CFTR null mutation altered cAMP-sensitive and swelling-activated Cl currents in - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：HervéBarrière, RadiaBelfodil, IsabelleRubera, MichelTauc, ChantalPoujeol, MichelBidet,  PhilippePoujeol作者单位：Unité Mixte de Recherche Centre National de la RechercheScientifique 654 Université de Nice-Sophia Antipolis, 06108 Nice Cedex  France 3 D9 I5 B, D0 Z


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      【摘要】
   The role of cystic fibrosis transmembraneconductance regulator (CFTR) in the control of Cl currents was studied in mouse kidney. Whole cell clamp was used toanalyze Cl currents in primary cultures of proximal anddistal convoluted and cortical collecting tubules from wild-type (WT)and cftr knockout (KO) mice. In WT mice, forskolin activateda linear Cl current only in distal convoluted andcortical collecting tubule cells. This current was not recorded in KOmice. In both mice, Ca 2  -dependent Cl currents were recorded in all segments. In WT mice, volume-sensitive Cl currents were implicated in regulatory volume decreaseduring hypotonicity. In KO mice, regulatory volume decrease andswelling-activated Cl current were impaired but wererestored by adenosine perfusion. Extracellular ATP also restoredswelling-activated Cl currents. The effect of ATP oradenosine was blocked by 8-cyclopentyl-1,3-diproxylxanthine. Theecto-ATPase inhibitor ARL-67156 inhibited the effect of hypotonicity and ATP. Finally, in KO mice, volume-sensitive Cl currents are potentially functional, but the absence of CFTR precludestheir activation by extracellular nucleosides. This observationstrengthens the hypothesis that CFTR is a modulator of ATP release in epithelia. 6 Y2 M% s' e! S- g1 m4 X$ Q! \' R% w. T( c
      【关键词】 kidney cystic fibrosis cell volume regulatory volume decrease! _2 Z5 ~3 L0 \. g; S8 I
   INTRODUCTION

THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) protein has beendetected by electrophysiological techniques in a variety of culturedcells of the renal tubule, such as distal convoluted tubule (DCT)( 21 ), cortical collecting tubule (CCT) ( 2, 30 ), and inner medullary collecting duct ( 10 ). Inthese segments, the presence of CFTR is correlated with activation of acAMP-activated Cl current. However, along the nephron,CFTR is not always associated with these Cl currents. Forinstance, despite the presence of CFTR transcripts, CFTR expression,along with forskolin-induced conductance, was not detected in rabbitproximal tubule in primary culture ( 21 ). This observationhighlights the fact that CFTR could play an important role in thecontrol of different channels in kidney tissue. Such control is nowwell established in secretory epithelia. In these structures, besidesthe cAMP-sensitive Cl secretion, CFTR controls theepithelial Na channel ( 12, 14, 18, 28 ) andthe outwardly rectifying Cl channel ( 24 ).Moreover, CFTR is also needed for an effective volume regulation inairway and intestinal epithelia ( 31, 32 ), suggesting thatit could modulate K and Cl channelsimplicated in regulatory volume decrease (RVD). Indeed, these multiplefunctions of CFTR could explain the different phenotypes induced bycystic fibrosis (CF) in secretory epithelia. In contrast, the role ofCFTR in the kidney remains uncertain, inasmuch as there is no majordisruption of renal function in CF patients ( 27 ).Nevertheless, the reduced renal excretion of NaCl observed in CFindicates that Cl and Na channels could bedependent on CFTR expression, suggesting that mutation of CFTR couldinduce a primary defect in renal function. A better understanding ofthe function of CFTR in the kidney, therefore, seems to be necessary.For this reason, we chose to investigate the role of CFTR along thenephron using primary cultures of proximal convoluted tubules (PCT),DCT, and cortical collecting ducts microdissected from the kidney of cftr / and cftr  /  mice. The cftr / mice lack cAMP-activatedCl currents in the colon, airways, and exocrine pancreascells ( 6 ) and represent a useful model for studying thedifferent ion channel defects due to CF. In the present study, usingpatch-clamp methodology, we confirmed that cAMP-sensitiveCl conductances measured in primary cultures of DCT andcortical collecting tubule (CCT) cells are linked to CFTR integrity.Moreover, in contrast to the data reported in the literature on airways and endothelial cells ( 33 ), an increase inCa 2  -dependent Cl channels does notcompensate for the lack of CFTR Cl channels in renaltissue. The PCT, DCT, and CCT cells from cftr / mice lost their capacity to regulatetheir volume after a hypotonic shock because of the impairment ofswelling-activated Cl channels. In cftr / cells, the activity of these channelscould be restored by external application of adenosine. This suggests that CFTR controls the swelling-activated Cl channels bymodulating adenosine autocrine production in renal cells.

MATERIALS AND METHODS! z+ L+ L' c7 h# Z& t
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Animals
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Knockout CFTR mice were generated with the gene-targetingmethodology previously described ( 26 ) at Centre deDéveloppement des Techniques Avancées pourl'Expérimentation Animale (Orléans, France). Thisstrain of mice was originally derived from ES129/Sv cells injected intoC57BL/6 embryos. They were backcrossed with C57BL/6 mice for threegenerations and then intercrossed. Mice were allowed free access tofood and water in a facility at 25 ± 1°C with a 12:12-hlight-dark cycle. The 4- to 6-wk-old wild-type cftr  /  miceand cftr / mice homozygous for the disrupted cftr gene were killed by cervical dislocation, and thekidneys were removed. All experiments were performed in accordance withthe guidelines of the French Agricultural Office and the legislation governing animal studies.- c$ f0 G0 o/ j5 t2 n4 m0 f5 t5 i- E# [

Primary Cell Cultures

PCT, DCT, and collecting tubules were microdissected understerile conditions. Kidneys were perfused with Hanks' solution (GIBCO)containing 700 kU/l collagenase (Worthington), cut into small pyramidsthat were incubated for 1 h at room temperature in perfusionbuffer (160 kU/l collagenase, 1% Nuserum, and 1 mM CaCl 2 ),and continuously aerated. The pyramids were then rinsed thoroughly inthe same buffer devoid of collagenase. The individual nephrons weredissected by hand in this buffer under binoculars using stainless steelneedles mounted on Pasteur pipettes. The criteria used to identify thenephron segments have been described elsewhere ( 4 ).Briefly, PCT corresponded to the 1- to 1.5-mm segment of tissue locatedimmediately following the glomerulus. The DCT portion was the segmentbetween the macula densa and the first branching with another tubule[i.e., connecting tubule (CNT)]. The CNT segment was discarded. TheCCT was identified as the straight, poorly branched portion thatfollowed the CNT segment. After they were rinsed in dissecting medium,tubules were transferred to collagen-coated 35-mm petri dishes filledwith culture medium composed of equal quantities of DMEM and Ham'sF-12 (GIBCO) containing 15 mM NaHCO 3, 20 mM HEPES, pH 7.4, 1% serum, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodiumselenite, and 10 nM triiodothyronine. Cultures were maintained at37°C in a 5% CO 2 -95% air water-saturated atmosphere.The medium was removed 4 days after seeding and then every 2 days.
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Electrophysiological Studies

Whole cell currents were recorded from 6- to 20-day-old culturedcells grown on collagen-coated supports maintained at 33°C for theduration of the experiments. The ruptured-patch whole cellconfiguration of the patch-clamp technique was used. Patch pipettes (2- to 3-M resistance) were made from borosilicate capillary tubes (1.5 mm OD, 1.1 mm ID; Popper Manufacturing) using a two-stage verticalpuller (model PP 83, Narishige, Tokyo, Japan) and filled with asolution containing (in mM) 140 N -methyl- D -glucamine (NMDG) chloride, 1 or 5 EGTA, 5 MgATP, and 10 HEPES, pH 7.4. The bath solution contained (inmM) 140 NMDG chloride, 1 CaCl 2, 60 mannitol, and 10 HEPES, pH 7.4. Cells were observed using an inverted microscope; thestage of the microscope was equipped with a water robotmicromanipulator (model WR 89, Narishige). The patch pipette wasconnected via an Ag-AgCl wire to the head stage of a patch amplifier(model RK 400, Biologic). After formation of a gigaseal, thefast-compensation system of the amplifier was used to compensate forthe intrinsic input capacitance of the head stage and the pipettecapacitance. The membrane was ruptured by additional suction to achievethe conventional whole cell configuration. Settings available on the amplifier (model RK 400) were used to compensate for cell capacitance. No series resistance compensation was applied, but experiments 20 M were discarded. Solutions wereperfused in the extracellular bath using a four-channel glass pipette,with the tip placed as close as possible to the clamped cell.
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Data acquisition and analysis. Voltage-clamp commands, data acquisition, and data analysis werecontrolled via a computer equipped with a Digidata 1200 interface (AxonInstruments). pCLAMP software (versions 5.51 and 6.0, Axon Instruments)was used to generate whole cell current-voltage ( I-V ) relations, with the membrane currents resulting from voltage stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly on thecomputer hard disk. Cells were held at 50 mV, and 400-ms pulses from 100 to  120 mV were applied in 20-mV increments every 2 s.

Cell Volume Measurement

The relative cell volume was monitored by image analysis withfura 2 as fluorescent volume indicator, as previously reported ( 22 ). Six- to 20-day-old cell monolayers grown on petridishes were loaded with a solution of 2 µM fura 2 containing 0.01%pluronic acid for 20 min at 37°C and then washed with an NaClsolution. The fluorescence was monitored with 360-nm excitationwavelength. At 360 nm, the variations in the signal emitted by theprobe are directly proportional to the variations in cell volume. In atypical experiment, the cells were first perfused with an isotonic NaCl solution containing (in mM) 110 NaCl, 5 KCl, 1 CaCl 2, 90 mannitol, and 10 HEPES, pH 7.4 [osmotic pressure(P osm ) = 320 mosmol/kgH 2 O] at 30 ml/min, and images were averaged eight times and recorded every 5 s for 15 min. Once the fluorescence was stabilized, a hypotonic shockwas induced by perfusing the NaCl solution without mannitol(P osm = 200 mosmol/kgH 2 O). The relativechange in cell volume was estimated from the fluorescent signal byassuming that a 30% decrease in osmolarity caused a decrease in thefluorescent signal corresponding to a maximum swelling of 30% comparedwith the initial volume. The means of relative volume changes were obtained by analysis of 10-20 zones in each culture ( n )chosen with the software. Each zone delimited a cytoplasmic area chosen in individual cells.
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Image analysis. The optical system was composed of a Zeiss ICM-405 inverted microscopeand a Zeiss ×40 objective, which was used for epifluorescent measurement with a 75-W xenon lamp. The excitation beam was filtered through a narrow-band filter centered at 360 nm, mounted in a motorizedwheel (model Lambda 10-2, Sutter Instrument), and equipped with ashutter to control the exposure times. The incident and the emittedfluorescence radiation were separated through a Zeiss chromatic beamsplitter. Fluorescence emission was selected through a 510-nmnarrow-band filter (Oriel). The transmitted light images were viewed byan intensified camera (Extended ISIS, Photonic Science, Sussex, UK).The eight-bit Extended ISIS camera was equipped with an integrationmodule to maximize signal-to-noise ratio. The video signal from thecamera proceeded to an image processor integrated in a DT2867 imagecard (Data Translation) installed in a Pentium 100 personal computer.The processor converts the video signal to 512 lines by 768 squarepixels per line by 8 bits per pixel. The 8-bit information for eachpixel represents one of the 256 possible gray levels, ranging from 0 (for black) to 255 (for white). Image acquisition and analysis wereperformed with the AIW software (version 2.0, Axon Instruments). Thefinal calculations were made using Excel software (Microsoft).

Calibration. We used the methods described by Tauc et al. ( 29 ) using2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein andimproved more recently by Raat et al. ( 17 ) using fura 2. After cells were loaded with the fluorescent probe in the culturemedium, they were perfused with a solution adjusted to variousosmolarities (150-400 mosmol/kgH 2 O) by omittingmannitol. For each osmolarity, two images were stored, averaged, andsubsequently corrected for fading after background subtraction. Themean fluorescence (360 nm) of five areas was plotted against theinverse of P osm (in mosmol/kgH 2 O). Datashowed that when the cells were exposed to a hyposmotic solution,fluorescence decreased linearly with P osm according toBoyle's law. To verify that cells in culture behave as osmometersin a reversible manner, we performed experiments in which the cultureswere perfused successively and randomly with 200-300mosmol/kgH 2 O solutions. The fluorescent signal was relatedto P osm in a reversible way. In all calibrationexperiments, images were recorded 1-2 min after the beginning ofperfusion, at which time the swelling in hypotonic solutions reachedthe maximum value. These methods measure variations in the relative volume as a function of P osm of the perfusion medium( 29 ).

Intracellular Ca 2 Measurements) O1 A* H/ o1 A$ V6 W3 d

Intracellular Ca 2  concentration([Ca 2  ] i ) was measured in cells grown inpetri dishes and loaded for 45 min at room temperature with a solutionof 2 µM fura 2-AM containing 0.01% pluronic acid. The cells werewashed with NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgSO 4, 5 glucose, 20 HEPES, pH 7.40, and 1 Tris. Cells weresuccessively excited at 350 and 380 nm, with images digitized andstored on the computer hard disk for later analysis. Each raw image wasthe result of an integration of four to five frames averaged fourtimes. The acquisition rate was one image every 10 s. For eachmonolayer, [Ca 2  ] i was monitored in18-20 random cells. The equation of Grynkiewicz et al.( 9 ) was used to calculate[Ca 2  ] i from the dualwavelength-to-fluorescence ratio.; u# a5 D5 d0 |7 p
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Expression in Cultured Cells

The cDNA encoding CFTR was introduced into a polycistronicexpression vector derived from the pIRESneo plasmid (cytomegalovirus promoter; Clontech) in which the neomycin resistance gene had beenreplaced by cDNA encoding the chain of the human CD8 cell surfaceantigen. Cells were transfected using the DAC-30 method according tothe manufacturer's instructions (Eurogentec, Herstal, Belgium).Six-day-old cultured cells grown on 35-mm-diameter petri dishes wereserum starved for 24 h before transfection. Transfected cells with2 µg of CD8-CFTR coexpress CFTR and CD8 at their plasma membrane andcan be visualized using anti-CD8 antibody-coated beads (DynabeadsM-450, Dynal, Oslo, Norway) (11a). Cells were electrophysiologically tested 48 h after transfection.5 }6 X& c* A2 g: X0 i3 X1 R
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Chemicals

5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem)was prepared at 100 mM in DMSO and used at 0.1 mM in final solutions.4-4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) wasdirectly dissolved at a final concentration of 1 mM. Forskolin andionomycin were prepared at 10 and 2 mM, respectively, in ethanol andused at 10 and 2 µM, respectively, in bath medium. DIDS, forskolin, ARL-67156(6- N,N -diethyl- - -dibromomethylene- D -adenosine-5'-triphosphate trisodium), apamin, and ionomycin were obtained from Sigma (Saint Quentin Fallavier, France). Fura 2-AM (Molecular Probes) was dissolved at 3 mM in DMSO and added to the loading solution at a finalconcentration of 2 µM, along with 0.01% pluronic acid.

RESULTS, a3 d" ?/ _8 f5 ?! H! J6 c$ L

Cl Currents Activated by Forskolin) y0 H8 m3 S7 Z0 D. o. K

Experiments were performed in a hyperosmotic extracellularsolution (350 mosmol/kgH 2 O) to characterizeCl currents activated by forskolin in PCT, DCT, and CCTcells. Under these conditions, volume-activated Cl currents could not be detected. In the absence of forskolin, thevoltage-step protocol elicited small currents that changed linearlywith the membrane potential in PCT, DCT, and CCT cell cultures fromkidneys of cftr  /  and cftr / mice(data not shown). In cftr  /  mice, exposure of cultured DCTand CCT cells to 10 µM forskolin induced an increase in membranecurrent amplitudes (Fig. 1 A )that reached a peak value 3-4 min after the beginning of theperfusion. These activated currents exhibited a linear I-V relationship, with a reversal potential ( E rev )of 1.3 ± 2.3 mV and a conductance of 6.4 ± 0.6 nS in DCTcells and an E rev of 2.2 ± 2.9 mV and aconductance of 6.0 ± 1.2 nS in CCT cells ( n = 8 monolayers from 4 mice). In contrast, application of forskolin did notmodify the currents recorded in cultured PCT cells (Fig. 1 A ). The unstimulated whole cell current in these cellsreversed at 3.1 ± 1.2 mV, with a slope conductance of 1.1 ± 0.1 nS ( n = 9 monolayers from 4 mice). Asexpected, in cftr / mice, addition offorskolin did not stimulate Cl conductance in all thecultured segments studied. This observation clearly indicated that, inDCT and CCT cells from cftr  /  mice, the Cl conductance stimulated by forskolin was related to CFTR.
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Fig. 1. A : forskolin-induced whole cell Cl currents in proximal convoluted tubule (PCT), distal convoluted tubule(DCT), and cortical collecting tubule (CCT) cells in primary culture of cftr  /  and cftr / mice. Membranevoltage was held at 50 mV and stepped to test potential of 100 to 120 mV in 20-mV increments. Whole cell currents were recorded after 3 min of extracellular perfusion of 10 µM forskolin in the presence of1 mM EGTA and 5 mM MgATP in pipette solution and 1 mM CaCl 2 in extracellular bath. B : effects of 1 mM DIDS, 0.1 mM5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), andextracellular Cl substitution by I onforskolin-induced whole cell Cl currents measured at  100mV. Values are means ± SE; n, number of monolayersfrom 4 different mice.
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The forskolin-sensitive Cl currents measured at  100 mVare compared in primary cultures of PCT, DCT, and CCT from cftr  /  and cftr / mice in Fig. 1 B. Only DCT and CCT from wild-type mice exhibitedforskolin-activated Cl currents that were blocked by 0.1 mM NPPB and insensitive to 1 mM DIDS in the extracellular bath.Moreover, in these segments, replacing external Cl withI strongly inhibited the Cl currentsactivated by forskolin and caused E rev to shifttoward positive values: E rev forI = 37.5 ± 1.4 and 17.0 ± 6.8 mV for DCTand CCT, respectively ( n = 4 monolayers from 4 different mice).- r8 q7 r2 V0 G4 g* T  w: J

Ca 2 -InducedCl Currents
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Whole cell currents were recorded with Ca 2  -free (1 mMEGTA) solutions containing NMDG chloride as the major cation in thepipette and with extracellular solutions containing NMDG chloride and 1 mM CaCl 2. The extracellular solution was adjusted to 350 mosmol/kgH 2 O with mannitol to avoid inducingvolume-activated currents. The control macroscopic currents wererecorded, and 2 µM ionomycin was added to the NMDG chloride bathingsolution. Stimulated currents were recorded after 2 min. Figure 2 A shows the currents recorded in PCT, DCT, and CCT monolayers from cftr  /  and cftr / mice. In all cultured segments fromboth types of mice, addition of ionomycin stimulated Cl currents, which increased during depolarizing voltage pulses. Thekinetics of the macroscopic current were clearly time dependent fordepolarizing potentials with a slowly developing component. In culturedPCT cells from cftr  /  mice, currents reversed at 2.6 ± 0.3 mV ( n = 16 monolayers from 4 different mice).Instantaneous currents measured 5 ms after the beginning of thestimulation were almost linear, with an inward current of 485 ± 34 pA at 100 mV and an outward current of 635 ± 42 pA at  100mV. The steady-state current at 380 ms exhibited a marked outwardrectification, with an inward current of 352 ± 40 pA at 100 mVand an outward current of 834 ± 79 pA at  100 mV( n = 16 monolayers from 4 different mice). When thesteady-state current measurements were used to calculate theCl conductance, the maximal outward conductance wassignificantly different from the maximal inward conductance: 9.6 ± 0.8 and 2.4 ± 0.4 nS, respectively ( n = 16 monolayers from 4 different mice; P 2 B, 1 mM DIDS inhibited the ionomycin conductance by 85.6 ± 5.1% ( n = 16 monolayers from 4 different mice). In cultured DCT and CCT cells from cftr  /  mice, the Cl currents induced byionomycin strongly resembled those induced in cultured PCT cells: thesteady-state currents were outwardly rectifying and strongly blocked by1 mM DIDS (Fig. 2 B ). Moreover, as illustrated in Fig. 2, theionomycin-sensitive Cl conductances measured in cftr / mice exhibited characteristics roughlysimilar to those measured in cftr  /  mice, indicating thatCFTR does not participate in the Ca 2  -sensitiveCl conductance along the mouse nephron.

Fig. 2. A : Ca 2  -induced whole cell Cl currents in PCT, DCT, and CCT cells in primary culture of cftr  /  and cftr / mice. Membranevoltage was held at 50 mV and stepped to test potential of 100 to 120 mV in 20-mV increments. Whole cell currents were recorded after 2 min of extracellular perfusion of 2 µM ionomycin in the presence of 1 mM EGTA and 5 mM MgATP in pipette solution and 1 mM CaCl 2 in extracellular bath. B : effects of 1 mM DIDS onCa 2  -induced whole cell Cl currents.Steady-state currents at  100 mV were measured 380 ms after onset ofpulse. Values are means ± SE; n, number of monolayersfrom 4 different mice.

Cl Currents Induced by a HypotonicShock  u0 @7 F4 H3 N2 b7 T) x

To study the effects of changes in P osm on thedevelopment of Cl conductance, currents were induced byosmotic shock. Whole cell currents were recorded withCa 2  -free (5 mM EGTA) pipette solutions containing NMDGchloride and maintained at 290 mosmol/kgH 2 O. Moreover, toeliminate any participation of cations in the inward current,experiments were carried out after Na in the bath solutionwas replaced with NMDG chloride and in the presence of 1 mMCaCl 2. In cftr  /  mice, the control currents were first measured in cultured PCT, DCT, and CCT cells with an extracellular solution osmolarity of 350 mosmol/kgH 2 O.Under this condition, the voltage-step protocol elicited smalltime-independent currents that changed linearly with the membranevoltage and had E rev of 6.2 ± 1.5, 1.5 ± 1.6, and  1.9 ± 0.8 mV for PCT, DCT, and CCT cells,respectively ( n = 4 monolayers from 4 different mice). Because of their small amplitude, the nature of these currents was not analyzed further.
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The monolayers were then perfused with a 290 mosmol/kgH 2 Osolution. Figure 3 A gives thecurrents recorded in PCT, DCT, and 95% of the cftr  /  cells, an increase in the whole cell current wasobserved within 1 min. In all epithelial cell types, the currentsreached a maximum after 4-5 min. Under these conditions, theinitial currents recorded at  100 mV were ~2.5 times the amplitude ofthe currents recorded at 100 mV. These large, outwardly rectifyingcurrents showed a small time-dependent inactivation at depolarizingpotentials 60 mV in cultured PCT and CCT cells and 40 mV incultured DCT cells. In most cases, the time course of this inactivationcould be well fitted with a single exponential irrespective of therecording time. When the cells were reexposed to the hyperosmoticsolution, the currents returned to the control level within 2-3min (Fig. 3 B ). In the three cultured segments, thecurrents induced by hypotonicity were strongly blocked by 1 mM DIDS(Fig. 3 B ).
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Fig. 3. A : characteristics of swelling-induced whole cellCl currents in PCT, DCT, and CCT cells in primary cultureof cftr  /  and cftr / mice.Membrane voltage was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mV increments. Whole cell currents were recordedafter 4-5 min of extracellular perfusion of a 30% hypotonicsolution in the presence of 5 mM EGTA and 5 mM MgATP in pipettesolution and 1 mM CaCl 2 in extracellular bath. B : effects of 1 mM DIDS and hyperosmotic solution (350 mosmol/kgH 2 O) on swelling-induced whole cellCl currents. Steady-state currents at  100 mV weremeasured 20 ms after onset of pulse. Values are means ± SE; n, number of monolayers from 4 different mice.; p& G! o& G3 Q9 A# ]0 O
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In cftr / mice, hypotonic shock was completelyinefficient for increasing Cl conductance in thethree different cultured segments (Fig. 3, A and B ). In all nephron segments studied, an absence of response to hypotonic shock was observed in 100% of the recorded cells. Thisresult implicates CFTR in the control of the swelling-activated Cl conductance in renal epithelium.4 d8 B! A! ]6 ^% U# z$ A

The results reported above clearly show that Cl conductances developed in the presence of forskolin, Ca 2 ,or hypotonic shock in DCT cells were roughly similar to those recordedin CCT cells under the same experimental conditions. Therefore, in thefollowing experimental series, no distinction was made between DCT andCCT cells.
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Cl Currents in Cultured PCT and DCTCells From cftr / Mice Transfected With CFTR cDNA7 m: u5 W" f; H- E

The cftr / PCT and DCT cells in primaryculture were transfected with CD8-CFTR plasmid, which allowsvisualization of transfected cells using anti-CD8 antibody-coatedbeads. After 48 h of transfection, whole cell currents of coatedcells were recorded and compared with whole cell currents of controlunlabeled cells. Figures 4 and 5 illustrate the currents recorded in cftr / -transfected PCT and DCT cells in thepresence of 10 µM forskolin. As expected, addition of forskolininduced an increase in membrane current amplitudes in PCT (Fig. 4 Ab ) and DCT (Fig. 5 Ab ) cells coated with beadsonly. Figures 4 Ae and 5 Ae show that theforskolin-activated currents exhibited a linear I-V relation, with an E rev of 0.2 ± 0.1 mV anda conductance of 7.6 ± 0.4 nS for PCT cells ( n = 5 cells) and an E rev of 0.16 ± 0.5 mV anda conductance of 8.3 ± 0.5 nS for DCT cells ( n = 7 cells). Currents in both cell types were insensitive to 1 mM DIDS(Figs. 4 Ac and 5 Ac ) and blocked by 77 ± 3 and 85 ± 2% for PCT and DCT, respectively, when Cl was replaced by I (Figs. 4 Ad and 5 Ad ). Moreover, this substitution shifted the E rev toward the more positive value: E rev for I = 36.4 ± 8 and 25.7 ± 9 mV in PCT and DCT, respectively. Overall, theseforskolin-sensitive Cl currents were identical to thosemeasured in cftr  /  DCT cells, indicating that transfectionwith CFTR plasmid could restore the normal CFTR currents in PCT and DCT cftr / cells.
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Fig. 4. Restoration of CFTR currents and swelling-activatedCl currents by transitory transfection ofpIRES-CD8 -cftr in PCT cells from cftr / mice. Transfected cells were visualizedusing anti-CD8 antibody-coated beads. Membrane potential was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mVincrements. A : CFTR currents in cells labeled withanti-CD8-coated beads. a : Control; b : 10 µMforskolin in bath solution; c : 10 µM forskolin 1 mMDIDS; d : 10 µM forskolin with extracellular substitutionof Cl by I. e : Averagecurrent-voltage ( I-V ) relationships measured 200 ms afteronset of pulse, obtained from the same cell at rest, during forskolinstimulation alone and after Cl substitution byI. Values are means ± SE of 5 cells from 3 transfected monolayers. B : swelling-activatedCl currents recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 5 mM EGTA and5 mM MgATP in pipette solution and 1 mM CaCl 2 inextracellular bath. a-c : Whole cell currents in cellslabeled with anti-CD8-coated beads. d : Average I-V relationships measured 20 ms after onset of pulse,obtained from the same cell at rest ( B ), during perfusionwith hyposmotic solution, and after perfusion with hyperosmoticsolution. Values are means ± SE of 4 cells obtained from 3 transfected monolayers.( k/ ]6 Q, b2 k4 B5 x7 n7 P
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Fig. 5. Restoration of CFTR currents and swelling-activatedCl currents by transitory transfection ofpIRES-CD8 -cftr in DCT cells from cftr / mice. Transfected cells were visualizedusing anti-CD8 antibody-coated beads. Membrane potential was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mVincrements. A : CFTR currents in cells labeled withanti-CD8-coated beads. a : Control; b : 10 µMforskolin in bath solution; c : 10 µM forskolin 1 mMDIDS; d : 10 µM forskolin with extracellular substitutionof Cl by I. e : Average I-V relations measured 200 ms after onset of pulse, obtainedfrom the same cell at rest, during forskolin stimulation alone andafter Cl substitution by I. Values aremeans ± SE of 7 cells from 3 transfected monolayers. B and C : swelling-activated Cl currents recordedafter 4-5 min of extracellular perfusion of a 30% hypotonicsolution in the presence of 5 mM EGTA and 5 mM MgATP in pipettesolution and 1 mM CaCl 2 in extracellular bath. Ba,Bb, and Bc : whole cell currents in cells labeled withanti-CD8-coated beads. Bd : average I-V relationships measured 20 ms after onset of pulse, obtained from thesame cell at rest ( B ), during perfusion with hyposmoticsolution, and after perfusion with hyperosmotic solution. Values aremeans ± SE of 4 cells from 3 transfected monolayers. C : whole cell currents in cells not labeled withanti-CD8-coated beads.' }2 y, v( U6 D2 B% ~# l- g& d3 |

In another experimental series, the effect of a hypotonic shock wasstudied in cftr / PCT and DCT cellstransfected with the cftr plasmid. In both cell types, afterthe hypotonic shock, the coated cells developed Cl currents within 3 min (Figs. 4 B and 5 B ). Theinitial currents measured 20 ms after the onset of the voltage pulserectified in the outward direction (Figs. 4 Bd and 5 Bd ). For PCT cells, they reversed at  0.6 ± 0.4 mV( n = 4 cells), and the total current at  100 mV was 3.8 times that at 100 mV: 1,555 ± 150 vs. 405 ± 18 pA( n = 4 cells). For DCT cells, they reversed at 0.9 ± 0.3 mV ( n = 4 cells), and the totalcurrent at  100 mV was 2.2 times that at 100 mV: 1,123 ± 155 vs. 508 ± 86 pA ( n = 4 cells). These largeoutwardly rectifying currents showed time-dependent 40 mV. Finally, replacement of thehypotonic bath solution by a hypertonic solution inhibited theCl currents by 74 ± 3 and 80 ± 4% for PCTand DCT cells, respectively ( n = 4). As expected, theuncoated cells remained insensitive to the hypotonic shock (Fig. 5 B ). Therefore, transfection of CFTR also restores theswelling-activated Cl conductance in cftr / PCT and DCT cells.- W0 J5 M! c+ {/ U

Regulation of the Cl ConductanceInduced by Hypotonic Shock incftr / andcftr / DCT and CCT Cells/ y( |1 z4 s, L: ^/ [. R; h
: {6 W2 e  Z; v% Z
Role of extracellular Ca 2 in thepresence of high EGTA concentration in the pipette solution. In cftr  /  cells, to eliminate the implication of cytosolicCa 2  in the development of hypotonicity-inducedCl currents, experiments were generally performed usingpipette solutions containing 5 mM EGTA without additionalCa 2 . The effects of extracellular Ca 2  on thedevelopment of hypotonicity-induced Cl currents were alsotested in cftr  /  DCT and CCT cells. When the hypotonicshock was carried out in the absence of bath Ca 2 ,development of the Cl current was significantly impaired(Fig. 6 A ). As previouslyreported in rabbit distal bright convoluted tubule (DCTb) in primaryculture ( 20 ), these experiments confirm that extracellularCa 2  was required to activate the swelling-activatedCl conductance in DCT and CCT cells cultured from cftr  /  mice. Using this information, we therefore decidedto study the effect of an influx of Ca 2  onswelling-activated Cl conductance in cftr / DCT and CCT cells. For this purpose,the effects of ionomycin were tested on whole cell Cl currents recorded in the absence of intracellular freeCa 2 . Whole cell currents were recorded in the presence of20 mM EGTA in the pipette solution and 1 mM free Ca 2  inthe bath (Fig. 6 B ). In the absence of ionomycin in the bath solution, the hypotonic shock remained inefficient for triggering Cl currents in cftr / cells(Fig. 6 Ba ). In contrast, when the hypotonic shock wasperformed in the presence of 2 µM ionomycin, Cl currents were activated within 5 min (Fig. 6 Bb ). Thesecurrents showed time-dependent inactivation at depolarizing steppotentials 60 mV and displayed an outwardly rectified instantaneous I-V plot (Fig. 6 Be ) with an E rev of 1.1 ± 0.3 mV( n = 7). When the cells were reexposed to thehyperosmotic solution, the currents returned toward control levelwithin 2-3 min (Fig. 6, Bc and Be ). Alternatively, addition of DIDS rapidly reduced the Cl currents (89.7 ± 4% inhibition at  100 mV, n = 5; Fig. 6 Bd ). Overall, the ionomycin-inducedCl currents developed during hypotonicity in DCT and CCTcells from cftr / mice were quite similar tothe swelling-activated Cl currents measured in cftr  /  mice.

Fig. 6. A : effect of extracellular Ca 2  ondevelopment of hypotonicity-induced Cl currents incultured DCT cells from cftr  /  mice. Membrane voltage washeld at 50 mV and stepped to test potential of 100 to  120 mV in20-mV increments. Whole cell currents were recorded after 4-5 minof extracellular perfusion of a 30% hypotonic solution in the presenceof 5 mM EGTA in pipette solution and in the absence of extracellularCa 2  in bath solution. B : effects of ionomycinon development of Cl currents in cultured DCT cells from cftr / mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 20 mM EGTAand 5 mM MgATP in pipette solution and 1 mM CaCl 2 inextracellular bath. Whole cell currents were measured during hypotonicshock. a : Control cells; b : 2 µM ionomycin; c : 2 min of replacement of extracellular solution with ahypertonic solution; d : 1 mM DIDS. e : Average I-V relationships measured 20 ms after onset of pulse,obtained from the same cell at rest. Values are means ± SE; n, number of cells from 4 monolayers.3 H$ V  x" z$ d' t  e% `( q8 V

Very similar results were obtained with PCT cells in primary culture.Briefly, in cftr  /  cells, the swelling-sensitiveCl conductance depended on external Ca 2 , andin cftr / cells, this conductance could bereactivated by addition of ionomycin to the bath solution (data not shown).3 S- X  K; g+ `
/ K2 D  \& F/ w
Role of extracellular Ca 2 in theabsence of EGTA in the pipette solution. The experiments described above indicate that Ca 2  influxinduced by ionomycin could restore the swelling-activatedCl currents in cftr / cells. Tofurther analyze this phenomenon, the effect of ionomycin was tested inthe absence of EGTA in the pipette solution. Two successive, increasedexternal Ca 2  concentrations were applied to the same cftr / DCT cells. The results are reported inFig. 7 A. Control currents wererecorded, and the cells were perfused with a Ca 2  -freesolution containing 2 µM ionomycin. After 2 min, raising theCa 2  concentration to 0.1 µM induced Cl currents that were identical to the swelling-activated Cl currents (Fig. 7 Ab ). A further new increase inCa 2  concentration to 1 µM enhanced the currents (Fig. 7 Ac ). These currents showed virtually no inactivation duringthe 400-ms voltage pulse. Currents obtained by subtracting the currentrecorded at 0.1 µM external Ca 2  from that recorded at 1 µM Ca 2  are shown in Fig. 7 Ad. The resultingcurrents exhibited the characteristic profile of theCa 2  -sensitive Cl currents.

Fig. 7. A : effect of extracellular Ca 2  concentration ([Ca 2  ] ext ) on development ofCl currents in cultured DCT cells from cftr  / mice. Whole cell currents were recorded in the absence of EGTA inpipette solution. a : Control; b : 2 min ofextracellular perfusion of 0.1 µM Ca 2  in the presence ofionomycin; c : 2 min of extracellular perfusion of 1 µMCa 2  in the presence of ionomycin; d : currentsobtained by subtraction of c from b usingpCLAMPFIT 6.0 software. e : Average I-V relationships measured 20 ms after onset of pulse, obtained from thesame cell at rest, during perfusion of ionomycin in the presence ofdifferent Ca 2  concentrations. Values are means ± SEof 6 cells obtained from 6 monolayers. B : effect ofhypotonic shock on intracellular free Ca 2  concentration([Ca 2  ] i ) in fura 2-loaded DCT cells from cftr  /  and cftr / mice. Fura 2 fluorescencewas monitored and converted to [Ca 2  ] i as described in MATERIALS AND METHODS. Hypotonic shock wasinduced by perfusion of a hypotonic NaCl solution (200 mosmol/kgH 2 O). Values are means ± SE of 20 randomcells from 3 monolayers.
% Z; S6 D* K) N' W
On the basis of these results, it appears that Ca 2  entryis an important step in the development of swelling-activatedCl conductance. Fluorescence experiments using fura2-loaded DCT cells were therefore carried out to follow cytosolicCa 2  variations during hypotonic shock. The effect ofhypotonic solution on [Ca 2  ] i in DCT cellsfrom cftr  /  and cftr / mice isshown in Fig. 7 B. In both types of mice, when the cells werebathed with an isotonic NaCl solution (300 mosmol/kgH 2 O)containing 1 mM CaCl 2, the resting[Ca 2  ] i averaged 30.8 ± 5.2 nM( n = 20). Swelling the cftr  /  DCT cells with hypotonic NaCl solution (200 mosmol/kgH 2 O) induced atransient increase of [Ca 2  ] i that reached amaximum value of 120.1 ± 25.1 nM and returned close to thecontrol value within 5 min (Fig. 7 B ). In contrast, swellingthe cftr / DCT cells did not significantlymodify [Ca 2  ] i.1 p* e/ R: `2 f5 u' m$ l4 C2 g
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Role of extracellular adenosine. We previously demonstrated that stimulation of A 1 adenosinereceptors could be implicated in the control of swelling-induced Cl currents in rabbit DCT ( 20 ), andexperiments were therefore performed to determine the role of adenosinein Cl permeability of PCT and DCT cells from cftr  /  and cftr / mice. Results ofwhole cell experiments performed in cftr / PCTand DCT cells are illustrated in Fig. 8.These results were strictly identical to those obtained with cftr  /  PCT and DCT cells. In both types of primarycultures, 10 µM adenosine activated an outwardly rectifyingCl conductance with a time-dependent inactivation atdepolarizing potentials and with a maximal effect at 3-4 min (Fig. 8 A ). E rev of the stimulated currentwere 3.8 ± 3.7 mV ( n = 5 monolayers) and 0.3 ± 2.9 mV ( n = 4) for PCT and DCT cells, respectively. In the presence of adenosine, the maximal slope conductances reached 19 ± 9 nS ( n = 5) and 11 ± 4 nS( n = 4) in PCT and DCT cells, respectively. Theseadenosine-sensitive Cl currents were decreased in thepresence of 1 mM DIDS by 90 and 78% in PCT and DCT cells,respectively. To determine whether the response to adenosine occurredvia receptor-mediated mechanisms, we examined the effect of aP 1 -selective receptor antagonist, 8-cyclopentyl-1,3-diproxylzanthine (DPCPX). Treatment of DCT cells with10 µM DPCPX completely inhibited the development of outward Cl currents first induced by 10 µM adenosine in PCT andDCT cells (Fig. 8 ).

Fig. 8. Effects of adenosine on development of Cl currents incultured PCT ( A ) and DCT ( B ) cells from cftr / mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 5 mM EGTA and5 mM MgATP in pipette solution and 1 mM CaCl 2 10 µM adenosine in extracellular bath. DIDS (1 mM) was perfusedafter development of Cl currents. Cells were treated withantagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX) beforeexposure to adenosine.
: r: d0 [. S/ g/ a; J
The effect of adenosine was concentration dependent. The dose-responsecurve in cultured DCT cells from cftr  /  mice is shown inFig. 9. The half-maximal effect from thiscurve occurred at 5.0 × 10 7 10 5 M.7 `7 m2 u9 l6 r9 ~
2 @2 d. s+ |3 c/ l* r0 E5 M3 S+ j. l* l
Fig. 9. Adenosine dose-response curve. Membrane voltage was heldat 50 mV and stepped to test potential of 100 to  120 mV in 20-mVincrements. Whole cell currents were recorded after 3 min ofextracellular perfusion of adenosine at different concentrations inisotonic N -methyl- D -glucamine solutions in thepresence of 5 mM EGTA and 5 mM MgATP in pipette solution and 1 mMCaCl 2 in extracellular bath. Values at  100 mV wereconverted to percent activation. Values are means ± SE of 6 cellsfrom 3 monolayers.& W' W2 K  g, R

Role of extracellular ATP. In addition to adenosine, it has been postulated that ATP couldactivate a volume-sensitive-like Cl conductance inimmortalized rabbit distal cells ( 20 ). To check thispossibility in PCT and DCT cells from cftr  /  and cftr / mice, we studied the role of ATP in thecontrol of whole cell Cl currents in the presence of 5 mMEGTA in the pipette solution. In PCT and DCT monolayers, addition of 10 µM ATP to the bath solution induced activation of Cl currents within 4-5 min. This ATP-activated Cl current showed time-dependent inactivation at depolarizing step 60 mV (Fig. 10, A and B ) and displayed an outwardly rectifiedinstantaneous I-V plot (data not given) with E rev close to 0 mV. DIDS (1 mM) stronglydecreased ATP-activated currents in both types of monolayers. Overall,these currents were quite similar to those induced by adenosine.Moreover, the effect of ATP was completely blocked by 10 µM DPCPX,indicating that the action was triggered via P 1, ratherthan P 2, receptors. Such results suggested that stimulationof Cl currents in the presence of ATP was most probablydue to an action of adenosine generated by degradation of ATP.Experiments were therefore carried out to check this hypothesis. Forthis purpose, DCT cells from cftr  /  mice were subjected toa hypotonic shock in the presence of the selective ecto-ATPaseinhibitor ARL-67156. ARL-67156 (100 µM) completely blocked theswelling-activated Cl currents (Fig. 10 C ).Adenosine (10 µM) restored a swelling-activated Cl conductance, which displayed an outwardly rectified instantaneous I-V plot with E rev close to 0 mV(Fig. 10 Cd ) and was strongly inhibited by DIDS (Fig. 10 Cb ).

Fig. 10. Effects of ATP on development of Cl currents incultured PCT ( A ) and DCT ( B ) cells from cftr / mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 5 mM EGTA and5 mM MgATP in pipette solution and 1 mM CaCl 2 10 µM ATP in extracellular bath. DIDS (1 mM) was perfused afterdevelopment of Cl currents. Cells were treated with DPCPXbefore exposure to adenosine. Values are means ± SE; n, number of cells from 3 monolayers. C : effectsof ecto-ATPase antagonist ARL-67156 on swelling-activatedCl currents in DCT cells from cftr  /  mice.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a hypotonic solution in the presence of 100 µM ARL-67156( a ), 10 µM adenosine ( b ), or 1 mM DIDS( c ). d : Average I-V relationshipsmeasured 20 ms after onset of pulse obtained from the same cell atrest. Values are means ± SE of 5 cells from 3 monolayers.

RVD in PCT and DCT Cells Fromcftr / andcftr / Mice

To confirm the role of swelling-activated Cl currents in cell volume regulation, we measured relative cell volume inmonolayers by fluorescence-video microscopy in PCT and DCT cells from cftr  /  and cftr / mice. Asexpected, PCT and DCT cells swelled in response to a hypotonic shock(Fig. 11 ). This cell swelling wasfollowed by an RVD in PCT and DCT cells from cftr  /  mice.At 2 min after the hypotonic shock, the relative cell volume reached130 ± 2% ( n = 3 monolayers) and 120 ± 1%( n = 8 monolayers) of the initial volume in PCT and DCTcells, respectively. PCT cells returned to 105 ± 1% of theiroriginal volume within 4 min (Fig. 11 A ), whereas DCT cellsrecovered 104 ± 1% of their volume within only 2 min (Fig. 11 B ). The RVD phenomenon in both cell types was inhibited inthe presence of 100 µM NPPB (Fig. 11 ). Contrary to observations incells from cftr  /  mice, the RVD mechanism was completelyimpaired in PCT and DCT cells from cftr / mice. Whenperfused with the hypotonic solutions, these cells never returned totheir initial volume. Addition of 10 µM adenosine during hypotonicshock restored RVD in DCT cells from cftr / mice (Fig. 11 B ).' |! U5 [3 e* v7 t& n

Fig. 11. Effects of hypotonic shock on cell volume in PCT and DCTcells from cftr  /  and cftr / mice.Cultures were loaded with 2 µM fura 2 and rinsed in an isotonic NaClsolution (300 mosmol/kgH 2 O) for 3 min. A hypotonic shockwas induced by reducing osmolarity of NaCl solution to 200 mosmol/kgH 2 O. Images were recorded every 15 s. Afteranalysis, relative volume change as percentage of initial volume wasplotted against time. A : regulatory volume decrease (RVD) in3 monolayers from PCT cells (25 random cells each) in the presence orabsence of 100 µM 5-nitro-2-(3-phenylpropylamino)-benzoic acid(NPPB). B : RVD in 10 monolayers from DCT cells (25 randomcells each) from cftr / mice and 8 monolayers (25 randomcells each) from DCT cells from cftr  /  mice in the presenceor absence of 100 µM NPPB or adenosine., z- C1 }% O; c9 G( m; a
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DISCUSSION8 |. [7 N3 ?- x, Q" F) g

The aim of the present study was to investigate the putative roleof CFTR in the control of Cl conductances along thedifferent segments of the mouse nephron. Using the patch-clamptechnique to measure whole cell conductance, we analyzed three distincttypes of Cl currents in primary cultures of PCT, DCT, andCCT segments obtained by microdissection of kidney cortex fromwild-type cftr  /  and cftr / mice. TheseCl conductances consisted of forskolin-activated,volume-sensitive, and Ca 2  -activated Cl currents.
: o& d4 }2 ^( f, F, N; W
In the first series of experiments, the effect of forskolin onCl conductance was tested in PCT, DCT, and CCT cells. Forthis purpose, swelling-activated currents were blocked by exposing thecells to a hyperosmotic solution, and Ca 2  -activatedconductances were impaired by the use of high EGTA concentrations inthe pipette solution. In cftr  /  mice, external applicationof forskolin activated a linear Cl current in DCT andCCT, but not PCT, cells. The halide selectivity was consistent with lowrelative I permeability and with an inhibitory effect ofI. Moreover, this forskolin-stimulated conductance wasblocked by NPPB but was quite insensitive to DIDS. Thesecharacteristics are very similar to those reported previously in rabbitdistal bright convoluted tubule (DCTb) in primary culture( 21 ). In contrast, in cultured DCT and CCT cells from cftr / mice, addition of forskolin remained completelyinefficient for increasing Cl conductances. Takentogether, the results obtained in primary cultures from cftr  /  and cftr / mice clearly demonstratethat, at least in DCT and CCT cells, the activity offorskolin-activated Cl channels is consistent with CFTR.In other words, as we concluded in a previous study ( 21 ),the channel involved in the Cl currents activated byforskolin in DCT and CCT is the small-conductance CFTR Cl channel.
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Interestingly, application of forskolin did not stimulate anyCl current in primary cultures of PCT cells from cftr  /  mice. Such an observation was reported in primaryculture of rabbit PCT, in which no CFTR expression and noforskolin-activated Cl currents were detected in theapical membrane, despite the presence of CFTR mRNA ( 21 ).

The presence of CFTR in the mammalian kidney is now well documented( 2, 15, 16 ), but the absence of detectable renal diseasein CF patients led several authors to postulate that an increase ofanother type of Cl channel might compensate for the lackof cAMP-activated Cl channels in renal tissue ( 6, 11 ). On the other hand, the cftr / miceused in the present study did not present significant pulmonarydisease. Moreover, in these mice, it has been shown that theCa 2  -activated Cl channels could becandidates for compensation of the missing CFTR Cl channels ( 6 ). To determine whether this possibility could arise in the renal epithelium, we studied theCa 2  -activated conductance in PCT, DCT, and CCT cells. Asexpected, in cftr  /  mice, extracellular application ofionomycin rapidly activated currents in all types of monolayers. ThisCa 2  -sensitive conductance was similar to that previouslydescribed in rabbit PCT and DCTb cells under identical experimentalconditions ( 21, 22 ). In cftr / mice, theincrease of Cl conductance triggered by ionomycin wasstrikingly identical to that observed in wild-type mice, eliminatingthe hypothesis that Ca 2  -activated conductance couldsubstitute for CFTR Cl conductance in renal epithelium.

In cftr  /  mice, cultured PCT, DCT, and CCT cells developeda volume-sensitive Cl current when exposed to a hypotonicshock. The biophysical and pharmacological characteristics of thisCl conductance show strong similarities to the propertiesof swelling-activated Cl currents described in many otherepithelial cells, including rabbit PCT and DCTb in primary culture( 7, 25 ). Null mutation of the cftr genestrongly impaired the swelling-activated Cl currents inthe three different nephron segments. Moreover, cftr / DCT or CCT cells transfected with cftr cDNA displayedcomplete restoration of cAMP-dependent and swelling-activatedCl currents. Transfection of PCT cells with cftr cDNA also restored both conductances. These observations indicatethat PCT cells have maintained their ability to insert exogenous CFTRinto the apical membrane. Therefore, the lack of forskolin-inducedCl conductance in wild-type PCT cells is probably due toa difference in the protein function, rather than a modification of theintracellular trafficking leading to protein retention in intracellular membranes.: }  j9 {, H" d5 W3 n* E' S6 W

It is well established that the swelling-activated Cl channels participate in the RVD phenomenon, which is induced byexposure of cells to hyposmotic solutions. In the present study, todetermine whether cultured PCT, DCT, and CCT cells develop RVD after ahypotonic shock, we used a simple fluorescence method for studyingrelative cell volume variations ( 29 ). The findingsindicate that cultured cells from cftr  /  mice are sensitiveto osmolarity changes in the bathing medium and that they are capableof RVD after hypotonic shock. RVD was also examined in cultured cellsfrom cftr / mice. These cells exhibited a defectivevolume regulation after a hyposmotic shock. This observation confirmsthe results in the literature ( 31 ) and indicates that CFTRcould play a role in the RVD of epithelia. Obviously, this defectiveRVD is due to the fact that the hypotonic shock is completelyinefficient for increasing Cl conductances. Theintervention of CFTR in the control of swelling-activated Cl conductances has been proposed by Chan et al.( 5 ), who demonstrated, in the human colonic cell line T84,that an antibody against CFTR inhibited the cAMP- as well as theswelling-induced whole cell Cl conductances but did notaffect the Ca 2  -activated Cl channel. Inprevious studies, we found that development of Cl conductance after a hypotonic shock in rabbit DCT cells was related toan influx of external Ca 2  through Ca 2  channels ( 21, 22 ). Such a hypothesis could also apply to the data obtained in DCT cells from cftr  /  mice, becauseremoval of external Ca 2  just before the hypotonic shockcompletely impaired the increase in Cl current,suggesting that Ca 2  influx could participate in activationof the Cl channels in mouse kidney. It is now proposedthat CFTR could regulate other ion channel proteins ( 23 )and could also be implicated in different cell functions such asapoptosis ( 1, 8, 13 ) or cytosolic Ca 2  regulation ( 19 ). Moreover, a recent study by Braunstein et al. ( 3 ) clearly demonstrated that CFTR participates incell volume regulation via control of ATP release. Taken together, these observations led us to propose the hypothesis that the absence ofswelling-activated Cl conductance in DCT cells from cftr / could be related to an alteration of theCa 2  entry. This hypothesis is strengthened by two mainresults: 1 ) The hypotonic shock induced an increase of[Ca 2 ] i in cftr  /  DCT cells, butnot in cftr / cells. 2 ) In cftr / cells, addition of ionomycin in the presence ofhigh intracellular EGTA concentration restored the ability of the cellsto respond to the hypotonic shock by increasing swelling-sensitiveCl conductance. It remains to be shown how intracellularCa 2  can increase in the presence of a high EGTAconcentration. The observations of Evans and Marty (6a) shed light onthis problem by indicating that, with EGTA as a buffer, a whole regionof the cell could escape control by the Ca 2  buffer.Because this region could extend to a large part of the plasma membrane( 10 ), a local transient increase of Ca 2  couldarise in the presence of EGTA. In accordance with the model proposed byBraunstein et al. ( 3 ), the defect in cell volume regulation that we observed in cftr / renal cells couldbe due to a defect in the ATP release pathway. We have proposed( 20 ) that hypotonic shock stimulates ATP release fromrabbit DCT cells. A 1 receptors are then activated byadenosine generated by the degradation of ATP by membrane ectoenzymes,and this stimulation of A 1 receptors induces an influx ofextracellular Ca 2 . Finally, this Ca 2  influxactivates the Cl channel. The observation that adenosinerestores swelling-activated Cl conductance and RVD in cftr / cells confirms that adenosine is a mediator ofRVD, at least in renal epithelium. Therefore, in cftr / cells, there is no volume-sensitive ATP release, and the cascade ofevents that triggers the final increase in Cl conductanceno longer occurs.

The present results confirm that autocrine ATP release is probably anessential step in the cell volume regulation phenomenon. However, itcould be questioned why adenosine or ATP did not activate a currentconsistent with a Ca 2  -activated Cl currentduring a hypotonic shock. We have demonstrated that swelling-activated and Ca 2  -sensitive Cl currents could beadditive but also that their thresholds of activation byCa 2  were quite different. Thus the former was activated at0.1 µM Ca 2 , whereas the latter was activated at 1 µMCa 2 . The cytosolic Ca 2  concentration inducedby hypotonic shock never exceeded 0.15 µM. This small increase isconsistent with the fact that CFTR control of ATP release duringswelling involved probably low ATP concentration and, consequently, lowadenosine production. In the present study, the effect of adenosine inincreasing swelling Cl currents was concentrationdependent, with a half-maximal effect at 5.0 × 10 7 M. As previously reported in rabbit DCT cells, this adenosine concentration raised cell Ca 2  to 0.11 nM, which wassufficient to trigger swelling-activated Cl currents buttoo low to induce Ca 2  -dependent Cl currents.

The RVD process involves Cl and K efflux.Previous data indicate that impairment of RVD in jejunal crypts of cftr / mice was due to a defective K channel( 31, 32 ). However, the nature of the K channels stimulated during hypotonic shock remains very uncertain andappears to depend on the tissue under investigation ( 32 ). Therefore, in the companion article (1a), we investigate theK conductances along the different nephron segments of cftr / and cftr  /  mice.
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1a. Belfodil, R,Barrière H,Rubera I,Tauc M,Poujeol C,Bidet M,andPoujeol P. CFTR-dependent and -independent swelling-activated K currents in primary cultures of mouse nephron. Am J Physiol Renal Physiol 284:F000-F000,2003.
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