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1.
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.

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2.
The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(ex) 350 nm, emission wavelength (em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (ex 380 nm,em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M1)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.  相似文献   

3.
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.

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4.
An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityPx/PCl 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.

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5.
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.

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6.
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.

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7.
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.

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8.
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.

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9.
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.

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10.
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.

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11.
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.

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12.
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.

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13.
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(Ki) 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.

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14.
Previous studies have indicated thatCa2+-dependentCl secretion acrossmonolayers of T84 epithelial cells is subject to a variety of negativeinfluences that serve to limit the overall extent of secretion.However, the downstream membrane target(s) of these inhibitoryinfluences had not been elucidated. In this study, nuclide effluxtechniques were used to determine whether inhibition ofCa2+-dependentCl secretion induced bycarbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor,or insulin reflected actions at an apical Cl conductance, abasolateral K+ conductance, orboth. Pretreatment of T84 cell monolayers with carbachol or acell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced theability of subsequently added thapsigargin to stimulate apical125I,but not basolateral86Rb+,efflux. These data suggested an effect on an apicalCl channel. Conversely,epidermal growth factor reducedCa2+-stimulated86Rb+but not125Iefflux, suggesting an effect of the growth factor on aK+ channel. Finally, insulininhibited125Iand86Rb+effluxes. Thus effects of agents that inhibit transepithelial Cl secretion are alsomanifest at the level of transmembrane transport pathways. However, theprecise nature of the membrane conductances targeted are agonistspecific.

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15.
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.

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16.
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 (Isc) 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 toIsc, CPT-cAMP stimulated a small DNDS-sensitive Isc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedIsc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2.

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17.
Human trabecular meshwork cell volume regulation   总被引:1,自引:0,他引:1  
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.

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18.
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.

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19.
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.

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20.
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.

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