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.

     

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.

     

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.

     

Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

The F508 mutationreduces the amount of cystic fibrosis transmembrane conductanceregulator (CFTR) expressed in the plasma membrane of epithelial cells.However, a reduced temperature, butyrate compounds, and "chemicalchaperones" allow F508-CFTR to traffic to the plasma membrane andincrease Cl permeability in heterologous and nonpolarizedcells. Because trafficking is affected by the polarized state ofepithelial cells and is cell-type dependent, our goal was to determinewhether these maneuvers induce F508-CFTR trafficking to the apicalplasma membrane in polarized epithelial cells. To this end, wegenerated and characterized a line of polarized Madin-Darby caninekidney (MDCK) cells stably expressing F508-CFTR tagged with greenfluorescent protein (GFP). A reduced temperature, glycerol, butyrate,or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP(CPT-cAMP)-stimulated transepithelial Cl secretion acrosspolarized monolayers. However, when the basolateral membrane waspermeabilized, butyrate, but not the other experimental maneuvers,increased the CPT-cAMP-stimulated Cl current across theapical plasma membrane. Thus butyrate increased the amount offunctional F508-CFTR in the apical plasma membrane. Butyrate failedto stimulate transepithelial Cl secretion because ofinhibitory effects on Cl uptake across the basolateralmembrane. These observations suggest that studies on heterologous andnonpolarized cells should be interpreted cautiously. The GFP tag onF508-CFTR will allow investigation of F508-CFTR trafficking inliving, polarized MDCK epithelial cells in real time.

     

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.

     

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.

     

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes

We investigated the regulation ofATP-sensitive K⁺ (K_ATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(10⁵ M) activated inward currents in the presence of highexternal K⁺ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (10⁵ M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 10⁷ M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of K_ATP channels wasincreased by pinacidil, and PDBu suppressed openings ofK_ATP channels. When cells were pretreated withchelerythrine (10⁶ M) or calphostin C (10⁷M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (10⁶ M).Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(10⁷ M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on K_ATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh onK_ATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates K_ATP in murine colonic myocytes.

     

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.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

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.     

Myosin regulation of NKCC1: effects on cAMP-mediated Cl- secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstratedpreviously to regulate Clsecretion from intestinal epithelia via its effects on theNa⁺-K⁺-2Clcotransporter (NKCC1). In nontransporting epithelia, inhibition ofmyosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the roleof myosin in the regulation of secretagogue-stimulated Cl secretion in intestinalepithelia. The human intestinal epithelial cell line T84 was used forthese studies. Prevention of myosin light chain phosphorylation withthe MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase withbutanedione monoxime (BDM) attenuated cAMP but notCa²⁺-mediatedCl secretion. Both ML-9 andBDM diminished cAMP activation of NKCC1. Neither apicalCl channel activity,basolateral K⁺ channel activity,norNa⁺-K⁺-ATPasewere affected by these agents. Cytochalasin D prevented suchattenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin lightchain and subsequent contraction of basal actin-myosin bundles arecrucial to the cAMP-driven activation of NKCC1 and subsequent apicalCl efflux.

     

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.

     

Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells

The functionalrole of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell(VSMC) apoptosis remains unknown. In this study, VSMC fromp53^/ and p53^+/+ murine aortas were exposedto exogenous or endogenous sources of NO. Unexpectedly,p53^/ VSMC were much more sensitive to theproapoptotic effects of NO than were p53^+/+ VSMC.Furthermore, this paradox appeared to be specific to NO, because otherproapoptotic agents did not demonstrate this differential effect onp53^/ cells. NO-induced apoptosis inp53^/ VSMC occurred independently of cGMP generation.However, mitogen-activated protein kinase (MAPK) pathways appeared toplay a significant role. Treatment of the p53^/ VSMCwith S-nitroso-N-acetylpenicillamine resulted ina marked activation of p38 MAPK and, to a lesser extent, of c-JunNH₂-terminal kinase, mitogen-activated protein kinasekinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase,ERK). Furthermore, basal activity of the MEK-p42/44 (ERK)pathway was increased in the p53^+/+ VSMC. Inhibition of p38MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-inducedapoptosis. Therefore, p53 may protect VSMC against NO-mediatedapoptosis, in part, through differential regulation of MAPK pathways.

     

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.

     

Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells

Monocyte-endothelial cell interactions havebeen implicated in the pathogenesis of a number of vascular diseasesthat target arterial and aortic endothelium, including atherosclerosis.Many different adhesion molecules, such as intercellular adhesionmolecule (ICAM)-1, are thought to mediate monocyte binding toendothelial cells during the development of these diseases. However,conflicting results have been reported regarding the specific role ofICAM-1 in these events. In this study, we used a genetic approach to determine the contribution of ICAM-1 in mediating monocyte adhesion tomouse aortic endothelial cells (MAEC) derived from both wild-type andICAM-1^/ mice. Treatment of wild-type MAEC with oxidizedlow-density lipoprotein significantly induced both WEHI 274.1 and wholeblood monocyte adhesion, whereas similarly treatedICAM-1^/ MAEC showed a complete inhibition of monocytebinding. Dose-response treatment with tumor necrosis factor- alsoincreased monocyte adhesion to wild-type MAEC, but significant adhesionwas only observed at higher doses for ICAM-1^/ MAEC.These data demonstrate a crucial role for ICAM-1-mediated monocyte-endothelial cell interactions in response to specific stimuliinvolved in inflammatory vascular diseases.

     

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.

     

Duality of G protein-coupled mechanisms for beta-adrenergic activation of NKCC activity in skeletal muscle

Skeletal muscleNa⁺-K⁺-2Cl cotransporter (NKCC)activity provides a potential mechanism for regulated K⁺uptake. -Adrenergic receptor (-AR) activation stimulatesskeletal muscle NKCC activity in a MAPK pathway-dependent manner. Weexamined potential G protein-coupled pathways for -AR-stimulatedNKCC activity. Inhibition of G_s-coupled PKA blockedisoproterenol-stimulated NKCC activity in both the slow-twitch soleusmuscle and the fast-twitch plantaris muscle. However, thePKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP(8-BrcAMP) were not sufficient to activate NKCC in the plantaris andpartially stimulated NKCC activity in the soleus.Isoproterenol-stimulated NKCC activity in the soleus was abolished bypretreatment with pertussis toxin (PTX), indicating aG_i-coupled mechanism. PTX did not affect the8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded theisoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus,consistent with NKCC's MAPK dependency. Inhibition ofisoproterenol-stimulated ERK activity by PTX treatment was associatedwith an increase in Akt activation and phosphorylation of Raf-1 on theinhibitory residue Ser²⁵⁹. These results demonstrate anovel, muscle phenotype-dependent mechanism for -AR-mediated NKCCactivation that involves both G_s and G_iprotein-coupled mechanisms.

     

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.

     

PGE(2), Ca(2+), and cAMP mediate ATP activation of Cl(-) channels in pigmented ciliary epithelial cells

Purines regulate intraocular pressure. Adenosine activatesCl channels of nonpigmented ciliary epithelial cellsfacing the aqueous humor, enhancing secretion. Tamoxifen and ATPsynergistically activate Cl channels of pigmented ciliaryepithelial (PE) cells facing the stroma, potentially reducing netsecretion. The actions of nucleotides alone on Cl channelactivity of bovine PE cells were studied by electronic cell sorting,patch clamping, and luciferin/luciferase ATP assay. Clchannels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca²⁺, prostaglandin (PG)E₂,and cAMP activated Cl channels without enhancing effectsof 100 µM ATP. Buffering intracellular Ca²⁺activity with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acidor blocking PGE₂ formation with indomethacininhibited ATP-triggered channel activation. The Rp stereoisomerof 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited proteinkinase A activity but mimicked 8-bromoadenosine 3',5'-cyclicmonophosphate. We conclude that nucleotides can act at >1 P2Yreceptor to trigger a sequential cascade involving Ca²⁺,PGE₂, and cAMP. cAMP acts directly on Clchannels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Basal lamina of ovarian follicle regulates an inward Cl- current in differentiated granulosa cells

Patch-clampexperiments were conducted to study the effects of basal lamina(basement membrane) of preovulatory chicken ovarian follicle onmembrane currents in differentiated chicken granulosa cells in ahomologous system. The membrane capacitance (measure of total membranearea) was smaller in cells cultured on intact basal lamina than that ofcontrol cells. The granulosa cells expressed outward and two inwardcurrents. A small fraction of the cells (3%) expressed only atransient fast-activating and -inactivating inward current carried byCa²⁺. The majority of the cells, however, expressed aslowly activating and inactivating inward current (carried byCl) that was superimposed on the transientCa²⁺ current. All cells expressed an outward currentcharacteristic of the delayed-rectifier K⁺ current. Theremoval of extracellular Ca²⁺ led to elimination of theslow inward Cl current, indicating that it is aCa²⁺-dependent Cl current. Both peakamplitude and current density of the inward Cl currentwere significantly lower in cells cultured on freshly isolated intactbasal lamina (or basal lamina stored at 4°C for 12 mo) than those ofcontrol cells; however, basal lamina had no significant effect on thedensity of the outward current. Similar to the observations made forintact basal lamina, solubilized basal lamina suppressed the inwardCl current in differentiated granulosa cells. These datashow that homologous basal lamina modulates aCa²⁺-dependent Cl current in differentiatedgranulosa cells. These findings provide a partial explanation for themechanisms that subserve the reported effects of basal lamina (basementmembrane) on the metabolic functions of differentiated granulosa cells.