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.

Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in ß-cells?

NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat ß-cells. Exogenous H(2)O(2) (100 to 200 µM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native ß-cells to extracellular hypotonicity.

Glycerol metabolism alteration in adipocytes from n3-PUFA-depleted rats, an animal model for metabolic syndrome.

Aquaglyceroporin 7 (AQP7) is a glycerol transporter expressed in adipocytes. Its expression has been shown to be modulated in obesity. Metabolic syndrome is characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension. An animal model displaying several features of metabolic syndrome was used to study the AQP7 expression at both mRNA and protein level and glycerol flux in adipocytes. Second generation n3-PUFA depleted female rats is a good animal model for metabolic syndrome as it displays characteristic features such as liver steatosis, visceral obesity, and insulin resistance. Our data show a reduced expression of AQP7 at the protein level in adipose tissue from n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-(14)C]-Glycerol uptake was not modified in adipocytes from n3-PUFA-depleted animals.

Functional role of aquaglyceroporin 7 expression in the pancreatic beta-cell line BRIN-BD11.

Both mouse and rat pancreatic islet beta-cells were recently found to express aquaglyceroporin 7 (AQP7). In the present study, the expression and role of AQP7 in the function of BRIN-BD11 cells were investigated. AQP7 mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. In an isoosmolar medium, the net uptake of [2-(3)H]glycerol displayed an exponential time course reaching an equilibrium plateau value close to its extracellular concentration. Within 2 min of incubation in a hypotonic medium (caused by a 50 mM decrease in NaCl concentration), the [2-(3)H]glycerol uptake averaged 143.2 +/- 3.8% (n = 24; P < 0.001) of its control value in isotonic medium, declining thereafter consistently with previously demonstrated volume regulatory decrease. When isoosmolarity was restored by the addition of 100 mM urea to the hypotonic medium, [2-(3)H]glycerol uptake remained higher (112.1 +/- 2.8%, n = 24; P < 0.001) than its matched control under isotonic conditions, indicating rapid entry of urea and water. Insulin release by BRIN-BD11 cells was 3 times higher in hypotonic than in isotonic medium. When glycerol (100 mM) or urea (100 mM) were incorporated in the hypotonic medium, the insulin release remained significantly higher than that found in the control isotonic medium, averaging respectively 120.2 +/- 4.2 and 107.0 +/- 3.8% of the paired value recorded in the hypotonic medium. These findings document the rapid entry of glycerol and urea in BRIN-BD11 cells, likely mediated by AQP7.

Evidence for concerted effects of aldosterone on a target sodium-transporting epithelium.

The sodium-transporting activity of toad skin is stimulated in vitro with aldosterone in the absence of energy-providing substrate; it can be stimulated further upon addition of glucose after prolonged (overnight) incubation. The magnifying effect exerted by glucose in these conditions could be blocked by inhibitors of ribonucleic acid and protein biosynthesis. In addition, exposure to cycloheximide prevented the increase in thermodynamic affinity resulting from aldosterone treatment. A synthetic 19-nor steroid, (RU 24411), dimethyl-2,2-hydroxy-21-nor-19-pregnene-4-dione-3,20, also stimulated sodium transport by toad skin incubated in the absence of glucose, but there was no magnifying effect of this substrate. Furthermore, there was no change in thermodynamic affinity with RU 24411. Therefore, the magnifying effect seen with glucose and the increase in thermodynamic affinity are not necessarily integral parts of the response of sodium-transporting epithelial to "mineralocorticoids."

Expression and intracellular processing of chimeric and mutant CFTR molecules.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the DeltaF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR DeltaF508 and to increase activity of both wild-type and DeltaF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain).

Active H+ transport in the turtle urinary bladder. Coupling of transport to glucose oxidation.

The turtle urinary bladder acidifies the contents of its lumen by actively transporting protons. H+ secretion by the isolated bladder was measured simultaneously with the rate of 14CO2 evolution from [14C]glucose. The application of an adverse pH gradient resulted in a decline in the rate of H+ secretion (JH) and in the rate of glucose oxidation (JCO2). The changes in JH and JCO2 were linear functions of the pH difference across the membrane. Hence, JH and JCO2 were linearly related to each other. The slope, deltaJH/deltaJCO2 was found to be similar in half-bladders from the same animal but was seen to vary widely in a population of turtles. To investigate the effect of pH gradients on deltaJH/deltaJCO2, two experiments were performed in each of 14 hemibladders. In one, JH and JCO2 were altered by changing the luminal pH. In the other, they were altered by changing the ambient pCO2 while the luminal pH was kept constant. The average slope, deltaJH/deltaJCO2, in the presence of pH gradients was 14.45 eq-mol-1. In the absence of gradients in the same hemibladders it was 14.72, delta = 0.27 +/- 1.46. The results show that H+ transport is organized in such a way that leaks to protons in parallel to the pump are negligible. Analysis of the transport system by use of the Essig-Caplan linear irreversible thermodynamic formalism shows that the system is tightly coupled. The degree of coupling, q, given by that analysis was measured and found to be at or very near the maximum theoretical value.

[Insulin and arterial hypertension: the role of the kidney]. / Insuline et hypertension artérielle: le rôle du rein.

We have demonstrated that insulin stimulates sodium reabsorption in the distal nephron by stimulating the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that any stimulation of this enzyme (e.g. by EGF, by H2O2 or by exogenous PIP3, added apically) leads to a parallel increase in sodium reabsorption. We therefore suggest that hyperinsulinemia leads to hypertension through increased renal sodium reabsorption in the distal nephron.

Anion selectivity by the sodium iodide symporter.

The iodide transporter of the thyroid (NIS) has been cloned by the group of Carrasco. The NIS-mediated transport was studied by electrophysiological methods in NIS-expressing Xenopus oocytes. Using this method, the anion selectivity of NIS was different from that previously reported for thyroid cells, whereas perchlorate and perrhenate were found not transported. In this study we compared the properties of human NIS, stably transfected in COS-7 cells to those of the transport in a thyroid cell line, the FRTL5 cells, by measuring the transport directly. We measured the uptake of (125)I(-), (186)ReO(4)(-), and (99m)TcO(4)(-) and studied the effect on it of known competing anions, i.e. ClO(4)(-), SCN(-), ClO(3)(-), ReO(4)(-), and Br(-). We conclude that the properties of the NIS transporter account by themselves for the properties of the thyroid iodide transporter as described previously in thyroid slices. The order of affinity was: ClO(4)(-) > ReO(4)(-) > I(-) >/= SCN(-) > ClO(3)(-) > Br(-). NIS is also inhibited by dysidenin (as in dog thyroid).

Stimulation of sodium transport by toad skin incubated with natural derivatives of corticosterone and deoxycorticosterone.

The effects on sodium transport of several steroids physiologically secreted or possibly involved in pathological disorders were compared with those of aldosterone in the isolated toad skin. The 18-hydroxylated derivatives of deoxycorticosterone and corticosterone, in contrast to their parent compounds, significantly enhanced sodium transport at a concentration of 50 nmol/l. In the presence of glucose, 18-hydroxydeoxycorticosterone increased transepithelial potential difference, as did aldosterone. The 19-nor derivative of deoxycorticosterone, recently implicated in the aetiology of adrenal regeneration hypertension, stimulated sodium transport, unlike 19-nor-corticosterone and 16-oxo-androstenediol. Insulin significantly increased sodium transport in aldosterone-treated skin and lowered the resistance. The natriferic response to vasopressin was potentiated fivefold by exposure of the skin to aldosterone and was doubled in skin exposed to 19-nor-deoxycorticosterone. We conclude that 18-hydroxylated adrenocortical steroids can play a physiological role in salt retention; furthermore, these steroids, as well as 19-nor-deoxycorticosterone, could be involved in pathological conditions such as low renin hypertension. Caution should be exercised in evaluating mineralocorticoid potency solely in terms of the urinary sodium to potassium ratio.

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) increases iodide trapping, inhibits thyroperoxidase and antagonizes the TSH-induced apical iodide efflux in porcine thyroid cells.

4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC50 of 5 x 10(-5) M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC50 being also of 5 x 10(-5) M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10(-3) M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10(-3) M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter.

NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2 (.-) and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

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 (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega 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 (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 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 significance of changes in thermodynamic affinity induced by aldosterone in sodium-transporting epithelia.

The energetics of sodium transport were examined in toad (and occasionally frog) skin, with particular emphasis on the effect of aldosterone. Thermodynamic affinity was computed according to Essig and Caplan. Following treatment with antidiuretic hormone or drugs believed to affect only the apical membrane barrier, no change in thermodynamic affinity was observed either acutely (after one to two hours) or chronically (after 18-odd hours hours). By contrast, following treatment with aldosterone overnight, thermodynamic affinity was considerably increased, whether or not incubation was conducted in the presence of sodium in the outer solution; addition of glucose at the end of incubation, whereby sodium transport was stimulated further, failed to influence affinity as measured. The stoichiometry between sodium transport and oxygen consumption was, however, unchanged by aldosterone treatment in short-circuit conditions, neither was that fraction of aerobic metabolism unrelated to sodium transport influenced. It is concluded that the change observed with aldosterone can be directly ascribed to the hormone, as it is independent of glucose availability and of sodium transport. Aldosterone action, at least following prolonged incubation, therefore does not involve only an increase in apical conductance for sodium.

Effects of vanadate on the functional properties of the isolated toad bladder.

1. Vanadate, considered by some as a candidate for physiological modulation of Na pumping activity, was studied in the isolated toad urinary bladder. It produced a decrease in Na transport activity that was reversible and could be partially antagonized by pretreatment with a disulphonic distilbene derivative, SITS. This suggests an intracytoplasmic site of action for vanadate. Other transition metal salts, prepared from Ta and Nb, produced instead a transient rise in Na transport and this was unaffected by SITS. 2. Comparison between ouabain and vanadate showed clear-cut differences: inhibition of Na transport by the former, not the latter, was partially overcome by increasing of Na transport by the former, not the latter, was partially overcome by increasing cell Na or serosal K. Unexpectedly intracellular K did not decrease appreciably following vanadate treatment, in contrast to what occurs when ouabain was used instead. 3. Vasopressin-induced hydro-osmotic flow was irreversibly inhibited by vanadate but not by ouabain; pretreatment with SITS attenuated this effect. Moreover, vanadate blocked urinary acidification in this epithelium, thus supporting the hypothesis that proton flow occurs through an enzyme distinct from the mitochondrial ATPase, insensitive to vanadate.

Opposite effects of indacrinone (MK-196) on sodium and chloride conductance of amphibian skin.

Indacrinone, a drug chemically related to ethacrynic acid, usually stimulated reversibly short circuit current and sodium influx when applied to the epithelial surface of amphibian skin. Concomitantly, transepithelial conductance, gt, decreased, provided chloride was the main anion in the incubation fluid. Electrophysiological analysis including microelectrode impalement indicated that the drug increased the sodium-conductance at the apical border of the impaled (most likely granular) cells. The decrease in gt thus points at shunt conductance being reduced with indacrinone, sometimes drastically. Decrease of transepithelial chloride flux with the drug as well as lack of effect of the drug on gt in the absence of chloride on the epithelial side demonstrate the influence of indacrinone on a chloride specific pathway. Whether this is along a paracellular route or through a cellular compartment not coupled to granular cells (mitochondria-rich cells?) cannot be decided on the basis of the present data.

Evidence for a transcellular component to the transepithelial sodium efflux in toad skin.

The transepithelial efflux of sodium, from the inner to the outer surface was measured across the isolated toad skin, mostly after abolition of the electrochemical gradient. The effects on this efflux of several agents and manipulations were studied in order to make a distinction between the paracellular component and a hypothetical transcellular one. Amiloride decreased the transepithelial efflux, while ouabain and cyanide increased it. From the known mode of action of those agents, it was inferred that part of the efflux occurred across the cell. Removal of sodium from the external solution interfered apparently with both components of the transepithelial efflux, while the action of external hypertonicity seemed to be restricted to the paracellular shunt pathway. Access of sodium from the internal solution to the active transport pool is thus suggested, with consequent increase in metabolic cost of transport. Yet, compared with the net influx, the amounts involved are very small; consequently, they escape detection by oxygen consumption measurements.