ andCl efflux pathways.Stabilization of actin with phalloidin blunted cotransporter activationby hypotonicity and abolished hypotonic activation ofK⁺ andCl efflux. However,phalloidin did not prevent activation of cotransport by hypertonicityor isosmotic reduction of extracellularCl. Conversely, hypertonicbut not hypotonic activation was attenuated by the microfilamentdisassembler cytochalasin D. The results emphasize the complexinterrelationship among intracellularCl activity, cell volume,and the actin cytoskeleton in the regulation of epithelialCl transport.

     

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells

The function of the apicalNa⁺-K⁺-2Clcotransporter in mammalian choroid plexus (CP) is uncertain andcontroversial. To investigate cotransporter function, we developed anovel dissociated rat CP cell preparation in which single, isolatedcells maintain normal polarized morphology. Immunofluorescencedemonstrated that in isolated cells theNa⁺-K⁺-ATPase,Na⁺-K⁺-2Clcotransporter, and aquaporin 1 water channel remained localized to thebrush border, whereas theCl/HCO₃(anion) exchanger type 2 was confined to the basolateral membrane. Weutilized video-enhanced microscopy and cell volume measurementtechniques to investigate cotransporter function. Application of 100 µM bumetanide caused CP cells to shrink rapidly. Elevation ofextracellular K⁺ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling wasblocked completely by Na⁺ removalor by addition of 100 µM bumetanide. Exposure of CP cells to 5 mMBaCl₂ induced rapid swelling thatwas inhibited by 100 µM bumetanide. We conclude that the CPcotransporter is constitutively active and propose that it functions inseries with Ba²⁺-sensitiveK⁺ channels to reabsorbK⁺ from cerebrospinal fluid to blood.

     

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⁺].

     

Regulation of Na(+)-K(+)-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

Na⁺-K⁺- ATPase -subunitsin basolateral membrane vesicles (BLMVs) purified from rat parotidglands were ³²P-labeled within 5 s by incubation with[-³²P]ATP at 37°C in the presence of cAMP, but nolabeling occurred without cAMP. Phosphorylation ofNa⁺-K⁺-ATPase was associated with a decrease inits activity. This -subunit phosphorylation disappeared when BLMVswere briefly incubated with cAMP and subsequent washing before theincubation with [-³²P]ATP, indicating that catalyticsubunit of protein kinase A (PKA) associated to BLMVs via binding withits RII regulatory subunit anchored on the membrane. In theabsence of cAMP, a PKA catalytic subunit readily reassociated with themembrane-bound RII subunit. HT-31 peptide inhibited theNa⁺-K⁺-ATPase phosphorylation by membrane-boundendogenous PKA, indicating an involvement of A-kinase anchoring protein(AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and anRII overlay assay and was coimmunoprecipitated by anti-RII antibody.These results show that Na⁺-K⁺-ATPase of ratparotid gland acinar cells is regulated in vivo by membrane-anchoredPKA via AKAP rather than by free cytosolic PKA.

     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

K+/Na+ antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation

Tissue-distinct interactions of theNa⁺-K⁺-ATPasewith Na⁺ andK⁺, independent ofisoform-specific properties, were reported previously (A. G. Therien,N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol.Chem. 271: 7104-7112, 1996). In this paper, wedescribe a detailed analysis of tissue-specific kinetics particularlyrelevant to regulation of pump activity by intracellularK⁺, namelyK⁺ inhibition at cytoplasmicNa⁺ sites. Our results show thatthe order of susceptibilities of ₁ pumps of various rat tissuestoK⁺/Na⁺antagonism, represented by the ratio of the apparent affinity forNa⁺ binding at cytoplasmicactivation sites in the absence ofK⁺ to the affinity constant forK⁺ as a competitive inhibitor ofNa⁺ binding at cytoplasmic sites,is red blood cell < axolemma  rat₁-transfected HeLa cells < small intestine < kidney < heart. In addition, we havecarried out an extensive analysis of the kinetics ofK⁺ binding and occlusion to thecytoplasmic cation binding site and find that, for most tissues, thereis a relationship between the rate ofK⁺ binding/occlusion and theapparent affinity for K⁺ as acompetitive inhibitor of Na⁺activation, the order for both parameters being heart  kidney > small intestine  rat₁-transfected HeLa cells. Thenotion that modulations in cytoplasmicK⁺/Na⁺antagonism are a potential mode of pump regulation is underscored byevidence of its reversibility. Thus the relatively highK⁺/Na⁺antagonism characteristic of kidney pumps was reduced when rat kidneymicrosomal membranes were fused into the dog red blood cell.

     

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     

CFTR upregulates the expression of the basolateral Na+-K+-2Cl- cotransporter in cultured pancreatic duct cells

The purpose ofthe current experiments was 1) toassess basolateralNa⁺-K⁺-2Clcotransporter (NKCC1) expression and2) to ascertain the role of cysticfibrosis transmembrane conductance regulator (CFTR) in the regulationof this transporter in a prototypical pancreatic duct epithelial cellline. Previously validated human pancreatic duct celllines (CFPAC-1), which exhibit physiological features prototypical ofcystic fibrosis, and normal pancreatic duct epithelia (stablerecombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown toconfluence before molecular and functional studies. High-stringencyNorthern blot hybridization, utilizing specific cDNA probes, confirmedthat NKCC1 was expressed in both cell lines and its mRNA levels weretwofold higher in CFPAC-WT cells than in CFPAC-1 cells(P < 0.01, n = 3).Na⁺-K⁺-2Clcotransporter activity, assayed as the bumetanide-sensitive, Na⁺- andCl-dependentNH⁺₄ entry into the cell (withNH⁺₄ acting as a substitute forK⁺), increased by ~115% inCFPAC-WT cells compared with CFPAC-1 cells(P < 0.01, n = 6). Reducing the intracellularCl by incubating the cellsin a Cl-free mediumincreasedNa⁺-K⁺-2Clcotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cellsand mediates the entry ofCl. NKCC1 activity isenhanced in the presence of an inwardCl gradient. The resultsfurther indicate that the presence of functional CFTR enhances theexpression of NKCC1. We speculate that CFTR regulates this process in aCl-dependent manner.

     

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

We evaluated theeffects of acute hyperoxic exposure on alveolar epithelial cell (AEC)active ion transport and on expression ofNa⁺ pump(Na⁺-K⁺-ATPase)and rat epithelial Na⁺ channelsubunits. Rat AEC were cultivated in minimal defined serum-free medium(MDSF) on polycarbonate filters. Beginning on day5, confluent monolayers were exposedto either 95% air-5% CO₂(normoxia) or 95% O₂-5%CO₂ (hyperoxia) for 48 h.Transepithelial resistance(R_t) andshort-circuit current(I_sc) weredetermined before and after exposure.Na⁺ channel -, -, and-subunit andNa⁺-K⁺-ATPase₁- and₁-subunit mRNA levels werequantified by Northern analysis.Na⁺ pump₁- and₁-subunit protein abundance wasquantified by Western blotting. After hyperoxic exposure,I_sc across AECmonolayers decreased by ~60% at 48 h relative to monolayersmaintained under normoxic conditions.Na⁺ channel -subunit mRNAexpression was reduced by hyperoxia, whereas - and -subunit mRNAexpression was unchanged. Na⁺ pump₁-subunit mRNA was unchanged,whereas ₁-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in₁-subunit protein. Becausekeratinocyte growth factor (KGF) has recently been shown to upregulateAEC active ion transport and expression ofNa⁺-K⁺-ATPaseunder normoxic conditions, we assessed the ability of KGF to preventhyperoxia-induced changes in active ion transport by supplementingmedium with KGF (10 ng/ml) from day2. The presence of KGF prevented theeffects of hyperoxia on ion transport (as measured byI_sc) relativeto normoxic controls. Levels of₁ mRNA and protein wererelatively preserved in monolayers maintained in MDSF and KGF comparedwith those cultivated in MDSF alone. These results indicate that AECnet active ion transport is decreased after 48 h of hyperoxia, likelyas a result of a decrease in the number of functionalNa⁺ pumps per cell. KGF largelyprevents this decrease in active ion transport, at least in part, bypreserving Na⁺ pump expression.

     

Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate

Na⁺-K⁺-2Clcotransporters are important in renal salt reabsorption and in saltsecretion by epithelia. They are also essential in maintenance andregulation of ion gradients and cell volume in both epithelial andnonepithelial cells. Expression ofNa⁺-K⁺-2Clcotransporters in brain tissues is high; however, little is known abouttheir function and regulation in neurons. In this study, we examinedregulation of theNa⁺-K⁺-2Clcotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitiveK⁺ influx, and protein expressionwas evaluated by Western blot analysis. Glutamate was found to induce adose- and time-dependent stimulation ofNa⁺-K⁺-2Clcotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonistN-methyl-D-asparticacid (NMDA) and the metabotropic receptor agonist(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantlystimulated the cotransport activity in these cells.NMDA-mediated stimulation of theNa⁺-K⁺-2Clcotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate.trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)--methyl-(4-carboxyphenyl)glycine. The results demonstrate thatNa⁺-K⁺-2Clcotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake andNa⁺ efflux via the Na⁺-K⁺ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa⁺-K⁺ pump. To analyze specifically the effectof insulin on the ₁-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa⁺-K⁺ pump ₁-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of ₁-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of ₁-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of₁ (at serine-18). These results suggest that insulinregulates the Na⁺-K⁺ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of ₁-subunits via a signalingpathway involving PI 3-kinase and PKC.

     

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV)infection on theNa⁺-K⁺-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]_i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO₃-free media) was reduced inthe absence of external Na⁺ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]_iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa⁺ sensitivities of[Cl]_iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb

Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa⁺-K⁺(NH⁺₄)-2Clcotransport. ANG II at 10¹⁶to 10¹² M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the10¹² M ANG II levelcotransport activity; eventually,10⁶ M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 10¹²M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 10¹² M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 10⁶ M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa²⁺ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus10¹² M ANG II inhibitsvia 20-HETE, whereas 5 × 10¹¹ M ANG II stimulatesvia PKCNa⁺-K⁺(NH⁺₄)-2Clcotransport; all ANG II effects involveAT₁ receptors and PLC activation.