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.

     

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.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.

     

Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes

Na⁺/Ca²⁺exchange has been investigated in squid(Loligopealei) rhabdomeric membranes.Ca²⁺-containing vesicles have beenprepared from purified rhabdomeric membranes by extrusion throughpolycarbonate filters of 1-µm pore size. After removal of externalCa²⁺, up to 90% of the entrappedCa²⁺ could be specificallyreleased by the addition of Na⁺;this finding indicates that most of the vesicles containedNa⁺/Ca²⁺exchanger. The Na⁺-inducedCa²⁺ efflux had a half-maximumvalue (K_1/2) of~44 mM and a Hill coefficient of ~1.7. The maximalNa⁺-inducedCa²⁺ efflux was ~0.6 nmolCa²⁺ · s¹ · mgprotein¹. SimilarNa⁺-inducedCa²⁺ effluxes were measured ifK⁺ was replaced withLi⁺ orCs⁺. Vesicles loaded withCa²⁺ byNa⁺/Ca²⁺exchange also released this Ca²⁺byNa⁺/Ca²⁺exchange, suggesting thatNa⁺/Ca²⁺exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate ofCa²⁺ efflux enhanced byapproximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin,Na⁺/Ca²⁺exchange was characterized by aK_1/2 of ~25 mMand a Hill coefficient of 1.6. For these vesicles, the maximalNa⁺-inducedCa²⁺ efflux was about twice asgreat as in control vesicles. We conclude thatNa⁺/Ca²⁺exchange proteins localized in rhabdomeric membranes mediate Ca²⁺ extrusion in squid photoreceptors.     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

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.

     

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.

     

Extracellular Cl(-) modulates shrinkage-induced activation of Na(+)/H(+) exchanger in rat mesangial cells

To examine theeffect of hyperosmolality on Na⁺/H⁺ exchanger(NHE) activity in mesangial cells (MCs), we used apH-sensitive dye,2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH_i) in a single MC from ratglomeruli. All the experiments were performed inCO₂/HCO₃-free HEPESsolutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH₂O) treated with mannitol caused cellalkalinization. The hyperosmolality-induced cell alkalinization wasinhibited by 100 µM ethylisopropylamiloride, a specific NHEinhibitor, and was dependent on extracellular Na⁺. Thehyperosmolality shifted the Na⁺-dependent acid extrusionrate vs. pH_i by 0.15-0.3 pH units in thealkaline direction. Removal of extracellular Cl byreplacement with gluconate completely abolished the rate of cellalkalinization induced by hyperosmolality and inhibited the Na⁺-dependent acid extrusion rate, whereas, under isosmoticconditions, it caused no effect on Na⁺-dependentpH_i recovery rate or Na⁺-dependent acidextrusion rate. The Cl-dependent cell alkalinizationrate under hyperosmotic conditions was partially inhibited bypretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS,and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acidextrusion rate via NHE is greater under hyperosmotic conditions thanunder isosmotic conditions at a wide range of pH_i,3) the NHE activation under hyperosmotic conditions, but notunder isosmotic conditions, requires extracellularCl, and 4) theCl-dependent NHE activation under hyperosmoticconditions partly occurs via Cl channel andmicrotubule-dependent processes.

     

Direct estimate of 1:1 stoichiometry of K(+)-Cl(-) cotransport in rabbit erythrocytes

This work was undertaken toobtain a direct measure of the stoichiometry ofNa⁺-independent K⁺-Cl cotransport(KCC), with rabbit red blood cells as a model system. To determinewhether ⁸⁶Rb⁺ can be used quantitatively as atracer for KCC, ⁸⁶Rb⁺ and K⁺effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulatedK⁺ efflux into isosmotic NaCl was smaller than that for⁸⁶Rb⁺ by a factor of 0.68 ± 0.11 (SD,n = 5). This correction factor was used in all otherexperiments to calculate the K⁺ efflux from the measured⁸⁶Rb⁺ efflux. To minimize interference from theanion exchanger, extracellular Cl was replaced withSO, and4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clampednear 0 mV with the protonophore 2,4-dinitrophenol. The Clefflux at 25°C under these conditions is ~100,000-fold smaller thanthe uninhibited Cl/Cl exchange flux and isstimulated ~2-fold by NEM. The NEM-stimulated ³⁶Cl flux is inhibited by okadaic acid andcalyculin A, as expected for KCC. The ratio of the NEM-stimulatedK⁺ to Cl efflux is 1.12 ± 0.26 (SD,n = 5). We conclude thatK⁺-Cl cotransport in rabbit red blood cellshas a stoichiometry of 1:1.

     

Serotonin-elicited inhibition of Cl(-) secretion in the rabbit conjunctival epithelium

The effects of serotonin[5-hydroxytryptamine (5-HT)] on the transepithelial electricalproperties of the short-circuited rabbit conjunctiva were examined.With this epithelium, the short-circuit current(I_sc) measures Cl secretion plusan amiloride-resistant Na⁺ absorptive process. Apicaladdition of 5-HT (10 µM) elicited a prompt I_screduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm² and increased transepithelial resistance from0.89 ± 0.05 to 1.03 ± 0.06 k · cm²(means ± SE, n = 21, P < 0.05).Similar changes were obtained with conjunctivae bathed withoutNa⁺ in the apical bath, as well as with conjunctivaepreexposed to bumetanide with the Cl-dependentI_sc sustained by the parallel activities ofbasolateral Na⁺/H⁺ andCl/HCO exchangers. In contrast, the5-HT-evoked effects were attenuated by the absence of Cl(I_sc = 0.5 ± 0.2, n = 5), suggesting that reduced Clconductance(s) is an effect of 5-HT exposure. In amphotericin B-treatedconjunctiva and in the presence of a transepithelial K⁺gradient, 5-HT addition reduced K⁺ diffusion across thepreparation by 13% and increased transepithelial resistance by 4%(n = 6, P < 0.05), indicating that aninhibition in K⁺ conductance(s) was also detectable.Significant electrical responses also occurred under physiologicalconditions when 5-HT was introduced to epithelia pretreated withadrenergic agonists or protein kinase C, phospholipase C,phosphodiesterase, or adenylyl cyclase inhibitors or after perturbationof Ca²⁺ homeostasis. Briefly, the conjunctiva harbors theonly known Cl-secreting epithelium in which 5-HT evokesCl transport inhibition; receptor subtype and signaltransduction mechanism were not determined.

     

Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmalNa⁺ transport, we measuredouabain-sensitive electrogenicNa⁺-K⁺pump current(I_p) involtage-clamped ventricular myocytes and intracellularNa⁺ activity(aⁱ_Na) in right ventricularpapillary muscles. Aldosterone (10 nM) induced an increase in bothI_p and the rateof rise of aⁱ_Na duringNa⁺-K⁺pump blockade with the fast-acting cardiac steroid dihydroouabain. Thealdosterone-induced increase inI_p and rate ofrise of aⁱ_Na was eliminated bybumetanide, suggesting that aldosterone activates Na⁺ influx through theNa⁺-K⁺-2Clcotransporter. To obtain independent support for this, theNa⁺,K⁺, andCl concentrations in thesuperfusate and solution of pipettes used to voltage clamp myocyteswere set at levels designed to abolish the inward electrochemicaldriving force for theNa⁺-K⁺-2Clcotransporter. This eliminated the aldosterone-induced increase inI_p. We concludethat in vitro exposure of cardiac myocytes to aldosterone activates theNa⁺-K⁺-2Clcotransporter to enhance Na⁺influx and stimulate theNa⁺-K⁺pump.

     

UTP inhibits Na+ absorption in wild-type and Delta F508 CFTR-expressing human bronchial epithelia

Ca²⁺-mediated agonists,including UTP, are being developed for therapeutic use in cysticfibrosis (CF) based on their ability to modulate alternativeCl conductances. As CF isalso characterized by hyperabsorption ofNa⁺, we determined the effect ofmucosal UTP on transepithelial Na⁺transport in primary cultures of human bronchial epithelia (HBE). Insymmetrical NaCl, UTP induced an initial increase in short-circuit current (I_sc)followed by a sustained inhibition. To differentiate between effects onNa⁺ absorption andCl secretion,I_sc was measuredin the absence of mucosal and serosal Cl(I_Na). Again,mucosal UTP induced an initial increase and then a sustained decreasethat reduced amiloride-sensitiveI_Na by 73%. TheCa²⁺-dependent agonists histamine,bradykinin, serosal UTP, and thapsigargin similarly induced sustainedinhibition (62-84%) ofI_Na. Mucosal UTPinduced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) ofI_Na in primarycultures of human CF airway homozygous for the F508 mutation.BAPTA-AM blunted UTP-dependent inhibition ofI_Na, butinhibitors of protein kinase C (PKC) and phospholipaseA₂ had no effect. Indeed, directactivation of PKC by phorbol 12-myristate 13-acetate failed to inhibitNa⁺ absorption. Apyrase, a tri-and diphosphatase, did not reverse inhibitory effects of UTP onI_Na, suggesting along-term inhibitory effect of UTP that is independent of receptoroccupancy. After establishment of a mucosa-to-serosaK⁺ concentration gradient andpermeabilization of the mucosal membrane with nystatin, mucosal UTPinduced an initial increase in K⁺current followed by a sustained inhibition. We conclude that increasingcellular Ca²⁺ induces a long-terminhibition of transepithelial Na⁺transport across normal and CF HBE at least partly due todownregulation of a basolateral membraneK⁺ conductance. Thus UTP may havea dual therapeutic effect in CF airway:1) stimulation of aCl secretory response and2) inhibition ofNa⁺ transport.     

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

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.

     

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

Human trabecular meshwork cell volume regulation

The volume ofcertain subpopulations of trabecular meshwork (TM) cells may modifyoutflow resistance of aqueous humor, thereby altering intraocularpressure. This study examines the contribution thatNa⁺/H⁺, Cl/HCOexchange, and K⁺-Cl efflux mechanisms have onthe volume of TM cells. Volume, Cl currents, andintracellular Ca²⁺ activity of cultured human TM cells werestudied with calcein fluorescence, whole cell patch clamping, and fura2 fluorescence, respectively. At physiological bicarbonateconcentration, the selective Na⁺/H⁺ antiportinhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicitytriggered a regulatory volume decrease (RVD), which could be inhibitedby the Cl channel blocker5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K⁺channel blockers Ba²⁺ and tetraethylammonium, and theK⁺-Cl symport blocker[(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism inisotonic conditions was dependent on bicarbonate; at physiologicallevels, the Na⁺/H⁺ exchange inhibitordimethylamiloride reduced cell volume, whereas at low levels theNa⁺-K⁺-2Cl symport inhibitorbumetanide had the predominant effect. Patch-clamp measurements showedthat hypotonicity activated an outwardly rectifying, NPPB-sensitiveCl channel displaying the permeability rankingCl > methylsulfonate > aspartate.2,3-Butanedione 2-monoxime antagonized actomyosin activity and bothincreased baseline [Ca²⁺] and abolishedswelling-activated increase in [Ca²⁺], but it did notaffect RVD. Results indicate that human TM cells display aCa²⁺-independent RVD and that volume is regulated byswelling-activated K⁺ and Cl channels,Na⁺/H⁺ antiports, and possiblyK⁺-Cl symports in addition toNa⁺-K⁺-2Cl symports.

     

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (I_sc) averaged 20.8 ± 0.7 µA/cm² (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, I_sc averaged 14.4 ± 1.1 µA/cm² (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (K_i) of 0.68 µM. The remainingamiloride-insensitive I_sc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO₃. It was further stimulated bybasolateral application of forskolin (10⁵ M), which increasedI_sc by 3.1 ± 0.3 µA/cm² (n=65) in epididymis and 0.9 ± 0.1 µA/cm² (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na⁺ viaamiloride-sensitive epithelial Na⁺ channels (ENaC) andsecrete Cl and HCO₃via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA.

     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR

The rubidium efflux from hypothermic rat hearts perfused by theLangendorff method at 20°C was studied. At thistemperature ⁸⁷Rb-NMR efflux experiments showed theexistence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial.Rat heart mitochondria showed a very slow exchange of mitochondrialRb⁺ for cytoplasmic K⁺. After washout ofcytosolic Rb⁺, mitochondria kept a stable Rb⁺level for >30 min. Rb⁺ efflux from mitochondria wasstimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmalpermeabilization and concomitant cellular energy depletion by saponin(0.01 mg/ml for 4 min) in the presence of a perfusate mimickingintracellular conditions, or by ATP-sensitive K (K_ATP)channel openers. DNP, a mitochondrial uncoupler, caused the onset ofmitochondrial Rb⁺ exchange; however, the washout was notcomplete (80 vs. 56% in control). Energy deprivation by saponin, whichpermeabilizes the sarcolemma, resulted in a rapid and completeRb⁺ efflux. The mitochondrial Rb⁺ efflux rateconstant (k) decreased in the presence of glibenclamide, aK_ATP channel inhibitor (5 µM;k = 0.204 ± 0.065 min¹; n = 8),or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,respectively; k = 0.134 ± 0.021 min¹;n = 4) in the saponin experiments (saponin only;k = 0.321 ± 0.079 min¹; n = 3),indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed theprobing of the mitochondrial K⁺ pool in whole heartswithout mitochondrial isolation.

     

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC₅₀ values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca²⁺concentration([Ca²⁺]_o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of⁴⁵Ca²⁺,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca²⁺ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca²⁺]_o.Veratridine induced oscillations of cytosolicCa²⁺ concentration([Ca²⁺]_i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca²⁺]_ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca²⁺]_iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa⁺ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na⁺channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na⁺ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca²⁺ channels, andCa²⁺ entry and secretion.