Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells

HCO-dependentfluid secretion by the corneal endothelium controls corneal hydrationand maintains corneal transparency. Recently, it has been shown thatmRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed asingle band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirectimmunofluorescence confocal microscopy indicated that CFTR locates tothe apical membrane. Relative changes in apical and basolateralchloride permeability were estimated by measuring the rate offluorescence quenching of the halide-sensitive indicator6-methoxy-N-ethylquinolinium iodide during Clinflux in the absence and presence of forskolin (FSK). Apical andbasolateral Cl permeability increased 10- and 3-fold,respectively, in the presence of 50 µM FSK. FSK-activated apicalchloride permeability was unaffected by H₂DIDs (250 µM);however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 µM) and glibenclamide (100 µM) inhibited activated Clfluxes by 45% and 30%, respectively. FSK-activated basolateral Cl permeability was insensitive to NPPB, glibenclamide,or furosemide but was inhibited 80% by H₂DIDS.HCO permeability was estimated by measuring changesin intracellular pH in response to quickly lowering bath[HCO]. FSK (50 µM) increased apicalHCO permeability by twofold, which was inhibited42% by NPPB and 65% by glibenclamide. BasolateralHCO permeability was unaffected by FSK. Genistein(50 µM) significantly increased apical HCO andCl permeability by 1.8- and 16-fold, respectively. When50 µM genistein was combined with 50 µM FSK, there was no furtherincrease in Cl permeability; however,HCO permeability was reduced to the control level.In summary, we conclude that CFTR is present in the apical membrane ofbovine corneal endothelium and could contribute to transendothelialCl and HCO transport. Furthermore,there is a cAMP-activated Cl pathway on the basolateralmembrane that is not CFTR.

     

Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway

Expression ofendothelial nitric oxide synthase (eNOS) in transfected U-937 cellsupregulates phorbol 12-myristate 13-acetate (PMA)-induced tumornecrosis factor- (TNF-) production through a superoxide(O)-dependent mechanism. Because mitogen-activatedprotein kinases (MAPK) have been shown to participate in both reactiveoxygen species signaling and TNF- regulation, their possible role ineNOS-derived O signal transduction was examined. Aredox-cycling agent, phenazine methosulfate, was found to bothupregulate TNF- (5.8 ± 1.0 fold; P = 0.01) andincrease the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. AlthoughS-nitroso-N-acetylpenicillamine, a nitric oxide(NO) donor, also increased TNF- production, NO exposure led tophosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-production by eNOS transfection was associated with increases inactivated p42/44 MAPK (P = 0.001), whereas levels ofphosphorylated p38 MAPK were unaffected. Furthermore, cotransfectionwith Cu/Zn superoxide dismutase, which blocks TNF- upregulation byeNOS, also abolished the effects on p42/44 MAPK. Expression ofGln³⁶¹eNOS, a mutant that produces O but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, twoNADPH binding site deletion mutants of eNOS that lack oxidase activityhad no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathwayinhibitor, blocked TNF- upregulation by eNOS (P = 0.02).Thus O produced by eNOS increases TNF- productionvia a mechanism that involves p42/44 MAPK activation.

     

Contribution of chloride channels to volume regulation of cortical astrocytes

The objective of this study was todetermine the relative contribution of Cl channels tovolume regulation of cultured rat cortical astrocytes after hypotoniccell swelling. Using a Coulter counter, we showed that corticalastrocytes regulate their cell volume by ~60% within 45 min afterhypotonic challenge. This volume regulation was supported whenCl was replaced with Br,NO, methanesulfonate, oracetate but was inhibited when Cl wasreplaced with isethionate or gluconate.Additionally, substitution of Cl with Icompletely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl channel blockers, including5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumicacid. Surprisingly, the combination of Cd²⁺ with NPPB,DIDS, or niflumic acid inhibited regulation to a greater extent thanany of these drugs alone. Volume regulation did not differ amongastrocytes cultured from different brain regions, as cerebellar andhippocampal astrocytes exhibited behavior identical to that of corticalastrocytes. These data suggest that Cl flux through ionchannels rather than transporters is essential for volume regulation ofcultured astrocytes in response to hypotonic challenge.

     

Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea

Airway surface liquid (ASL) pH hasbeen proposed to be important in the pathophysiology of cysticfibrosis, asthma, and cough. Ratio image analysis was used to measurepH in the ASL after staining with the fluorescent pH indicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at anair-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO/CO₂. Steady-state ASL pH changed in parallel to changes in bath pH and wasacidified by Na⁺ or Cl replacement but wasnot affected by the inhibitors amiloride, glibenclamide, or4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to suddenacidification or alkalization of the ASL by ~0.4 pH units byHCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pHunits/min (HCO) and 0.060 pH units/min(+HCO); the pH recovery rate was reduced byamiloride and H₂DIDS. In anesthetized mice in which thetrachea was surgically exposed for measurement of BCECF-dextranfluorescence through the translucent tracheal wall, ASL pH was7.14 ± 0.01. ASL pH was sensitive to changes in blood pH createdby metabolic (HCl or NaHCO₃ infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, andH⁺/OH transport between the ASL andbasolateral fluid involves amiloride-sensitive Na⁺/H⁺ exchange and stilbene-sensitiveCl/HCO exchange. The rapid response ofASL pH to changes in systemic acid-base status may contribute to airwayhypersensitivity in asthma and other airway diseases.

     

Localization of endogenous and recombinant Na+-driven anion exchanger protein NDAE1 from Drosophila melanogaster

Na⁺-dependent Cl/HCOexchange activity helps maintain intracellular pH (pH_i)homeostasis in many invertebrate and vertebrate cell types. Ourlaboratory cloned and characterized a Na⁺-dependentCl/HCO exchanger (NDAE1) fromDrosophila melanogaster (Romero MF, Henry D, Nelson S, HartePJ, and Sciortino CM. J Biol Chem 275:24552-24559, 2000). In the present study we usedimmunohistochemical and Western blot techniques to characterize thedevelopmental expression, subcellular localization, and tissue distribution of NDAE1 protein in D. melanogaster. We haveshown that a polyclonal antibody raised against the NH₂terminus of NDAE1 (CWR57) recognizes NDAE1 electrophysiologicallycharacterized in Xenopus oocytes. Moreover, our resultsbegin to delineate the NDAE1 topology, i.e., both the NH₂and COOH termini are intracellular. NDAE1 is expressed throughoutDrosophila development in the central and peripheral nervoussystems, sensilla, and the alimentary tract (Malpighian tubules, gut,and salivary glands). Coimmunolabeling of larval tissues with NDAE1antibody and a monoclonal antibody to theNa⁺-K⁺-ATPase -subunit revealed that themajority of NDAE1 is located at the basolateral membranes of Malpighiantubule cells. These results suggest that NDAE1 may be a keypH_i regulatory protein and may contribute to basolateralion transport in epithelia and nervous system of Drosophila.

     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage,cAMP, and calyculin A

In this report, wedescribe the cloning, cellular localization, and functionalcharacteristics of Na⁺/H⁺ exchanger 1 (NHE1)from red blood cells of the winter flounder Pseudopleuronectesamericanus (paNHE1). The paNHE1 protein localizes primarily to themarginal band and exhibits a 74% similarity to the trout -NHE, and65% to the human NHE1 (hNHE1). Functionally, paNHE1 sharescharacteristics of both -NHE and hNHE1 in that it is activated bothby manipulations that increase cAMP and by cell shrinkage,respectively. In accordance, the paNHE1 protein exhibits both proteinkinase A consensus sites as in -NHE and a region of high homology tothat required for shrinkage-dependent activation of hNHE1. Aftershrinkage-dependent activation of paNHE1 and resulting activation of aCl/HCO exchanger, their paralleloperation results in net uptake of NaCl and osmotically obliged water.Activation of paNHE1 by cAMP is at least additive to that elicited byosmotic shrinkage, suggesting that these stimuli regulate paNHE1 bydistinct mechanisms. Finally, exposure to the serine/threoninephosphatase inhibitor calyculin A potently activates paNHE1, and thisactivation is also additive to that induced by shrinkage or cAMP.

     

Alterations in airway ion transport in NKCC1-deficient mice

Airways of Na⁺-K⁺-2Cl(NKCC1)-deficient mice (/) were studied in Ussing chambers todetermine the role of the basolateral NKCC1 in transepithelial anionsecretion. The basal short-circuit current (I_sc)of tracheae and bronchi from adult mice did not differ betweenNKCC1/ and normal mice, whereas NKCC1/ tracheae from neonatalmice exhibited a significantly reduced basalI_sc. In normal mouse tracheae, sensitivity tothe NKCC1 inhibitor bumetanide correlated inversely with the age of themouse. In contrast, tracheae from NKCC1/ mice at all ages wereinsensitive to bumetanide. The anion secretory response to forskolindid not differ between normal and NKCC1/ tissues. However, whenlarger anion secretory responses were induced with UTP, airways fromthe NKCC1/ mice exhibited an attenuated response. Ion substitutionand drug treatment protocols suggested that HCOsecretion compensated for reduced Cl secretion inNKCC1/ airway epithelia. The absence of spontaneous airway diseaseor pathology in airways from the NKCC1/ mice suggests that theNKCC1 mutant mice are able to compensate adequately for absence of theNKCC1 protein.

     

Functional and molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia

This study focused on the role ofsodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated iontransport in porcine vas deferens epithelium. Ion substitutionexperiments in modified Ussing chambers revealed that cAMP-mediatedstimulation was dependent on the presence of Na⁺,HCO, and Cl for a full response.HCO-dependent current was unaffected byacetazolamide, bumetanide, or amiloride but was inhibited bybasolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.Na⁺-driven, HCO-dependent,stilbene-inhibitable anion flux was observed across the basolateralmembrane of selectively permeabilized monolayers. Results ofradiotracer flux studies suggest a4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 baseequivalents per Na⁺. Antibodies raised against rat kidneyNBC epitopes (rkNBC; amino acids 338-391 and 928-1035)identified a single band of ~145 kDa. RT-PCR detected NBC1 message inporcine vas deferens epithelia. These results demonstrate that vasdeferens epithelial cells possess the proteins necessary for thevectoral transport of HCO and that these mechanismsare maintained in primary culture. Taken together, the results indicatethat vas deferens epithelia play an active role in male fertility andhave implications for our understanding of the relationship betweencystic fibrosis and congenital bilateral absence of the vas deferens.

     

Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells

The -adrenergic antagonisttimolol reduces ciliary epithelial secretion in glaucomatous patients.Whether inhibition is mediated by reducing cAMP is unknown. Elementalcomposition of rabbit ciliary epithelium was studied by electron probeX-ray microanalysis. Volume of cultured bovine pigmented ciliaryepithelial (PE) cells was measured by electronic cell sizing;Ca²⁺ activity and pH were monitored with fura 2 and2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 µM) produced similar K and Cl losses fromciliary epithelia in HCO/CO₂ solutionbut had no effect in HCO/CO₂-free solution or in HCO/CO₂ solutioncontaining the carbonic anhydrase inhibitor acetazolamide. Inhibitionof Na⁺/H⁺ exchange by dimethylamiloride inHCO/CO₂ solution reduced Cl and Kcomparably to timolol. cAMP did not reverse timolol's effects. Timolol(100 nM, 10 µM) and levobunolol (10 µM) produced cAMP-independentinhibition of the regulatory volume increase (RVI) in PE cells andincreased intracellular Ca²⁺ and pH. IncreasingCa²⁺ with ionomycin also blocked the RVI. The resultsdocument a previously unrecognized cAMP-independent transport effect oftimolol. Inhibition of Cl/HCO exchangemay mediate timolol's inhibition of aqueous humor formation.

     

Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains

The solute carrier gene family SLC26consists of tissue-specific anion exchanger genes, three of themassociated with distinct human recessive disorders. By a genome-drivenapproach, several new SLC26 family members have been identified,including a kidney- and pancreas-specific gene, SLC26A6. We report thefunctional characterization of SLC26A6 and two new alternativelyspliced variants, named SLC26A6c and SLC26A6d. Immunofluorescencestudies on transiently transfected cells indicated membranelocalization and indicated that both NH₂- and COOH-terminaltails of the SLC26A6 variants are located intracellularly, suggesting atopology with an even number of transmembrane domains. Functionalexpression of the three proteins in Xenopus oocytesdemonstrated Cl and SO transportactivity. In addition, the transport of SO andCl was inhibited by DIDS and HCO. We demonstrated also that the COOH terminus of SLC26A6 binds to the firstand second PDZ domains of the Na⁺/H⁺ exchanger(NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor(NHERF) proteins in vitro. Truncation of the last three amino acids(TRL) of SLC26A6 abrogated the interaction but did not affect transportfunction. These results demonstrate that SLC26A6 and its two splicevariants can function as anion transporters linked to PDZ-interactionpathways. Our results support the general concept of microdomainorganization for ion transport and suggest a mechanism for cysticfibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulationin pancreatic duct cells.

     

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.

     

Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

Regulation of glutamate transport and transport proteins in a placental cell line

We utilized HRP.1 cells derived from midgestation ratplacental labyrinth to determine that the primary pathway for glutamate uptake is via system X, a Na⁺-dependenttransport system. Kinetic parameters of system X activity were similar to those previously determined in rat and humanplacental membrane vesicle preparations. Amino acid depletion caused asignificant upregulation of system X activity at 6, 24, and 48 h. This increase was reversed by the addition ofglutamate and aspartate but not by the addition of -(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with systemX activity indicated a trend toward an increase inGLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h;cell surface expression of the same was enhanced by 24 h.Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basalanionic amino acid transfer, with an enhanced role for GLT1 underconditions of amino acid depletion. In summary, amino acid availabilityas well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line.

     

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.

     

Ca(2+) regulation of gap junctional coupling in lens epithelial cells

The quantitative effects of Ca²⁺signaling on gap junctional coupling in lens epithelial cells have beendetermined using either the spread of Mn²⁺ that is imagedby its ability to quench the fluorescence of fura 2 or the spread ofthe fluorescent dye Alexa Fluor 594. Gap junctional coupling wasunaffected by a mechanically stimulated cell-to-cell Ca²⁺wave. Furthermore, when cytosolic Ca²⁺ concentration(Ca) increased after the addition of the agonistATP, coupling was unaffected during the period thatCa was maximal. However, coupling decreasedtransiently ~5-10 min after agonist addition whenCa returned to resting levels, indicating that thistransient decrease in coupling was unlikely due to a direct action ofCa on gap junctions. An increase inCa mediated by the ionophore ionomycin that wassustained for several minutes resulted in a more rapid and sustaineddecrease in coupling (IC₅₀ ~300 nM Ca²⁺, Hillcoefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca²⁺ waves were too transient to mediate a sustaineduncoupling of lens epithelial cells.

     

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes

To investigate theeffects of reactive oxygen species (ROS) on NHpermeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (V_m) and intracellular pH(pH_i) induced by a 20 mM NH₄Cl-containingsolution. Under control conditions, NH₄Cl exposure induceda large membrane depolarization (to V_m = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pH_i(pH_i) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpH_i/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H₂O₂ or -amyloid protein had nomarked effect on the NH₄Cl-induced pH_i. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pH_i anddpH_i/dt. These increases in pH_iand dpH_i/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies ¹gO₂ and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (G_m). ThisRB-induced G_m increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na⁺concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway.

     

CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts

To study the potentialinfluence of cystic fibrosis conductance regulator (CFTR) onintracellular pH regulation during apoptosis induction, we usedPS120 Chinese hamster lung fibroblasts devoid of theNa⁺/H⁺ exchanger (NHE1 isoform) transfectedwith constructs, allowing the expression of CFTR and/or NHE1. Kineticsof lovastatin-induced apoptosis were measured by orceinstaining, double staining with Hoechst-33258, propidium iodide, DNAfragmentation, and annexin V labeling. In PS120 control cells, thepercentage of apoptotic cells after 40 h of lovastatintreatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells,this percentage was 40 ± 4%. In PS120 NHE1 cells, thetransfection with CFTR did not modify the percentage of apoptoticcells after 40 h (control: 19 ± 3%, n = 8;CFTR: 17 ± 1%, n = 8), indicating that blockingintracellular acidification by overexpressing theNa⁺/H⁺ exchanger inhibited the enhancement ofapoptosis induced by CFTR. In all cell lines, the initial pHvalues were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification.However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of thisincreased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitableCl/HCO exchanger was studied.8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted inCl/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, ourresults suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl/HCO exchanger, resulting in a more efficient intracellular acidification.

     

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29

The patch-clamp technique was used to study the effects ofcarbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to theequilibrium potential forCl(;48 mV) and the membrane potential then started to oscillate(16/23 cells). In voltage-clamp experiments, similar oscillations inwhole cell currents could be demonstrated. The whole cell conductanceincreased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). Insubstitution experiments (22 mMCl in bath solution, = 0 mV), the reversal potential changed from 41.6 ± 2.2 mV(means ± SE, n = 9) to 3.2 ± 2.0 mV (means ± SE, n = 7).When the cells were loaded with the calcium-sensitive fluorescent dye,fluo 3, and simultaneously patch clamped, CCh caused a synchronousoscillating pattern of fluorescence and membrane potential. Incell-attached patches, the CCh-activated currents reversed at arelative membrane potential of 1.9 ± 3.7 mV (means ± SE,n = 11) with control solution in thepipette and at 46.2 ± 5.3 mV (means ± SE,n = 10) with a 15 mMCl solution in the pipette.High K⁺ (144 mM) did not changethe reversal potential significantly (P  0.05, n = 8). In inside-out patches,calcium-dependent Clchannels could be demonstrated with a conductance of 19 pS(n = 7). It is concluded that CChcauses oscillations in membrane potential that involvecalcium-dependent Clchannels and a K⁺ permeability.

     

Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells

The purpose of this study was to examinethe role of the serum- and glucocorticoid-induced kinase (SGK) in theactivation of the epithelial sodium channel (ENaC) by aldosterone,arginine vasopressin (AVP), and insulin. We used atetracycline-inducible system to control the expression of wild-type(SGK), constitutively active (S425Dmutation; SGK), or inactive (K130Mmutation; SGK) SGK in A6 cellsindependently of hormonal stimulation. The effect of SGK expression onENaC activity was monitored by measuring transepithelialamiloride-sensitive short-circuit current (I_sc) of transfected A6 cell lines. Expression ofSGK orSGK and aldosterone stimulation haveadditive effects on I_sc. Although SGK could playsome role in the aldosterone response, our results suggest that othermechanisms take place. SGK abrogatesthe responses to AVP and insulin; hence, in the signaling pathways ofthese hormones there is a shared step that is stimulated by SGK.Because AVP and insulin induce fusion of vesicles to the apicalmembrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane.