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.

     

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.

     

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice

Alterations in the competency of the creatine kinase systemelicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular andkinetic changes in AMP deaminase from skeletal muscles of micedeficient in either cytosolic creatine kinase alone(M-CK^/) or alsodeficient in mitochondrial creatine kinase(CK^/) comparedwith wild type. We found that predominantly fast-twitch muscle, but notslow-twitch muscle, from bothM-CK^/ andCK^/ mice had muchlower AMP deaminase; the quantity of AMP deaminase detected by Westernblot was correspondingly lower, whereas AMP deaminase-1(AMPD1) gene expressionwas unchanged. Kinetic analysis of AMP deaminase from mixed musclerevealed negative cooperativity that was significantly greater increatine kinase deficiencies. Treatment of AMP deaminase with acidphosphatase abolished negative cooperative behavior, indicating that aphosphorylation-dephosphorylation cycle may be important in theregulation of AMP deaminase.

     

Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts

The substitution of gluconate forCl is commonly used tocharacterize Cl transportor Cl-dependent transportmechanisms. We evaluated the effects of substituting gluconate forCl on the transport of theP-glycoprotein substrate rhodamine 123 (R123). The replacement ofRinger solution containingCl(Cl-Ringer)with gluconate-Ringer inhibited R123 efflux, whereas the replacement ofCl by other anions (sulfateor cyclamate) had no effect. The inhibition of R123 efflux bygluconate-Ringer was absent after chloroform extraction of the sodiumgluconate salt. The readdition of the sodium gluconate-chloroformextract to the extracted gluconate-Ringer or to cyclamate-Ringerinhibited R123 efflux, whereas its addition toCl-Ringer had no effect.These observations indicate that the inhibition ofP-glycoprotein-mediated R123 transport by gluconate is due to one ormore chloroform-soluble contaminants and that the inhibition is absentin the presence of Cl. Theresults are consistent with the fact that P-glycoprotein substrates arehydrophobic. Care should be taken when replacing ions to evaluatemembrane transport mechanisms because highly pure commercialpreparations may still contain potent contaminants that affect transport.

     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

     

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells

Cystic fibrosis iscaused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) Clchannel, which mediates transepithelialCl transport in a varietyof epithelia, including airway, intestine, pancreas, and sweat duct. Insome but not all epithelial cells, cAMP stimulatesCl secretion in part byincreasing the number of CFTRCl channels in the apicalplasma membrane. Because the mechanism whereby cAMP stimulates CFTRCl secretion is cell-typespecific, our goal was to determine whether cAMP elevates CFTR-mediatedCl secretion across serousairway epithelial cells by stimulating the insertion of CFTRCl channels from anintracellular pool into the apical plasma membrane. To this end westudied Calu-3 cells, a human airway cell line with a serous cellphenotype. Serous cells in human airways, such as Calu-3 cells, expresshigh levels of CFTR, secrete antibiotic-rich fluid, and play a criticalrole in airway function. Moreover, dysregulation of CFTR-mediatedCl secretion in serouscells is thought to contribute to the pathophysiology of cysticfibrosis lung disease. We report that cAMP activation of CFTR-mediatedCl secretion across humanserous cells involves stimulation of CFTR channels present in theapical plasma membrane and does not involve the recruitment of CFTRfrom an intracellular pool to the apical plasma membrane.

     

Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells

Corneal endothelial function is dependent onHCO₃ transport. However, the relativeHCO₃ permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO₂-HCO₃ (at constant pH) or removing HCO₃alone (at constant CO₂) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO₃ permeabilities and their dependencies on Na⁺ andCl. Removal ofCO₂-HCO₃from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO₃ at constantCO₂ indicated that the basolateralHCO₃ fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa⁺ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO₃ flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO₃ fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO₂ conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO₂-HCO₃apically suggests that CO₂diffusion may contribute to apicalHCO₃ flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO₂ fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa⁺-dependent basolateralHCO₃ permeability is consistent withNa⁺-nHCO₃cotransport. Changes inHCO₃ flux in the absence ofCl are most likely due toNa⁺-nHCO₃cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO₃ permeabilityis relatively low, but may be augmented byCO₂ diffusion in conjunction witha CAIV.

     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells

Limitations of available indicators [such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ)] for measurement of intracellular Cl are their relatively dimfluorescence and need for ultraviolet excitation. A series oflong-wavelength polar fluorophores was screened to identify compoundswith Cl and/orI sensitivity, brightfluorescence, low toxicity, uniform loading of cytoplasm with minimalleakage, and chemical stability in cells. The best compound found was7-(-D-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation andemission maxima at 400-470 and 490-560 nm, molar extinction11,100 M¹ · cm¹(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched byI by a collisionalmechanism (Stern-Volmer constant 60 M¹) and is not affectedby other halides, nitrate, cations, or pH changes (pH 5-8). AfterLZQ loading into cytoplasm by hypotonic shock or overnight incubation,LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasmuniformly, remained chemically inert, did not bind to cytoplasmiccomponents, and was photobleached by <1% during 1 h of continuousillumination. Cytoplasmic LZQ fluorescence was quenched selectively byI (50% quenching at 38 mMI). LZQ was used tomeasure forskolin-stimulatedI/ClandI/NO₃exchange in cystic fibrosis transmembrane conductance regulator(CFTR)-expressing cell lines by fluorescence microscopy and microplatereader instrumentation using 96-well plates. The substantially improvedoptical and cellular properties of LZQ over existing indicators shouldpermit the quantitative analysis of CFTR function in gene deliverytrials and high-throughput screening of compounds for correction of thecystic fibrosis phenotype.

     

Direct inhibitory effect of CCCP on the Cl--H+ symporter of the guinea pig ileal brush-border membrane

The effect of carbonylcyanide-m-chlorophenylhydrazone (CCCP)on Cl uptake across thebrush-border membrane (BBM) was quantified using³⁶Cl and BBM vesicles from guineapig ileum. CCCP inhibited only partially both the pH gradient-activatedCl uptake andCl/Clexchange activities present in these vesicles. In contrast, CCCP had noeffect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the mainaction of CCCP does not consist of dissipating any imposed pH gradientbut rather in inhibiting directly the pH gradient-activated Cl uptake andCl/Clexchange activities characterizing the intestinal BBM. Because thesetwo activities can be explained in terms of a single (homogeneous) random, nonobligatory two-siteCl-H⁺symporter, in whichCl/Clexchange occurs by counterflow [F. Alvarado and M. Vasseur.Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628,1996], we developed a new, more general three-site symport modelthat fully explains the Cluptake inhibitions caused by CCCP. This new model postulates theexistence of a third, allosteric, inhibitory CCCP-binding site separatefrom either of the two substrate-binding sites of theCl-H⁺symporter, the Cl-bindingand the H⁺-binding sites. Finally,we show that, to explain the partial inhibitions observed, it isnecessary to postulate that all the substrate-bound carrier complexes,=C-S, I=C-S, A=C-S, and IA=C-S, where C is carrier, I is inhibitor, Sis substrate, and A is activator, can form and be translocated.

     

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.

     

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studieshave indicated a role of the actin cytoskeleton in the regulation ofthe cystic fibrosis transmembrane conductance regulator (CFTR) ionchannel. However, the exact molecular nature of this regulation isstill largely unknown. In this report human epithelial CFTR wasexpressed in human melanoma cells genetically devoid of the filaminhomologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP() cells orcells genetically rescued with ABP-280 cDNA [ABP(+)] waswithout effect on whole cell Cl currents. InABP() cells expressing CFTR, cAMP was also without effect onCl conductance. In contrast, cAMP induced a 10-foldincrease in the diphenylamine-2-carboxylate (DPC)-sensitive whole cellCl currents of ABP(+)/CFTR(+) cells. Further, incells expressing both CFTR and a truncated form of ABP-280 unable tocross-link actin filaments, cAMP was also without effect on CFTRactivation. Dialysis of ABP-280 or filamin through the patch pipette,however, resulted in a DPC-inhibitable increase in the whole cellcurrents of ABP()/CFTR(+) cells. At the single-channel level,protein kinase A plus ATP activated single Clchannels only in excised patches from ABP(+)/CFTR(+) cells.Furthermore, filamin alone also induced Cl channelactivity in excised patches of ABP()/CFTR(+) cells. The presentdata indicate that an organized actin cytoskeleton is required forcAMP-dependent activation of CFTR.

     

Separate taurine and chloride efflux pathways activated during regulatory volume decrease

Organic osmolyte and halide permeability pathways activated inepithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl byanions that are more permeant through the volume-activated Cl channel, as indicated byelectrophysiological measurements, significantly decreasedtaurine efflux. In the presence of less-permeant anions, an increase intaurine efflux was observed. Simultaneous measurement of the¹²⁵I, used as a tracer forCl, and[³H]taurine effluxshowed that the time courses for the two effluxes differed. InCl-rich medium the increasein I efflux was transient,whereas that for taurine was sustained. OsmosensitiveCl conductance, assessed bymeasuring changes in cell volume, increased rapidly after hypotonicshock. The influx of taurine was able to counteractCl conductance-dependentcell shrinkage but only ~4 min after triggering cell swelling. Thistaurine-induced effect was blocked by DIDS. Differences in anionsensitivity, the time course of activation, and sensitivity to DIDSsuggest that the main cell swelling-activated permeability pathways fortaurine and Cl are separate.

     

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts

The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO₃exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pH_i) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO₂-25 mMHCO₃ were nearly the same. However,replacement of external Clwith gluconate caused anH₂DIDS-inhibitable (100 µM)increase in the pH_i ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO₂/HCO₃caused the pH_i of both cell typesto increase. The pH_i remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepH_i peaked and then recoveredtoward control values. This pH_irecovery phase was completely blocked by 100 µMH₂DIDS. In the presence ofCO₂/HCO₃, there was an H₂DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO₂/HCO₃),the H₂DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H₂DIDS-sensitive, netCl efflux decreased aspH_i increased, whereas forHCMV-infected cells, efflux increased aspH_i increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO₃exchanger activity.

     

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibitelectrogenic Cl secretionin response to the Ca²⁺ agonistthapsigargin and to the cAMP agonist forskolin. To evaluate directlythe regulation of apical Clconductance by these two agonists, we have utilized amphotericin B topermeabilize selectively the basolateral membranes of T84 cellmonolayers. We find that apical anion conductance is stimulated by bothforskolin and thapsigargin but that these conductances aredifferentially sensitive to the anion channel blocker DIDS. DIDSinhibits thapsigargin-stimulated responses completely but forskolinresponses only partially. Furthermore, the apical membrane anionconductances elicited by these two agonists differ in anion selectivity(for thapsigargin, I > Cl; for forskolin,Cl > I). However, theDIDS-sensitive component of the forskolin-induced conductance responseexhibits anion selectivity similar to that induced by thapsigargin(I > Cl). Thusforskolin-induced apical anion conductance comprises at least twocomponents, one of which has features in common with that elicited bythapsigargin.

     

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.

     

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.

     

Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines

The dominant routefor Cl secretion in mouse tracheal epithelium is viaCl channels different from the cystic fibrosis (CF)transmembrane conductance regulator (CFTR), the channel that isdefective in CF. It has been proposed that the use of purinergicagonists to activate these alternative channels in human airways may bebeneficial in CF. In the present study, two conditionally immortalepithelial cell lines were established from the tracheae of micepossessing the tsA58 T antigen gene, one of which [MTE18-(/)] washomozygous for a knockout of CFTR and the other [MTE7b-(+/)]heterozygous for CFTR expression. In Ussing chamber studies, amiloride(10⁴ M) and a cocktail of cAMP-activating agents(forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in theshort-circuit current (I_sc) and resistance ofboth cell lines, with larger increases in I_scbeing elicited by ionomycin (10⁶ M). Both cell linesexpressed P₂Y₂ receptors and responded to thepurinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10⁴ M) with an increase in I_sc.This response could be inhibited by DIDS and was abolished in thepresence of Cl-free Ringer solution. Reducing the mucosalCl concentration increased the response to UTP of bothcell lines, with a significantly greater increase in MTE18-(/)cells. Pretreatment of these cells with thapsigargin caused a directincrease in I_sc and inhibited the response toUTP. These data suggest that both cell lines expresspurinergic-regulated Cl currents and may prove valuabletools in studying the properties of this pathway.

     

Functional and molecular characterization of an anion exchanger in airway serous epithelial cells

Serous cells secreteCl and HCO₃ and play an importantrole in airway function. Recent studies suggest that aCl/HCO₃ anion exchanger (AE) maycontribute to Cl secretion by airway epithelial cells.However, the molecular identity, the cellular location, and thecontribution of AEs to Cl secretion in serous epithelialcells in tracheal submucosal glands are unknown. The goal of thepresent study was to determine the molecular identity, the cellularlocation, and the role of AEs in the function of serous epithelialcells. To this end, Calu-3 cells, a human airway cell line with aserous-cell phenotype, were studied by RT-PCR, immunoblot, andelectrophysiological analysis to examine the role of AEs inCl secretion. In addition, the subcellular location of AEproteins was examined by immunofluorescence microscopy. Calu-3 cellsexpressed mRNA and protein for AE2 as determined by RT-PCR and Westernblot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rattracheal serous cells in situ. InCl/HCO₃/Na⁺-containingmedia, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate(CPT-cAMP)-stimulated short-circuit anion current (I_sc) was reduced by basolateral but not byapical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na⁺ in thebath solutions, to eliminate the contributions of the Na⁺/HCO₃ andNa⁺/K⁺/2Cl cotransporters toI_sc, CPT-cAMP stimulated a small DNDS-sensitive I_sc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedI_sc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2.