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.

Interactions between Na+-dependent uptake of D-glucose, phosphate and L-alanine in rat renal brush border membrane vesicles.

D-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, D-glucose and L-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na+-dependent phosphate transport was inhibited in the presence of either D-glucose (39 mM) or L-alanine (2.4 mM). In this model, with D-glucose or with L-alanine the V value of the phosphate uptake was decreased, whereas the apparent Km for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of L-glucose, D-alanine or D-glucose after phlorizin preincubation. A 30% Na+-dependent L-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. D-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, D-glucose decreased the L-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of D-glucose by L-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na+ gradient insofar as the rate of the Na+ uptake was increased with D-glucose, L-alanine or phosphate and since the absence of variations in membrane potentials did not suppress these inhibitions.

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.

Effect of imidazolines on Na+ transport and intracellular pH in renal proximal tubule cells.

Recently, we characterized an imidazoline-guanidinium receptive site (IGRS) in the renal proximal tubule of rabbit kidney. Although recognized by a series of imidazoline and guanidinium alpha-2 adrenergic compounds, IGRS is insensitive to catecholamines and can be physically separated from alpha-2 adrenergic receptors after solubilization. In the present study, we investigated the effect of imidazoline derivatives on 22Na+ uptake and intracellular pH in isolated cells from rabbit renal proximal tubule. After 5 min of preincubation, idazoxan inhibited the total 22Na+ influx (-30%) in a dose-dependent manner, with a maximum effect at 10(-5) M. The effect of idazoxan was not competitive as shown by the decrease of the maximal velocity of 22Na+ entry (control: 3.80 +/- 0.42; idazoxan 10(-5) M: 3.23 +/- 0.33 nmol/30 s per mg protein, P less than 0.01). A series of imidazoline derivatives inhibited 22Na+ entry with an order of potency similar to that previously found for inhibition of [3H]idazoxan binding to IGRS (cirazoline greater than idazoxan greater than UK 14304 greater than rilmenidine much greater than cimetidine). The inhibition of 22Na+ uptake by these compounds does not appear to be related to interaction with alpha-adrenergic receptors since it was observed in the presence of saturating concentrations of the adrenergic antagonists rauwolscine (alpha-2) or prazosin (alpha-1). When tested on the regulation of intracellular pH by fluorimetric techniques, 10(-5) M cirazoline or idazoxan inhibited by 20% the velocity of the sodium-dependent H+ efflux in acidified cells (P less than 0.02). The concomitant inhibition of 22Na+ entry and of cell realkalinization suggests that imidazoline derivatives inhibit Na+/H(+)-exchanger. This effect could be mediated via the renal IGRS and intracellular second messengers that are not yet known.

Fluorescent video-microscopy study of regulatory volume decrease in primary culture of rabbit proximal convoluted tubule.

The ability of proximal convoluted tubules in primary culture to regulate volume after a hypotonic shock was investigated by a method based on the use of a fluorescent intracellular probe, (2,7-bis(carboxyethyl)-5,6-carboxyfluorescein: BCECF/AM). The fluorescent signal emitted by the trapped dye excited at 450 nm and analyzed by a video-microscopic set was used to measure the relative volume change. At this wavelength the pH indicator, BCECF, was pH-insensitive and the fluorescent signal related only to the intracellular dye concentration and reflected the variations of the cellular volume as calculated from calibration data. We first determined the fading characteristics of the probe. Second, we characterized the mechanism of regulatory volume decrease (RVD) in primary cultures. RVD occurred 1 min after hypotonic shock and was complete by 4 min. This process was blocked in the presence of barium and scorpion venom (Leiurus quinquestriatus Hebraeus). In the same way, lack of chloride in external medium inhibited RVD. The Cl- blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) at 1.10(-5) M also blocked the regulation. We conclude that RVD in primary cultures of rabbit proximal convoluted tubules involves the stimulation of a potassium conductance via the Ca2(+)-activated maxi K+ channel and that the accompanying anion is chloride via a conductive pathway and (or) a KCl cotransport.

Role of monocarboxylic acid transport in intracellular pH regulation of isolated proximal cells.

Isolated proximal cells were prepared from rabbit kidney cortex by mechanical dissociation. The intracytoplasmic pH (pHi) was measured in HCO3(-)-free media (external pH (pHe), 7.3) using the fluorescent dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Cells were acid-loaded by the nigericin technique. Addition of 70 mM Na+ to the cells caused a rapid pHi recovery, which was blocked by 0.5 mM amiloride. When the cells were exposed to 5 mM sodium butyrate in the presence of 1 mM amiloride, the H+ efflux was significantly increased and followed Michaelis-Menten kinetics. Increasing pHe from 6.4 to 7.6 at a constant pHi of 6.4 enhanced the butyrate activation of the H+ efflux. Increasing pHi from 6.5 to 7.2 at a constant pHe of 7.2 reduced the butyrate effect. 22Na uptake experiments in the presence of 1 mM amiloride showed that 1.5 mM butyrate increased the Na+ flux in the proximal cells (pHi 7.10). The efficiency of monocarboxylic anions in promoting a pHi recovery increased with the length of their straight chain (acetate less than propionate less than butyrate less than valerate). The data show that when the Na+/H+ antiporter is blocked, the proximal cells can regulate their pHi by a Na+-coupled absorption of butyrate followed by non-ionic diffusion of butyric acid out of the cell and probably also by OH- influx by means of the OH-/anion exchanger.

Nucleotides mobilize intracellular calcium stores of renal proximal cells in primary culture: existence of a suramin-sensitive mechanism.

Changes of intracellular calcium concentrations [Ca2+]i were measured in primary cultured rabbit proximal convoluted tubules (PCT). A dual-excitation, digital-imaging inverted microscope was used to monitor the fura-2 fluorescence. The basal calcium level was 106 +/- 11 nM (n = 36). The stimulatory effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine were studied. ATP and ADP induced transient increases of [Ca2+]i (1059 +/- 115% of the resting level (n = 29), and 659 +/- 134% (n = 10), respectively) by releasing calcium from cytoplasmic stores. Adenosine had less effect (279 +/- 48% of the resting level, n = 3). In the same conditions the ATP antagonist suramin (100 microM) inhibited the action of ATP and ADP to 231 +/- 52% (n = 3), and 308 +/- 29% (n = 4) of the resting level, respectively, but did not modify that of adenosine (281 +/- 72%, n = 3). A pretreatment (500 ng/ml for 2 h at 37 degrees C) of the culture with the toxin of Bordetella pertussis completely blocked the ATP response. Our results are evidence for the presence of a functional suramin-sensitive ATP and ADP puriceptor in cultured renal proximal cells. A pertussis-toxin-sensitive G protein is linked to the transduction mechanism. This receptor is distinct from an adenosine puriceptor also found in the proximal monolayer.

Apical membrane ionic channels in the rabbit cortical thick ascending limb in primary culture.

Cortical thick ascending limbs of Henle's loop (cTAL) were microdissected from rabbit kidneys and cultured in a hormonally-defined medium. The cultured cells grew as a monolayer and retained the morphological and biochemical characteristics of the original tubule. Cyclic AMP production of the cultured cells was increased by human calcitonin (x13) and parathyroid hormone (x2). The cultured epithelial developed a transepithelial potential of 4.1 +/- 1.3 mV that was orientated positively towards the apical compartment. The basolateral membrane of the cells exhibited a chloride conductance sensitive to diphenylamine 2-carboxylate (DPC) and the apical membrane a barium-sensitive K+ permeability. Patch clamp analysis conducted on the apical membrane of the cells revealed the presence of three types of ionic channel. The first is a large conductance Ca(2+)-activated K+ channel (95 pS). The second K+ channel has a much smaller conductance (18.3 pS) and is insensitive to Ca2+. It may represent the conductive pathway for K+ recycling into the lumen in the original tubule. The last channel is cation selective, does not discriminate between Na+ and K+ and was found to have a conductance of 20.5 pS. Channel activity required a high cytoplasmic calcium concentration (1 mM), and was blocked by ATP (10 microM) applied on its cytoplasmic face.

Zinc transport and metallothionein induction in primary cultures of rabbit kidney proximal cells.

Primary cultures of isolated rabbit renal proximal cells were grown on collagen-coated permeable supports. The confluent epithelia were polarized, making possible the measurement of uptakes and effluxes across the apical and the basolateral membranes. Uptakes of 65Zn were assessed under initial rate conditions, after 0.5 min incubation. The kinetic parameters of apical uptake were a Jmax of 25.1 +/- 5.3 pmol min-1 (micrograms DNA)-1, a Km of 43.3 +/- 7.3 microM and an unsaturable constant of 0.105 +/- 0.029 (n = 7) at 37 degrees C. Cadmium competitively inhibited the zinc uptake, with a Ki value of 24.5 +/- 7.3 microM. Basolateral uptake was characterized by a high capacity (Jmax = 227.9 +/- 46.6 pmol min-1 (micrograms DNA)-1) and an affinity similar to that of the apical uptake (Km = 35.4 +/- 14.2 microM). Cadmium had no effect on the basolateral zinc uptake. Effluxes across the basolateral face of the epithelium always exceeded those across the apical face. Excess zinc in the culture medium induced the synthesis of metallothionein in the epithelia, as judged by the rate of [35S]cysteine incorporation into a fraction of cytosolic proteins. Metallothionein induction did not appear to modify the kinetic parameters of the apical zinc uptake. These data suggest that separate saturable transport systems are responsible for the apical and basolateral zinc uptakes in proximal renal cells. Induction of metallothionein had no apparent effect on apical zinc uptake in this system.

Two types of K+ channels in the apical membrane of rabbit proximal tubule in primary culture.

The patch-clamp technique was used to investigate ionic channels in the apical membrane of rabbit proximal tubule cells in primary culture. Cell-attached recordings revealed the presence of a highly selective K+ channel with a conductance of 130 pS. The channel activity was increased with membrane depolarization. Experiments performed on excised patches showed that the channel activity depended on the free Ca2+ concentration on the cytoplasmic face of the membrane and that decreasing the cytoplasmic pH from 7.2 to 6.0 also decreased the channel activity. In symmetrical 140 mM KCl solutions the channel conductance was 200 pS. The channel was blocked by barium, tetraethylammonium and Leiurus quinquestriatus scorpion venom (from which charybdotoxin is extracted) when applied to the extracellular face of the channel. Barium and quinidine also blocked the channel when applied to the cytoplasmic face of the membrane. Another K+ channel with a conductance of 42 pS in symmetrical KCl solutions was also observed in excised patches. The channel was blocked by barium and apamin, but not by tetraethylammonium applied to the extracellular face of the membrane. Using the whole-cell recording configuration we determined a K+ conductance of 4.96 nS per cell that was blocked by 65% when 10 mM tetraethylammonium was applied to the bathing medium.

Polarized 86Rb+ effluxes in primary cultures of rabbit kidney proximal cells: role of calcium and hypotonicity.

Isolated proximal cells from rabbit kidney were seeded on collagen-coated permeable supports. After 8 days, the cultured cells became organized as a confluent monolayer. The proximal origin of the monolayer was confirmed by enzymatic, immunological, electrical and electron microscopical studies. The epithelia exhibited a morphological polarity that allowed for measurements of effluxes across the apical or the basolateral membranes. 86Rb was used as an isotopic tracer to indicate potassium movements. The 86Rb+ efflux across the basolateral face was 1.93-times that across the apical face, and both effluxes were pH dependent. Apical and basolateral 86Rb+ effluxes increased when the Ca2+ ionophore ionomycin (3 microM) was applied and when monolayers were exposed to a hypotonic medium. A pharmacological study revealed that BaCl2 (5 mM), tetraethylammonium (TEA, 20 mM) and Leiurus quinquestriatus hebraeus scorpion venom (from which charybdotoxin is extracted) abolished both ionomycin and hypotonically-stimulated effluxes, whereas apamin had no significant effect on the hypotonically-stimulated 86Rb+ efflux. This stimulated efflux was also abolished when monolayers were preincubated with pertussis toxin, but did not decrease in a Ca2(+)-free medium.

Evolution of a cortical collecting tubule primary culture.

The evolution of a primary culture of rabbit kidney cortical collecting duct (CCT) was followed with the electron microscope using two monoclonal antibodies directed against the principal (Mab 703) and intercalated (Mab 503) cells, respectively. As a result of the loss of the basement membrane surrounding the seeded tubule, the intercalated cells showed a tendency to be eliminated while the basal cytoplasm of the remaining cells consisting mainly of principal cells, quickly spread out at the surface of the filter. Between the first and the seventh hour, cells underwent rapid processes of both dedifferentiation and redifferentiation. At 48 h and later on, they started to proliferate with the production of many multinucleated cells. Normal mitotic divisions, in contrast, were rarely encountered. Whereas the number of intercalated cells as recognized by Mab 503 increased from the fourth day up to the tenth day corresponding to a fully mature culture, culture cells at all time intervals rather resembled principal cells found in the internal part of the cortex or in the outer stripe of the external medulla. It is suggested that in our experimental conditions, dedifferentiated principal cells give rise to both principal and intercalated cells as recognized by immunocytochemistry in the fully developed cell culture.

Conversion of a rabbit proximal convoluted tubule (PCT) into a cell monolayer: ultrastructural study of cell dedifferentiation and redifferentiation.

The evolution of a primary culture of kidney proximal convoluted tubule (PCT) cells was followed step by step from the plating time of an isolated tubule to the 39th day of culture. During the first 48 h, the structural remodeling of PCT, leading to the formation of a cell monolayer without cell division, is accompanied by intracytoplasmic changes indicating cell dedifferentiation. Numerous autophagic vacuoles are observed inside the cells, and the ultrastructural features characteristic of in situ PCT cells are progressively lost. Despite these drastic modifications, cell polarity, as observed by immunocytochemical detection of the leucine aminopeptidase, remains unaltered. Starting at 48 h, the peripheral cells divide, and the culture proliferates in a centrifugal direction while newly formed cells differentiate. From 6 days onwards, glycogen granules, never encountered in in situ PCT cells, appear in cultured cells and progressively accumulate. At the optimal stage of the culture (12-17 days old), cells somewhat resemble PCT cells, but their apical brush borders remain rudimentary, and basal cytoplasmic interdigitations surrounding densely packed mitochondria are poorly developed. Subsequently, the cells become overloaded with glycogen and lipid inclusions and resemble degenerating cells.

In young primary cultures of rabbit kidney cortical collecting ducts intercalated cells originate from principal or undifferentiated cells.

The evolution of a primary culture of rabbit kidney cortical collecting tubule was followed over a period of 10 to 11 days. The cell types of this segment were characterized by using monoclonal antibodies, specifically directed against principal (Mab 703) and intercalated (Mab 503) cells of the apical membrane. The activity of a H+ pump ATPase was revealed in Mab 503-labeled cells, confirming that these cultured cells present characteristics of intercalated cells. The primary culture was also stained with peanut agglutinin (PNA), a specific ligand of beta intercalated cells. During the first two days, some cells, mainly Mab 503-labeled cells, disappeared, and cell division did not occur. At 2 days, the culture showed 80% and 18% of Mab 703-labeled and Mab 503-labeled cells, respectively. The first mitoses were observed at 2 days. From two to four days, cell division was nestly in Mab 703-labeled cells and only rarely seen in Mab 503-labeled cells, although during this period the proportion of Mab 703-labeled cells decreased to 44% of cells and that of Mab 503-labeled cells increased to 30%. The labeling with PNA was curious. Up to 2 days, PNA stained Mab 503-labeled cells, but from 4 days it stained other cells, probably dedifferentiated ones. In our culture conditions types of cells other than Mab 703-labeled and Mab 503-labeled cells occurred. First, throughout the life of the culture, some cells were not recognized by any monoclonal antibody; their number varied between 10 and 28% of the total cell number.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

Effects of cadmium and copper on zinc transport kinetics by isolated renal proximal cells.

Zinc, cadmium, and copper are known to interact in many transport processes, but the mechanism of inhibition is widely debated, being either competitive or noncompetitive according to the experimental model employed. We investigated the mechanisms of inhibition of zinc transport by cadmium and copper using renal proximal cells isolated from rabbit kidney. Initial rates of 65Zn uptake were assessed after 0.5 min of incubation. The kinetics parameters of zinc uptake obtained at 20 degrees C were a Jmax of 208.0 +/- 8.4 pmol.min-1.(mg protein)-1, a Km of 15.0 +/- 1.5 microM and an unsaturable constant of 0.259 +/- 0.104 (n = 8). Cadmium at 15 microM competitively inhibited zinc uptake. In the presence of 50 microM cadmium, or copper at both 15 and 50 microM, there was evidence of noncompetitive inhibition. These data suggest that zinc and cadmium enter renal proximal cells via a common, saturable, carrier-mediated process. The mechanisms of the noncompetitive inhibition observed at higher concentrations of cadmium or with copper require further investigation, but may involve a toxic effect on the cytoskeleton.

[Interaction of rilmenidine with renal imidazoline-guanidine sites]. / Interaction de la rilmenidine avec les sites imidazoliniques-guanidiniques rénaux.

Several studies have suggested that clonidine, guanfacine and rilmenidine decrease systemic blood pressure by stimulating central alpha 2-adrenergic receptors. However, we have shown that these molecules interact not only with alpha 2-adrenergic but also a new type of "non catecholamine" receptor in rabbit and human renal proximal tubules. This receptor, which we have called the imidazoline-guanidium receptor site (IGRS) seems to be pharmacologically, biochemically and fractionally distinct from alpha 2-adrenergic receptors. In order to determine the relative affinity of rilmenidine for these two types of receptor, we studied its capacity to inhibit the liaison of (H3)-idazoxan, a ligand with a high affinity for the IGRS, and of (H3)-rauwolscine, a ligand selective for alpha 2-adrenergic receptors in the rabbit kidney. The results based on the apparent constants of inhibition (Ki) of the two radioligands [231 +/- 34 nM for (H3)-idazoxan and 2440 +/- 322 nM for (H3)-rauwolscine] showed that the selectivity of rilmenidine was 10 times greater for IGRS than for alpha 2-adrenergic receptors. This preferential activity on IGRS was confirmed by studies of the influx of Na22 into isolated renal proximal tubule cells of the rabbit. They showed that rilmenidine, in contrast to catecholamines, inhibited the transport of Na22 into the renal cells. In conclusion, the data from our studies shows that rilmenidine interacts with renal IGRS and inhibits cellular transport of sodium by a mechanism other than the stimulation of alpha 2-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Genetic abnormalities in cultured cutaneous fibroblasts in SHR rats]. / Anomalies génétiques dans les fibroblastes cutanés de rats SHR en culture.

Skin fibroblasts were isolated from newborn spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) to study their cell growth and reactivity in culture. SHR fibroblasts exhibited an enhanced growth rate in presence of 10 per cent fetal calf serum and a marked increase in 3H thymidine incorporation compared to WKY cells, when confluent quiescent fibroblasts were stimulated by 2, 5, 10 or 15 per cent serum as well as by 10 ng/ml EGF. Inositol phosphate formation determined by exchange chromatography in presence of 20 mM LiC1, was stimulated by serum, 1 microM angiotensin II, I microM bradykinin and 0.1 microM vasopressin in both type of cells labelled with 3H myoinositol. Significantly higher levels were produced in SHR cells by angiotensin II, serum and bradykinin compared to WKY fibroblasts. No difference between the two cell groups was observed with vasopressin. The intracellular pH (pHi) of isolated SHR and WKY fibroblasts was measured in bicarbonate-free medium using the fluorescent dye BCECF. The identical pHi values (7.03 +/- 0.10, n = 5 and 7.04 +/- 0.07, n = 6 for WKY and SHR respectively agree with an absence of Na+/H+ antiport activation in unstimulated cells. This study allows to conclude that skin fibroblasts isolated from newborn SHR, similarly to vascular smooth muscle cells, exhibit an hyperresponsiveness to serum, EGF and angiotensin II. These results demonstrate the presence of an intrinsic cellular developing capacity.

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.