Renaud Beauwens

P-GLYCOPROTEIN INHIBITION BY GLIBENCLAMIDE AND RELATED COMPOUNDS

Abstract Glibenclamide is well known to interact with the sulphonylurea receptor (SUR) and has been shown more recently to inhibit the cystic fibrosis transmembrane conductance regulator protein (CFTR), both proteins that are members of the ABC [adenosine 5′-triphosphate (ATP)-binding cassette] transporters. The effect of glibenclamide and two synthetic sulphonylcyanoguanidine derivatives (dubbed BM-208 and BM-223) was examined on P-glycoprotein, the major ABC transporter responsible for multidrug resistance (MDR) in cancer cells. To this end, we employed different cell lines that do or do not express P-glycoprotein, as confirmed by Western blotting: first, a tumour cell line (VBL600) selected from a human T-cell line (CEM) derived from an acute leukaemia; second, an epithelial cell line derived from a rat colonic adenocarcinoma (CC531mdr+) and finally, a non tumour epithelial cell line derived from the proximal tubule of the opossum kidney (OK). Glibenclamide and the two related derivatives inhibited P-glycoprotein because firstly, they acutely increased [3H]colchicine accumulation in P-glycoprotein-expressing cell lines only; secondly BM-223 reversed the MDR phenomenon, quite similarly to verapamil, by enhancing the cytotoxicity of colchicine, taxol and vinblastine and thirdly, BM-208 and BM-223 blocked the photoaffinity-labelling of P-glycoprotein by [3H]azidopine. Furthermore, glibenclamide is itself a substrate for P-glycoprotein, since the cellular accumulation of [3H]glibenclamide was low and substantially increased by addition of P-glycoprotein substrates (e.g., vinblastine and cyclosporine) only in the P-glycoprotein-expressing cell lines. We conclude that glibenclamide and two sulphonylcyanoguanidine derivatives inhibit P-glycoprotein and that sulphonylurea drugs would appear to be general inhibitors of ABC transporters, suggesting an interaction with some conserved motif.

K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon.

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock‐out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (Vte) (UTP: from −0.81 ± 0.23 to 3.11 ± 0.61 mV, n= 24), an increase of equivalent short circuit current (Isc) by 166.9 ± 22.8 μA cm−2 and a decrease of transepithelial resistance (Rte) from 29.4 ± 2.4 to 23.5 ± 2.0 Ω cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n= 5) and iberiotoxin (240 nm, n= 6), indicating UTP/ATP‐stimulated K+ secretion. RT‐PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP‐stimulated K+ secretion was still present in P2Y2 receptor knock‐out mice, but significantly reduced (ΔVte: 0.83 ± 0.26 mV) compared to wild‐type littermates (ΔVte: 2.08 ± 0.52 mV, n= 9). In P2Y4 receptor knock‐out mice the UTP‐induced K+ secretion was similarly reduced. Luminal UTP‐stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

The role of epithelial P2Y2 and P2Y4 receptors in the regulation of intestinal chloride secretion

UTP‐induced chloride secretion by the intestinal mucosa mounted in Ussing chambers was assessed by measurement of the short‐circuit current (Isc) in the presence of phloridzin in the case of jejunum or amiloride in the case of colon to eliminate any contribution of electrogenic Na+ movement to the net ionic transport. Since we have previously demonstrated the absence of chloride‐secretory response to apical UTP in the jejunum from P2Y4‐null mice, in the present study we studied the response to basolateral UTP in the jejunum and to either apical or basolateral UTP in the colon, in both P2Y2‐ and P2Y4‐deficient mice. In the jejunum, the chloride‐secretory response to basolateral UTP was partially reduced in both P2Y2‐ (40%) and P2Y4‐ (60%) null mice. In the colon, both apical or basolateral UTP increased the Isc. That response was abolished in a chloride‐free medium. The colonic chloride‐secretory response to either basolateral or apical UTP was abolished in P2Y4‐deficient mice, but not significantly affected in P2Y2‐deficient mice. The chloride‐secretory response to forskolin was potentiated by prior basolateral addition of UTP and this potentiation was abolished in P2Y4‐null mice. The jejunum of mice homozygous for the ΔF508 mutation of cystic fibrosis transmembrane conductance regulator was responsive to UTP, but the magnitude of that response was smaller than in the wild‐type littermates. In conclusion, the P2Y4 receptor fully mediates the chloride‐secretory response to UTP in both small and large intestines, except at the basolateral side of the jejunum, where both P2Y2 and P2Y4 receptors are involved.

Fluid transport and cystogenesis in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited nephropathy. The development and enlargement of cysts in ADPKD requires tubular cell proliferation, abnormalities in the extracellular matrix and transepithelial fluid secretion. Multiple studies have suggested that fluid secretion across ADPKD cyst-lining cells is driven by the transepithelial secretion of chloride, mediated by the apical CFTR channel and specific basolateral transporters. The whole secretory process is stimulated by increased levels of cAMP in the cells, probably reflecting modifications in the intracellular calcium homeostasis and abnormal stimulation of the vasopressin V2 receptor. This review will focus on the pathophysiology of fluid secretion in ADPKD cysts, starting with classic, morphological and physiological studies that were followed by investigations of the molecular mechanisms involved and therapeutic trials targeting these pathways in cellular and animal models and ADPKD patients. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Renal expression of parvalbumin is critical for NaCl handling and response to diuretics

The distal convoluted tubule (DCT) plays an essential role in the reabsorption of NaCl by the kidney, a process that can be inhibited by thiazide diuretics. Parvalbumin (PV), a Ca2+-binding protein that plays a role in muscle fibers and neurons, is selectively expressed in the DCT, where its role remains unknown. We therefore investigated the renal phenotype of PV knockout mice (Pvalb−/−) vs. wild-type (Pvalb+/+) littermates. PV colocalized with the thiazide-sensitive Na+-Cl− cotransporter (NCC) in the early DCT. The Pvalb−/− mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Acute furosemide administration increased diuresis and natriuresis/kaliuresis, but, surprisingly, did not increase calciuria in Pvalb−/− mice. NaCl supplementation of Pvalb−/− mice increased calciuria at baseline and after furosemide. The Pvalb−/− mice showed no significant diuretic response to hydrochlorothiazide, but an accentuated hypocalciuria. A decreased expression of NCC was detected in the early DCT of Pvalb−/− kidneys in the absence of ultrastructural and apoptotic changes. The PV-deficient mice had a positive Ca2+ balance and increased bone mineral density. Studies in mouse DCT cells showed that endogenous NCC expression is Ca2+-dependent and can be modulated by the levels of PV expression. These results suggest that PV regulates the expression of NCC by modulating intracellular Ca2+ signaling in response to ATP in DCT cells. They also provide insights into the Ca2+-sparing action of thiazides and the pathophysiology of distal tubulopathies.

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: effects of inhibitors of H+ ATPase

SummaryThe coupling between H+ transport (JH) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.JH was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2<0.5 mm Hg) and contained 10mm glucose. Under these conditions,JH was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measuredJH. The slope ofJlac onJH was 0.58±0.12 with an intercept forJlac atJH=0 of 0.55 μmol/hr. Values for δJlac/δJH were determined in groups of individual bladders whenJH was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of δJlac/δJH indicates a high degree of coupling betweenJH andJlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the δJlac/δJH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJH without change in δJlac/δJH or basalJlac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJH, but the inhibition was associated with an increase inJlac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2.

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

Contrasting effects of glycerol and urea transport on rat pancreatic beta-cell function.

Background/aims: Pancreatic β-cell function is influenced by changes in cell volume. Such volume changes depend on water permeability of the plasma membrane, conferred in part by aquaporins. Islet cells express aquaporin 7 (AQP7), which is permeable to urea and glycerol in addition to water. We therefore investigated the effects of glycerol and urea on rat pancreatic β-cell function. Methods: Electrical activity and whole-cell current were studied using the perforated patch technique. Cell volume was measured by video-imaging and insulin release by radioimmunoassay. Aquaporin 7 expression was studied by RT-PCR, Western blot and double fluorescent immunolabelling. Results: The isosmotic addition of glycerol and urea resulted in depolarization of the plasma membrane and electrical activity, accompanied by β-cell swelling, activation of the volume-regulated anion channel (VRAC) and insulin release. However, the effects of glycerol, in contrast to urea, persisted throughout exposure to the osmolyte. Glycerol also caused β-cell activation when added hyperosmotically. A non-metabolizable glycerol analogue had comparable effects to urea on β-cells. The expression of AQP7 was demonstrated in rat β-cells. Conclusion: Glycerol and urea can activate β-cells via their rapid uptake across the β-cell plasma membrane, possibly via AQP7. This results in cell swelling, VRAC activation, electrical activity and insulin release. Glycerol appears to exert an additional effect, possibly related to its intracellular metabolism.

Pendrin: the Thyrocyte Apical Membrane Iodide Transporter?

In the thyroid, the transport of iodide from the extracellular space to the follicular lumen requires two steps: the transport in the cell at the basal side and in the lumen at the apical side. The first step is mediated by the Na+/I- symporter (NIS). In most reviews and textbooks, the second step is presented as mediated by pendrin. In this review, we analyze this assumption. There are several arguments supporting the concept that indeed pendrin plays an important role in thyroid physiology. However, biochemical, clinical and histological data on the thyroid of a patient with Pendred syndrome do not suggest an essential role in iodide transport, which is corroborated by the lack of a thyroid phenotype in pendrin knockout mice. Experiments in vivo and in vitro on polarized and unpolarized cells show that iodide is transported transport of iodide at the apex of the thyroid cell. Moreover, ectopic expression of pendrin in transfected non-thyroid cells is capable of mediating iodide efflux. It is concluded that pendrin may participate in the iodide efflux into thyroid lumen but not as the unique transporter. Moreover, another role of pendrin in mediating Cl-/HCO3- exchange and controlling luminal pH is suggested.

A new role for aquaporin 7 in insulin secretion.

Bacgrouns/Aims: Several insulinotropic agents were recently reported to cause β-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7+/ + or AQP7-/- mice. Methods: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. Results: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in β-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7+/ + or AQP7-/- mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7-/- mice. Yet, exposure of β-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7+/ + and AQP7-/- mice. Both the cell swelling and change in membrane potential were only impaired in AQP7-/- cells when exposed to 50 mM glycerol. Conclusion: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.