Michel Bidet

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.

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.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.

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pHi) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pHi was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pHi values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pHi by means of the classic Na+/H+ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pHi recovery was mediated by a Na+/H+ exchange present in the basolateral side. Moreover, at physiological initial pHi values, chloride removal from the apical solution caused the pHi to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pHi recovery was induced in sodium-free media by 15 mM HCO3−in the presence of an outward chloride gradient. This pHi change was completely abolished by 4,4′-diisothiocyanostilbene 2,2′-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na+-independent Cl−/HCO3−exchanger.

Activation of calcium influx by ATP and store depletion in primary cultures of renal proximal cells

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 μM) induced a biphasic increase of [Ca2+]i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca2+]i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca2+]i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca2+ uptake, blocked by lanthanum (10 μM) and verapamil (100 μM) but not by the nifedipin (25 μM). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by86Rb+ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca2+-sensitive K+ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca2+ influx, which facilitates the replenishment of the internal stores.

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.

The Hedgehog Receptor Patched Functions in Multidrug Transport and Chemotherapy Resistance

Most anticancer drugs fail to eradicate tumors, leading to the development of drug resistance and disease recurrence. The Hedgehog signaling plays a crucial role during embryonic development, but is also involved in cancer development, progression, and metastasis. The Hedgehog receptor Patched (Ptc) is a Hedgehog signaling target gene that is overexpressed in many cancer cells. Here, we show a link between Ptc and resistance to chemotherapy, and provide new insight into Ptc function. Ptc is cleared from the plasma membrane upon interaction with its ligand Hedgehog, or upon treatment of cells with the Hedgehog signaling antagonist cyclopamine. In both cases, after incubation of cells with doxorubicin, a chemotherapeutic agent that is used for the clinical management of recurrent cancers, we observed an inhibition of the efflux of doxorubicin from Hedgehog-responding fibroblasts, and an increase of doxorubicin accumulation in two different cancer cell lines that are known to express aberrant levels of Hedgehog signaling components. Using heterologous expression system, we stringently showed that the expression of human Ptc conferred resistance to growth inhibition by several drugs from which chemotherapeutic agents such as doxorubicin, methotrexate, temozolomide, and 5-fluorouracil. Resistance to doxorubicin correlated with Ptc function, as shown using mutations from Gorlins syndrome patients in which the Ptc-mediated effect on Hedgehog signaling is lost. Our results show that Ptc is involved in drug efflux and multidrug resistance, and suggest that Ptc contributes to chemotherapy resistance of cancer cells. Mol Cancer Res; 10(11); 1496–508. ©2012 AACR.

Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, we expressed these human receptors in Drosophila Schneider 2 cells. We show here that, as its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. We also provide experiments showing that human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, we propose that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different.

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl−/HCO3−exchanger [1]. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl−/HCO3−exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.

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.

Ion Channels in the Distal Convoluted Tubule in Primary Culture

Cortical distal bright convoluted tubules (DCTb) 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. The cultured epithelia developed a transepithelial potential of 3.11±0.53 mV that was oriented negatively towards the apical compartment. The apical membrane of the cells exhibited a sodium conductance sensitive to 10−6 M amiloride and 10−7 M phenamil and a small K+ conductance which was blocked by 5 mM Ba2+. The basolateral membrane was highly permeable to K+. Patch clamp analysis conducted on the apical membrane of the cells revealed the presence of three types of ionic channel. The first was a small conductance cation channel (3 pS), sensitive to phenamil, which resembled the Na+ channel, but the specificity of which seemed to be altered in excised configuration. The second was a non selective cation channel, it did not discriminate between Na+ and K+ and had a conductance of 20.5 pS. It was strongly voltage-dependent and required a high calcium concentration (1 mM) to its cytoplasmic face. The last channel was a small conductance highly selective K+ channel (8 pS). These three channels could account for the macroscopic Na+ and K+ conductances found in the apical membrane of rabbit DCTb in primary culture.