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1.
J Intern Med ; 289(5): 709-725, 2021 05.
Article in English | MEDLINE | ID: mdl-33107650

ABSTRACT

PURPOSE: Pseudoxanthoma elasticum (PXE) is a recessive disorder involving skin, eyes and arteries, mainly caused by ABCC6 pathogenic variants. However, almost one fifth of patients remain genetically unsolved despite extensive genetic screening of ABCC6, as illustrated in a large French PXE series of 220 cases. We searched for new PXE gene(s) to solve the ABCC6-negative patients. METHODS: First, family-based exome sequencing was performed, in one ABCC6-negative PXE patient with additional neurological features, and her relatives. CYP2U1, involved in hereditary spastic paraplegia type 56 (SPG56), was selected based on this complex phenotype, and the presence of two candidate variants. Second, CYP2U1 sequencing was performed in a retrospective series of 46 additional ABCC6-negative PXE probands. Third, six additional SPG56 patients were evaluated for PXE skin and eye phenotype. Additionally, plasma pyrophosphate dosage and functional analyses were performed in some of these patients. RESULTS: 6.4% of ABCC6-negative PXE patients (n = 3) harboured biallelic pathogenic variants in CYP2U1. PXE skin lesions with histological confirmation, eye lesions including maculopathy or angioid streaks, and various neurological symptoms were present. CYP2U1 missense variants were confirmed to impair protein function. Plasma pyrophosphate levels were normal. Two SPG56 patients (33%) presented some phenotypic overlap with PXE. CONCLUSION: CYP2U1 pathogenic variants are found in unsolved PXE patients with neurological findings, including spastic paraplegia, expanding the SPG56 phenotype and highlighting its overlap with PXE. The pathophysiology of ABCC6 and CYP2U1 should be explored to explain their respective role and potential interaction in ectopic mineralization.


Subject(s)
Cytochrome P450 Family 2/genetics , Multidrug Resistance-Associated Proteins/genetics , Pseudoxanthoma Elasticum/genetics , Spastic Paraplegia, Hereditary/genetics , Calcinosis , Cytochrome P-450 Enzyme System/metabolism , Eye/pathology , HEK293 Cells , Humans , Mutation, Missense , Phenotype , Pseudoxanthoma Elasticum/metabolism , Pseudoxanthoma Elasticum/pathology , Retrospective Studies , Skin/pathology , Spastic Paraplegia, Hereditary/metabolism , Spastic Paraplegia, Hereditary/pathology
2.
Br J Pharmacol ; 171(15): 3716-27, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24758416

ABSTRACT

BACKGROUND AND PURPOSE: For decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh -172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl(-) -conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non-CFTR-expressing cells. EXPERIMENTAL APPROACH: Using patch-clamp technique, we tested the effects of CFTRinh -172 and GlyH-101 inhibitors on three distinct types of Cl(-) currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl(-) conductance (VSORC) and finally the Ca(2+) -dependent Cl(-) conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines. KEY RESULTS: We confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl(-) conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations. CONCLUSIONS AND IMPLICATIONS: Both inhibitors affected Cl(-) conductances apart from CFTR. Our results provided insights into their use in mouse models.


Subject(s)
Benzoates/pharmacology , Chlorides/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Glycine/analogs & derivatives , Hydrazines/pharmacology , Thiazolidines/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Cricetinae , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Glycine/pharmacology , Mice
3.
Cell Death Dis ; 4: e817, 2013 Oct 03.
Article in English | MEDLINE | ID: mdl-24091660

ABSTRACT

The clinical use of the antineoplastic drug cisplatin is limited by its deleterious nephrotoxic side effect. Cisplatin-induced nephrotoxicity is associated with an increase in oxidative stress, leading ultimately to renal cell death and irreversible kidney dysfunction. Oxidative stress could be modified by the cystic fibrosis transmembrane conductance regulator protein (CFTR), a Cl(-) channel not only involved in chloride secretion but as well in glutathione (GSH) transport. Thus, we tested whether the inhibition of CFTR could protect against cisplatin-induced nephrotoxicity. Using a renal proximal cell line, we show that the specific inhibitor of CFTR, CFTR(inh)-172, prevents cisplatin-induced cell death and apoptosis by modulating the intracellular reactive oxygen species balance and the intracellular GSH concentration. This CFTR(inh)-172-mediated protective effect occurs without affecting cellular cisplatin uptake or the formation of platinum-DNA adducts. The protective effect of CFTR(inh)-172 in cisplatin-induced nephrotoxicity was also investigated in a rat model. Five days after receiving a single cisplatin injection (5 mg/kg), rats exhibited renal failure, as evidenced by the alteration of biochemical and functional parameters. Pretreatment of rats with CFTR(inh)-172 (1 mg/kg) prior to cisplatin injection significantly prevented these deleterious cisplatin-induced nephrotoxic effects. Finally, we demonstrate that CFTR(inh)-172 does not impair cisplatin-induced cell death in the cisplatin-sensitive A549 cancer cell line. In conclusion, the use of a specific inhibitor of CFTR may represent a novel therapeutic approach in the prevention of nephrotoxic side effects during cisplatin treatment without affecting its antitumor efficacy.


Subject(s)
Cisplatin/adverse effects , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Oxidative Stress/drug effects , Animals , Benzoates/pharmacology , Biomarkers/metabolism , Body Weight/drug effects , Caspase 3/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , DNA Adducts/metabolism , Enzyme Activation/drug effects , Female , Glutathione/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Kidney/drug effects , Kidney/enzymology , Kidney/pathology , Kidney/physiopathology , Kidney Diseases/physiopathology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/enzymology , Kidney Tubules, Proximal/pathology , Kidney Tubules, Proximal/physiopathology , Mice , Platinum/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Thiazolidines/pharmacology
4.
Nephron Physiol ; 119(2): p22-9, 2011.
Article in English | MEDLINE | ID: mdl-21832858

ABSTRACT

The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.


Subject(s)
Kidney/metabolism , Nephrons/metabolism , Serine Proteases/metabolism , Sodium/metabolism , Animals , Biological Transport/physiology , Fibrinolysin/urine , Humans , Models, Biological , Nephrotic Syndrome/metabolism , Nephrotic Syndrome/physiopathology , Nephrotic Syndrome/urine , Plasminogen
5.
Am J Physiol Renal Physiol ; 293(5): F1450-60, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17686954

ABSTRACT

In the adult rat, chronic cadmium intoxication induces nephropathy with Fanconi-like features. This result raises the question of whether intoxication of pregnant rats has any deleterious effects on renal function in their offspring. To test this hypothesis, we measured the renal function of 2- to 60-day-old postnatal offspring from female rats administered cadmium chloride by the oral route (0.5 mg.kg(-1).day(-1)) throughout their entire gestation. Investigations of rat offspring from contaminated pregnant rats showed the presence of cadmium in the kidney at gestational day 20. After birth, the cadmium kidney concentration increased from postnatal day 2 to day 60 (PND2 to PND60), presumably because of 1) milk contamination and 2) neonatal liver cadmium content release. Although the renal parameters (glomerular filtration, U/P inulin, and urinary excretion rate) were not significantly affected until PND45, renal failure appeared at PND60, as demonstrated by a dramatic decrease of the glomerular filtration rate associated with increased excretion of the main ions. In parallel, an immunofluorescence study of tight-junction protein expression of PND60 offspring from contaminated rats showed a disorganization of the tight-junction proteins claudin-2 and claudin-5, specifically expressed in the proximal tubule and glomerulus, respectively. In contrast, expression of a distal claudin protein, claudin-3, was not affected. In conclusion, in utero exposure of cadmium leads to toxic renal effects in adult offspring. These results suggest that contamination of pregnant rats is a serious and critical hazard for renal function of their offspring.


Subject(s)
Cadmium Chloride/pharmacology , Kidney/drug effects , Kidney/physiology , Prenatal Exposure Delayed Effects , Aging/metabolism , Animals , Animals, Newborn , Blood Pressure/drug effects , Body Weight/drug effects , Cadmium Chloride/analysis , Cadmium Chloride/metabolism , Claudin-5 , Claudins , Female , Glomerular Filtration Rate/drug effects , Intercellular Junctions/metabolism , Ions/urine , Kidney/metabolism , Kidney Glomerulus/metabolism , Kidney Tubules, Proximal/metabolism , Liver/metabolism , Membrane Proteins/metabolism , Milk/chemistry , Pregnancy , Rats , Rats, Wistar , Renal Insufficiency/chemically induced , Renal Insufficiency/physiopathology
6.
Am J Physiol Renal Physiol ; 292(2): F628-38, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17003225

ABSTRACT

We have previously shown that K(+)-selective TASK2 channels and swelling-activated Cl(-) currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177-190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812-F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl(-) and K(+) currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K(+) currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pH(i)) and increased the external pH (pH(e)) in wild-type but not in cftr (-/-) cells. The inhibitory effect of DIDS suggests that the acidification of pH(i) and the alkalinization of pH(e) induced by hypotonicity in wild-type cells could be due to an exit of HCO(3)(-). In conclusion, these results indicate that Cl(-) influx will be the driving force for HCO(3)(-) exit through the activation of the Cl(-)/HCO(3)(-) exchanger. This efflux of HCO(3)(-) then alkalinizes pH(e), which in turn activates TASK2 channels.


Subject(s)
Chloride-Bicarbonate Antiporters/physiology , Hypotonic Solutions/pharmacology , Kidney Tubules, Proximal/metabolism , Potassium Channels, Tandem Pore Domain/physiology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Buffers , Cell Line , Cell Membrane/physiology , Cell Size/drug effects , Chloride Channels/physiology , Chlorides/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Hydrogen-Ion Concentration , Kidney Tubules, Proximal/cytology , Mice , Nitrobenzoates/pharmacology , Potassium Channels/physiology , Sodium/pharmacology
7.
J Membr Biol ; 193(3): 153-70, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12962276

ABSTRACT

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 -/- mice. In wild-type PCT cells in primary culture, a Cl- conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > gluconate. This conductance was sensitive to 1 mM 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 microM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 microM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl- and K+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.


Subject(s)
Chlorine/metabolism , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/physiology , Membrane Potentials/physiology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , Potassium/metabolism , Animals , Cell Size , Cells, Cultured , Chloride Channels/drug effects , Chloride Channels/physiology , Electric Conductivity , Hypotonic Solutions/pharmacology , Kidney Tubules, Proximal/drug effects , Membrane Potentials/drug effects , Mice , Mice, Knockout , Osmosis/drug effects , Osmosis/physiology , Osmotic Pressure/drug effects , Potassium Channels/deficiency , Potassium Channels/drug effects
8.
Pflugers Arch ; 445(4): 463-9, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12548390

ABSTRACT

The kidney plays a dominant role in maintaining sodium homeostasis. The control of a nearly constant electrolyte composition and osmotic pressure in the extracellular fluids is achieved by well-regulated vectorial salt and water transport processes. Derangement in function of Na(+) transporting proteins is likely to be responsible for a number of clinical disorders of fluid and electrolyte homeostasis. The identification of the genes implicated in sodium reabsorption in the kidney not only allows a detailed analysis of regulation and function of these proteins in vitro but also the generation of genetically engineered mice that constitute valuable mouse models for human diseases. Our review will focus on recent strategies for generating nephron segment-specific knock-outs for the main apical renal Na(+) transporters and channels.


Subject(s)
Kidney Diseases/metabolism , Kidney/metabolism , Sodium/metabolism , Animals , Biological Transport , Disease Models, Animal , Humans , Mice , Mice, Knockout
9.
Am J Physiol Renal Physiol ; 280(1): F126-45, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11133523

ABSTRACT

Cl(-) currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by (125)I(-) efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl(-) currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate. (125)I(-) efflux experiments showed that exposure of the monolayer to a hypotonic medium increased (125)I(-) loss. Preincubation of cells with LaCl(3) or GdCl(3) prevented the development of the response. The addition of 10 microM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A(1)-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl(3), or GdCl(3) and was activated by GTPgammaS. The selective A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on (125)I(-) efflux. The CPA-induced increase of (125)I(-) efflux was inhibited by DPCPX and external application of LaCl(3) or GdCl(3). Adenosine also enhanced Mn(2+) influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl(-) conductances that share identical characteristics. The activation of both conductances involved Ca(2+) entry into the cell, probably via mechanosensitive Ca(2+) channels. The effects of adenosine are mediated via A(1) receptors that could mediate the purinergic regulation of the volume-sensitive Cl(-) conductance.


Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Chloride Channels/physiology , Kidney Tubules/physiology , Receptors, Purinergic P1/physiology , Animals , Cell Line , Cell Membrane/physiology , Cell Size/drug effects , Chloride Channels/drug effects , Gadolinium/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Hypotonic Solutions , Iodine Radioisotopes/pharmacokinetics , Kidney Tubules/cytology , Lanthanum/pharmacology , Manganese/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Patch-Clamp Techniques , Rabbits , Receptors, Purinergic P1/drug effects , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Transfection , Xanthines/pharmacology
10.
Am J Physiol Renal Physiol ; 279(1): F92-F101, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10894791

ABSTRACT

Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca(2+) concentration ([Ca(2+)](i); EC(50) 3 and 6 microM, respectively). The order of potency for nucleotide analogs was ATP = UTP > adenosine 5'-O-[thiotriphosphate] >> ADP > UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca(2+)](i) responses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca(2+)](i) response to ATP. Thus ATP- and UTP-stimulated [Ca(2+)](i) mobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca(2+)](i) response to ATP.


Subject(s)
Adenosine Triphosphate/pharmacology , Calcium/metabolism , Kidney Tubules, Distal/drug effects , Receptors, Purinergic P2/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/antagonists & inhibitors , Animals , Calcium Channel Blockers/pharmacology , Calcium Signaling/drug effects , Cell Line , Cell Membrane Permeability/drug effects , Estrenes/pharmacology , Fura-2 , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , Heterotrimeric GTP-Binding Proteins/metabolism , Kidney Tubules, Distal/cytology , Kidney Tubules, Distal/metabolism , Male , Manganese/metabolism , Pertussis Toxin , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Pyrrolidinones/pharmacology , Rabbits , Receptors, Purinergic P2Y2 , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , Uridine Triphosphate/pharmacology , Virulence Factors, Bordetella/pharmacology , Xanthines/pharmacology
11.
Am J Physiol Renal Physiol ; 279(1): F102-11, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10894792

ABSTRACT

We characterized Cl(-) conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). (125)I(-) efflux experiments showed that ATP increased (125)I(-) loss with an EC(50) = 3 microM. Diphenylamine-2-carboxylate (10(-3) M) and NPPB (10(-4) M) abolished the (125)I(-) efflux. Preincubation with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or 10(-7) M thapsigargin inhibited the effect of ATP. Ionomycin (2 microM) increased (125)I(-) efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca(2+) concentration ([Ca(2+)](i)). The ATP agonist potency order was ATP >/= UTP > ATPgammaS. Suramin (500 microM) inhibited the ATP-induced (125)I(-) efflux, consistent with P2 purinoceptors. (125)I(-) effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 microM) activated outwardly rectifying Cl(-) currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl(-) currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca(2+) mobilization for apical Cl(-) conductance in a distal tubule cell line.


Subject(s)
Adenosine Triphosphate/pharmacology , Calcium/metabolism , Chloride Channels/metabolism , Chlorides/metabolism , Kidney Tubules, Distal/drug effects , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/antagonists & inhibitors , Animals , Calcium/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Signaling/drug effects , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Polarity , Chelating Agents/pharmacology , Chloride Channels/antagonists & inhibitors , Electric Conductivity , Iodine Radioisotopes/metabolism , Ionomycin/pharmacology , Kidney Tubules, Distal/cytology , Kidney Tubules, Distal/metabolism , Kinetics , Male , Models, Biological , Nitrobenzenes/pharmacology , Rabbits , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y2 , Suramin/pharmacology , Uridine Triphosphate/pharmacology , Xanthines/pharmacology
12.
Am J Physiol ; 276(1): F104-21, 1999 01.
Article in English | MEDLINE | ID: mdl-9887086

ABSTRACT

Cl- conductances were studied in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtained after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR experiments showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell line. Using the whole cell patch-clamp technique, we recorded a linear Cl- conductance activated by forskolin (FK). This conductance was insensitive to DIDS and corresponded to a CFTR-like channel conductance. Fluorescence experiments with 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) showed that FK induced an increase in Cl- efflux and influx in DC1 cells similar to that observed in cultured DCTb cells. 125I- efflux experiments performed on DC1 cells grown on collagen-coated filters showed that exposure of the monolayer to FK led to an increased 125I- loss through the apical membrane only. The addition of 10 microM adenosine activated a linear conductance identical to that recorded with FK and corresponding to the CFTR-like conductance. This conductance was also activated by 5'-(N-ethylcarboxamido)adenosine and CGS-21680 and inhibited in the presence of 8-cyclopentyl-1, 3-diproxylxanthine (DPCPX). This Cl- conductance could also be activated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by CGS-21680. Finally, 125I- efflux showed that adenosine induced an apical efflux mediated through basolateral A2 receptors. Overall, the data show that the DC1 cell line expressed an apical CFTR Cl- conductance that could be activated by adenosine via A2A receptors located in the basolateral membrane and involving G protein and PKA pathways.


Subject(s)
Chloride Channels/metabolism , Cyclic AMP/physiology , Kidney Tubules, Distal/metabolism , Adenosine/pharmacology , Animals , Cell Line, Transformed , Cell Membrane Permeability/drug effects , Chlorides/metabolism , Colforsin/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Fluorescent Dyes , Iodides/metabolism , Kidney Tubules, Distal/cytology , Male , Quinolinium Compounds , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/physiology
13.
Am J Physiol ; 275(5): F651-63, 1998 11.
Article in English | MEDLINE | ID: mdl-9815124

ABSTRACT

Cl- conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl- conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl- currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl- currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl- currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl- current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl- pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.


Subject(s)
Chloride Channels/physiology , Chlorides/metabolism , Kidney Tubules/physiology , Animals , Cells, Cultured , Ion Transport , Kidney Tubules/cytology , Patch-Clamp Techniques , Rabbits
14.
Am J Physiol ; 273(5): F680-97, 1997 11.
Article in English | MEDLINE | ID: mdl-9374831

ABSTRACT

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl- currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl- currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I- = Br- > Cl- >> glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+ but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 microM LaCl3, 10 microM nifedipine, 100 microM verapamil, 5 microM tamoxifen, and 50 microM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl- currents. Osmotic shock also activated K+ currents in Cl- free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl- and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.


Subject(s)
Chloride Channels/physiology , Kidney Tubules, Distal/cytology , Kidney Tubules, Distal/physiology , Potassium Channels/physiology , Animals , Anions/metabolism , Bromides/pharmacology , Cell Size , Cells, Cultured , Chlorides/metabolism , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Hypotonic Solutions , Kinetics , Meglumine/pharmacology , Membrane Potentials/drug effects , Patch-Clamp Techniques , Rabbits
15.
C R Acad Sci III ; 320(3): 223-32, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9183441

ABSTRACT

Ionic Cl- currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl- conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying 1-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > glutamate. The forskolin-activated Cl- conductance demonstrated a linear I-V relationship and its ion selectivity was Br- > Cl- > I- > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl- currents, and DIDS inhibited only the swelling-activated Cl- current. Forskolin had no effect on the activation of the swelling-activated Cl- current. In DCTb cells which exhibited swelling-activated Cl- currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl- currents. In the continuous presence of I- which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl- conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.


Subject(s)
Cell Size , Chlorides/physiology , Colforsin/pharmacology , Kidney Tubules, Distal/physiology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Cell Membrane Permeability/drug effects , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Electric Conductivity , Electrophysiology , Kidney Tubules, Distal/cytology , Osmolar Concentration , Patch-Clamp Techniques , Rabbits
16.
Am J Physiol ; 271(4 Pt 2): F940-50, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8898026

ABSTRACT

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.


Subject(s)
Calcium/pharmacology , Chlorides/physiology , Kidney Tubules, Distal/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Biological Transport , Calcium/metabolism , Cell Membrane/metabolism , Cell Membrane Permeability , Cells, Cultured , Chlorides/antagonists & inhibitors , Chlorides/metabolism , Colforsin/pharmacology , Electric Conductivity , Fluorescent Dyes , Intracellular Membranes/metabolism , Ionomycin/pharmacology , Ionophores/pharmacology , Kidney Tubules, Distal/cytology , Male , Quinolinium Compounds , Rabbits
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