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P Poujeol

Publications and source records attributed to P Poujeol.

At least 19 recordsLinked to original sources
PubMed2006

Extracellular pH alkalinization by Cl-/HCO3- exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney.

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2005

Zinc protects renal function during cadmium intoxication in the rat.

This study investigates the effect in the rat of chronic CdCl2 intoxication (500 microg Cd2+/kg, daily i.p. injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2 (500 microg Zn2+/kg) during chronic CdCl2 intoxication. Our results indicate that 1) most of the filtered Cd2+ is reabsorbed within the kidney; 2) chronic Cd2+ intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2 exposure; 4) epithelial integrity is altered by chronic Cd2+ intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+ protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+ toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+ supplementation may provide a basis for future treatments.

Animals↗
PubMed2004

Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo.

This study investigates the effect in rats of acute CdCl(2) (5 microM) intoxication on renal function and characterizes the transport of Ca(2+), Cd(2+), and Zn(2+) in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd(2+) injection resulted in renal losses of Na(+), K(+), Ca(2+), Mg(2+), PO(4)(-2), and water, but the glomerular filtration rate remained stable. (45)Ca microinjections showed that Ca(2+) permeability in the DT was strongly inhibited by Cd(2+) (20 microM), Gd(3+) (100 microM), and La(3+) (1 mM), whereas nifedipine (20 microM) had no effect. (109)Cd and (65)Zn(2+) microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of (109)Cd were taken up. (109)Cd fluxes were inhibited by Gd(3+) (90 microM), Co(2+) (100 microM), and Fe(2+) (100 microM) in all nephron segments. Bumetanide (50 microM) only inhibited (109)Cd fluxes in LH; Zn(2+) (50 and 500 microM) inhibited transport of (109)Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd(2+) intoxication are suggestive of proximal tubulopathy; 2) Cd(2+) inhibits Ca(2+) reabsorption possibly through the epithelial Ca(2+) channel in the DT, and this blockade could account for the hypercalciuria associated with Cd(2+) intoxication; 3) the PT is the major site of Cd(2+) reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd(2+) reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd(2+) in the DT; and 6) Zn(2+) is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1.

Animals↗
PubMed2003

Swelling-activated chloride and potassium conductance in primary cultures of mouse proximal tubules. Implication of KCNE1 protein.

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.

Animals↗
PubMed2001

Exploration of the pore structure of a peptide-gated Na+ channel.

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Amino Acid Sequence↗
PubMed2001

Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site.

On the basis of intracellular acidifications in the presence of 30 microM cariporide, we selected a fibroblast cell line termed CR5, expressing a mutated Na(+)/H(+) exchanger NHE-1 with a low affinity for cariporide (87 +/- 11.6 microM) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displays a Phe162Ser substitution in its fourth transmembrane segment. Using intracellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recovery for sodium affinity but were still resistant to cariporide. Sequencing the cDNAs encoding these revertants revealed the presence of two mutations situated at a distant location from Phe162 in the same fourth transmembrane segment (Ile169Ser and Ile170Thr). Interestingly, introducing these two mutations in the wild-type cDNA did not result in a significant increase in affinity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. These data suggest that the mutation resulting in the low sodium affinity and the two mutations responsible for the reversion of this phenotype affect the binding of sodium itself instead of the conformational changes triggering substrate translocation. Taken together, these results allow us to propose that optimal sodium binding by the Na(+)/H(+) exchangers requires the geometrical integrity of a highly constrained sodium coordination site.

Animals↗
PubMed2001

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

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

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

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Extracellular adenosine modulates a volume-sensitive-like chloride conductance in immortalized rabbit DC1 cells.

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.

Adenosine↗
PubMed2000

Potassium efflux triggered by P2Y purinoceptor activation in cultured pituicytes.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus-secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 microM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5'-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 microg/ml) and Buthus tamulus (BT) scorpion venom (20-200 microg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 pM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor.

Adenosine Triphosphate↗
PubMed2000

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K(+) channels.

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl(-) channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K(+) channel of 123 +/- 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K(+) over Na(+) and Ca(2+). The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 microM), and gadolinium (500 microM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K(+) channel activity is consistent with the hypothesis that stretch-activated K(+) channels could mediate an increase in K(+) conductance during cell swelling.

Animals↗
PubMed2000

Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock.

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K(+) channels with a conductance of 122 pS. The present report examines the involvement of K(+) channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K(+) currents were produced by exposing cells to a hypotonic solution or to 1 microM ionomycin. These K(+) currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using (86)Rb(+) as a tracer to indicate potassium movements. Apical and basolateral membrane (86)Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of (86)Rb efflux on the apical side of the monolayer was inhibited by 500 microM quinidine or 100 microM gadolinium but was insensitive to scorpion venom [Leirus quinquestriatus hebraeus (LQH)]. An increased (86)Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane (86)Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K(+) channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.

Animals↗
PubMed2000

Extracellular ATP increases [Ca(2+)](i) in distal tubule cells. II. Activation of a Ca(2+)-dependent Cl(-) conductance.

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Extracellular ATP increases [CA(2+)](i) in distal tubule cells. I. Evidence for a P2Y2 purinoceptor.

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.

Adenosine↗
PubMed1999

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes).

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

Adenosine Triphosphate↗
PubMed1999

Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture.

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.

Adenosine↗
PubMed1998

Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules.

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.

Animals↗