Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors.

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.

Role of CFTR in oxidative stress and suicidal death of renal cells during cisplatin-induced nephrotoxicity.

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.

New evidence of a dihydropyridine-activated cationic channel in the MDCK cell line.

Newborn rat distal cells express an apical Ca2+ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca2+i in a concentration-dependent manner (IC50=4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca2+ channel antagonist isradipine and agonist BayK8644 increased Ca2+i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at -50 mV with perfusion solution containing 10 mM CaCl2. Nifedipine (20 µM) induced an increase in current (1.2±0.3 nA), which was partially inhibited by Gd3+. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9±0.8 mV (n=21). EC50 to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.

Cadmium causes delayed effects on renal function in the offspring of cadmium-contaminated pregnant female rats.

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.

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.

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.

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.

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.

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.

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.

Embryonic renal collecting duct cell differentiation is influenced in a concentration-dependent manner by the electrolyte environment.

BACKGROUND: During kidney development, the embryonic collecting duct (CD) epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. It is unknown by which molecular mechanism the different cell types arise. We have experimental evidence that the electrolyte environment is involved in the process of terminal cell differentiation. METHODS: Embryonic CD epithelia from neonatal rabbit kidneys were microsurgically isolated and maintained in gradient perfusion culture for 13 days under serum-free conditions. Controls were maintained in the same medium (Iscove's modified Dulbecco's medium; IMDM) on basal and luminal sides. Experimental series were performed with IMDM only on the basal side, while on the luminal side IMDM with increasing concentrations of NaCl was used. Finally, the development of principal and intercalated cell features was registered by immunohistochemical labeling with markers specific for adult CD cells. RESULTS: Immunohistochemical markers show that the differentiation pattern is quite different when the embryonic CD epithelia are cultured in IMDM only as compared with specimens kept in IMDM supplemented with 3-24 mmol/l NaCl on the luminal cell side. First signs of changes in development were seen when low doses of 3-6 mmol/l NaCl were added. CONCLUSIONS: We conclude that facultative protein expression in embryonic CD epithelium is influenced by the electrolyte environment and starts to be upregulated after administration of unexpectedly low doses of 3-6 mmol/l NaCl added to the luminal perfusion culture medium and increases in a concentration-dependent manner.

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).

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.

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.

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.

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.

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.

The influence of culture media on embryonic renal collecting duct cell differentiation.

During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove's modified Dulbecco's medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams' medium E, McCoys 5A medium, and Dulbecco's modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.

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.