Philippe Poujeol

Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells

The aim of this study was to characterize the role of CFTR during Cd(2+)-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr(+/+) and cftr(-/-) mice were used. In cftr(+/+) cells, the application of Cd(2+) (5 microM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl(-) conductance sensitive to CFTR(inh)-172. Thereafter Cd(2+) induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl(-) and TASK2 K(+) conductances. By contrast, cftr(-/-) cells exposed to Cd(2+) were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr(+/+) cells, Cd(2+) enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR(inh)-172 or in cftr(-/-) cells. Additionally, Cd(2+) exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr(+/+) cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd(2+) exposure by modulating the intracellular content of glutathione.

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC50 of ∼2 nm, VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V1a receptor antagonist. This pharmacological profile matches the one for VP‐ and OT‐evoked calcium signals in pituicytes, consistent with the fact that VP‐induced taurine efflux is blocked by BAPTA‐AM. However, BAPTA‐AM also blocks the taurine efflux induced by a 270 mosmol l−1 challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium‐mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS) and 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB) suggests the same pathway for VP‐induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l−1). Moreover, it is likely that the osmosensitivity of the taurine channel is up‐regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.

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 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 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 positions 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.

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Abstract. Chloride (Cl−) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl− current activated by 10−5m forskolin, 10−3m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6m phorbol 12-myristate 13-acetate (PMA), 10−3m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br− > Cl−≫ I− > glutamate. This current was inhibited by 10−3m diphenylamine-2-carboxylate (DPC) and 10−4m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl− current blocked by DIDS. To determine the exact location of the Cl− conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl− removal from the apical solution induced a Cl− efflux which was stimulated by 10−5m forskolin, 10−7 calcitonin and inhibited by 10−5m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br− influx through the Cl− pathway. Forskolin and calcitonin had no effect on the basolateral Cl− permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl− conductance in the apical membrane through a cAMP-dependent mechanism.

Swelling-activated transport of taurine in cultured gill cells of sea bass: physiological adaptation and pavement cell plasticity.

We have investigated volume-activated taurine transport and ultrastructural swelling response of sea bass gill cells in culture, assuming that euryhaline fish may have developed particularly efficient mechanisms of salinity adaptation. In vivo, when sea basses were progressively transferred from seawater to freshwater, we noticed a decrease in blood osmotic pressure. When gill cells in culture were subjected to 30% hypotonic shock, we observed a five-fold stimulation of [(3)H]taurine efflux. This transport was reduced by various anion channel inhibitors with the following efficiency: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid > DIDS = diphenylamine-2-carboxylic acid. With polarized gill cells in culture, the hypotonic shock produced a five-fold stimulation of apical taurine transport, whereas basolateral exit was 25 times higher. Experiments using ionomycin, thapsigargin, BAPTA-AM, or removal of extracellular calcium suggested that taurine transport was regulated by external calcium. The inhibitory effects of lanthanum and streptomycin support Ca(2+) entry through mechanosensitive Ca(2+) channels. Branchial cells also showed hypotonically activated anionic currents sensitive to DIDS and NPPB. Similar pharmacology and time course suggested the potential existence of a common pathway for osmosensitive taurine and Cl(-) efflux through volume-sensitive organic osmolyte and anion channels. A three-dimensional structure study revealed that respiratory gill cells began to swell only 15 s after hypoosmotic shock. Apical microridges showed membrane outfoldings: the cell surface became smoother with a progressive disappearance of ridges. Therefore, osmotic swelling may not actually induce membrane stretch per se, inasmuch as the microridges may provide a reserve of surface area. This work demonstrates mechanisms of functional and morphological plasticity of branchial cells during osmotic stress.

In vivo Cre/loxP mediated recombination in mouse Clara cells

In small airways, Clara cells are the main epithelial cell type and play an important physiological role in surfactant production, protection against environmental agents, regulation of inflammatory and immune responses in the respiratory system. Thus, Clara cells are involved in lung homeostasis and pathologies like asthma, Chronic Obstructive Pulmonary Diseases (COPD) or cancers. To date, Clara cells implication in these pathological processes remains largely enigmatic. The engineering of a transgenic strain mouse allowing specific gene invalidation in Clara cells may be of interest to improve our knowledge about the genes involved in these diseases. By using the Cre/loxP strategy we report the engineering of a transgenic mouse strain with expression of Cre recombinase under the control of the Clara Cell Secretory Protein (CCSP) promoter. Specific staining and immuno-histochemistry performed after breeding with reporter mice revealed that CCSP drives a functional Cre expression specifically in Clara cells. This mouse strain is a powerful tool for Cre-loxP-mediated conditional recombination in the lung and represents a new tool to study Clara cell physiology.

KCNQ1 K+ channels are involved in lipopolysaccharide-induced apoptosis of distal kidney cells.

Most bacteria initiate host inflammatory responses through interactions with epithelial cells. Lipopolysaccharide (LPS), a component of the bacterial cell wall is a major cause of septic shock in emergency care units and in the pathogenesis of acute renal failure. Kidney cells exposed to LPS undergo apoptotic changes, including cell volume decrease, phosphatidylserine exposure, caspase-3- and membrane K+ conductance -activation. Whole-cell configuration was used to identify K+ channels in primary and immortalized culture of mice distal convoluted tubules. LPS exposure induced a 3 fold increase in intracellular cAMP concentration and the activation of an outwardly rectifying K+ conductance in both immortalized and primary culture of distal cells. This LPS-induced current exhibited KCNQ1 K+ channel characteristics, i.e. inhibition by quinidine, chromanol293B and low dose of HMR1556 (IC50<1 µM) and insensitive to TEA and charybdotoxin. The background-like biophysical properties of the current suggest that the KCNQ1 pore-forming subunit is associated with a KCNE2 or KCNE3 ancillary subunit. RT-PCR experiments confirmed the presence of KCNQ1 and KCNE3 mRNA transcripts in primary culture of distal segments. Activation of the KCNQ1/KCNE3 K+ current appeared to be an essential step in the LPS-induced apoptosis process since HMR1556 blocked the LPS-induced- cell volume decrease, -caspase-3 activation and -phosphatidylserine exposure.

Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule

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.