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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl- cotransporter

Previous studies from our laboratory have shown a closecorrelation between increasedNa⁺-K⁺-2Clcotransporter activity and increased cotransporter phosphorylation after -adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase inthe number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. Wealso show that the sensitivity of cotransporter fluxes to inhibition bybumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that -adrenergic stimulation and the accompanying phosphorylation result in the activation ofpreviously quiescent transporters rather than in a change in theproperties of already active proteins. In addition, we demonstrate thatthe increased phosphorylation on the cotransporter resulting from-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Westernblotting experiments demonstrate that this peptide is derived from theNH₂-terminal cytosolic tail of thecotransporter, which surprisingly does not contain the sole proteinkinase A consensus site on the molecule.

     

Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

To identify protein kinases (PK) and phosphatases (PP) involvedin regulation of theNa⁺-K⁺-2Clcotransporter in Ehrlich cells, the effect of various PK and PPinhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculinA (Cal-A) was a potent activator ofNa⁺-K⁺-2Clcotransport (EC₅₀ = 35 nM).Activation by Cal-A was rapid (<1 min) but transient. Inactivation isprobably due to a 10% cell swelling and/or the concurrentincrease in intracellularCl concentration. Cellshrinkage also activates theNa⁺-K⁺-2Clcotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation withCal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH andCa²⁺/calmodulin dependent.Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA byH-89 and KT-5720 inhibited cotransport activity induced by Cal-A and bycell shrinkage, with IC₅₀ values similar to reported inhibition constants of the respective kinases invitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of theNa⁺-K⁺-2Clcotransport activity during regulatory volume increase.

     

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.

     

Bumetanide blocks CFTR GCl in the native sweat duct

Bumetanide is wellknown for its ability to inhibit the nonconductiveNa⁺-K⁺-2Clcotransporter. We were surprised in preliminary studies to find thatbumetanide in the contraluminal bath also inhibited NaCl absorption inthe human sweat duct, which is apparently poor in cotransporteractivity. Inhibition was accompanied by a marked decrease in thetransepithelial electrical conductance. Because the cystic fibrosistransmembrane conductance regulator (CFTR) Cl channel is richlyexpressed in the sweat duct, we asked whether bumetanide acts byblocking this anion channel. We found that bumetanide1) significantly increased wholecell input impedance, 2)hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membranepotential, 4) increased the ratio ofapical-to-basolateral membrane resistance, and5) decreased transepithelialCl conductance(G_Cl).These results indicate that bumetanide inhibits CFTRG_Clin both cell membranes of this epithelium. We excluded bumetanideinterference with the protein kinase A phosphorylation activationprocess by "irreversibly" phosphorylating CFTR [by usingadenosine5'-O-(3-thiotriphosphate) in thepresence of a phosphatase inhibition cocktail] before bumetanideapplication. We then activated CFTRG_Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath(10³ M) inhibited ~71%of this ATP-activated CFTRG_Cl,indicating possible direct inhibition of CFTRG_Cl.We conclude that bumetanide inhibits CFTRG_Clin apical and basolateral membranes independent of phosphorylation. Theresults also suggest that>10⁵ M bumetanide cannotbe used to specifically block theNa⁺-K⁺-2Cl cotransporter.

     

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

AlthoughNa⁺-K⁺-2Cl cotransport has beendemonstrated in cultured bovine corneal endothelial cells, its presenceand role in the native tissue have been disputed. Using RT-PCR we havenow identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99%identical to that of the bovine isoform (bNKCC1).[³H]bumetanide binding shows that the cotransporter sitesare located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium.Immunocytochemistry confirms the basolateral location of thecotransporter. We calculate the turnover rate of the cotransporter tobe 83 s¹. Transendothelial fluid transport, determinedfrom deepithelialized rabbit corneal thickness measurements, ispartially inhibited (30%) by bumetanide in a dose-dependent manner.Our results demonstrate thatNa⁺-K⁺-2Cl cotransporters arepresent in the basolateral domain of freshly dissected bovine cornealendothelial cells and contribute to fluid transport across cornealendothelial preparations.

     

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.

     

Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtainedduring the last two decades show that spontaneously hypertensive rats,an acceptable experimental model of primary human hypertension, possessincreased activity of both ubiquitous and renal cell-specific isoformsof theNa⁺/H⁺exchanger (NHE) andNa⁺-K⁺-2Clcotransporter. Abnormalities of these ion transporters have been foundin patients suffering from essential hypertension. Recent geneticstudies demonstrate that genes encoding the - and -subunits ofENaC, a renal cell-specific isoform of theNa⁺-K⁺-2Clcotransporter, and 3-, 1-, and 2-subunits of theNa⁺-K⁺pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis ofthe homology of genome maps, several other genes encoding these transporters, as well as theNa⁺/H⁺exchanger andNa⁺-K⁺-2Clcotransporter, can be predicted in QTL related to the pathogenesis ofhypertension. However, despite their location within QTL, analysis ofcDNA structure did not reveal any mutation in the coding region of theabove-listed transporters in primary hypertension, with the exceptionof G276L substitution in the1-Na⁺-K⁺pump from Dahl salt-sensitive rats and a higher occurrence of T594Mmutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities ofsystems involved in the regulation of their expression and/or function.Further analysis of QTL in F₂hybrids of normotensive and hypertensive rats and in affected siblingpairs will allow mapping of genes causing abnormalities ofthese regulatory pathways.     

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC)

Our group recentlycloned the electrogenicNa⁺-HCO₃cotransporter (NBC) from salamander kidney and later from mammaliankidney. Here we report cloning an NBC isoform (hhNBC) from a humanheart cDNA library. hhNBC is identical to human renal NBC (hkNBC),except for the amino terminus, where the first 85 amino acids in hhNBCreplace the first 41 amino acids of hkNBC. About 50% of the amino acidresidues in this unique amino terminus are charged, compared with~22% for the corresponding 41 residues in hkNBC. Northern blotanalysis, with the use of the unique 5' fragment of hhNBC as aprobe, shows strong expression in pancreas and expression in heart andbrain, although at much lower levels. InXenopus oocytes expressing hhNBC,adding 1.5% CO₂/10 mMHCO₃ hyperpolarizes the membrane andcauses a rapid fall in intracellular pH(pH_i), followed by apH_i recovery. Subsequent removalof Na⁺ causes a depolarization anda reduced rate of pH_i recovery.Removal of Cl from the bathdoes not affect the pH_i recovery.The stilbene derivative DIDS (200 µM) greatly reduces thehyperpolarization caused by addingCO₂/HCO₃.In oocytes expressing hkNBC, the effects of addingCO₂/HCO₃and then removing Na⁺ were similarto those observed in oocytes expressing hhNBC. We conclude that hhNBCis an electrogenicNa⁺-HCO₃cotransporter and that hkNBC is also electrogenic.     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.

     

Hydrogen peroxide inhibits cAMP-induced Cl- secretion across colonic epithelial cells

We examined the effects ofH₂O₂on Cl secretion acrosshuman colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, ameasure of Cl secretion,was inhibited in a concentration-dependent fashion when monolayers werepretreated withH₂O₂for 30 min (30-100% inhibition between 500 µM and 5 mM).Moreover,H₂O₂inhibited 76% of the Clcurrent across monolayers when the basolateral membranes were permeabilized with nystatin (200 µg/ml). When the apical membrane waspermeabilized with amphotericin B,H₂O₂inhibited the Na⁺ current (ameasure ofNa⁺-K⁺-ATPaseactivity) by 68% but increased theK⁺ current more than threefold. Inaddition to its effects on ion transport pathways,H₂O₂also decreased intracellular ATP levels by 43%. We conclude that theprincipal effect ofH₂O₂on colonic Cl secretion isinhibitory. This may be due to a decrease in ATP levels followingH₂O₂treatment, which subsequently results in an inhibition of the apicalmembrane Cl conductance andbasolateral membraneNa⁺-K⁺-ATPaseactivity. Alternatively,H₂O₂may alter Cl secretion bydirect action on the transporters or alterations in signal transductionpathways.

     

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Cl- cotransport

The role ofNa⁺-K⁺-2Clcotransport in ion and fluid transport of the corneal endothelium wasexamined by measuring changes in corneal hydration and uptake of⁸⁶Rb by the endothelial celllayer. Isolated, intact rabbit corneas maintain normal hydration whenthey are superfused at the endothelial surface with bicarbonate()-Ringer solutions as aresult of equilibrium between active ion and fluid transport out of thestromal tissue and leak of fluid into stromal tissue from the aqueoushumor. Furosemide and bumetanide did not alter this equilibrium whenthey were added to the superfusion medium. Uptake of⁸⁶Rb by the endothelium of theincubated cornea was increased 25% by bumetanide, but uptake in thepresence of ouabain (70% less than that of controls) was not changedby bumetanide. In Na⁺-free medium,uptake of ⁸⁶Rb was reduced by58%, but it was unchanged inCl-free medium. CalyculinA, a protein phosphatase inhibitor and activator ofNa⁺-K⁺-Clcotransport, was without effect on⁸⁶Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes wassignificantly different when bumetanide was present in the media. It isconcluded thatNa⁺-K⁺-2Clcotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport ofthis cell layer. Its presence in cultured endothelial cells may reflectthe reported importance of this protein in growth, proliferation, anddifferentiation.