Iodide Efflux Research Articles

A Coherent Organization of Differentiation Proteins Is Required to Maintain an Appropriate Thyroid Function in the Pendred Thyroid

Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed. Three morphological zones were identified: nearly normal follicles with iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone 2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were localized at the apical pole, OS and cell apoptosis were absent, but ClC-5 expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly present and increased in the cytosol and associated with high OS, apoptosis, cell proliferation, and increased expression of peroxiredoxin-5, catalase, and dehalogenase-1 but moderate ClC-5 expression. In conclusion, the absence of pendrin is accompanied by increased ClC-5 expression that may transiently compensate for apical iodide efflux. In more affected follicles, Duox and TPO are relocated in the cytosol, leading to abnormal intracellular thyroid hormone synthesis, which results in cell destruction presumably because intracellular OS cannot be buffered by antioxidant defenses.

Sphingosine‐1‐Phosphate acutely modulates the CFTR (Cystic Fibrosis Transmembrane Regulator) transporter in an AMPK‐dependent manner

IntroductionSphingosine‐1‐Phosphate (S1P) is a primary modulator of resistance artery tone. Its bioavailability is controlled by a rheostat between sphingosine kinase 1 and intracellular S1P phosphohydrolase 1. We have found that the transport by CFTR is the rate‐limiting step for the intracellular hydrolysis of S1P. We hypothesized that S1P limits its own degradation by an AMPK‐dependent modulation of CFTR conductance.MethodsCFTR conductance was assessed using the iodide efflux technique (conventional and real‐time) in baby hamster kidney (BHK) cells, which stably express human wild type CFTR. AMPK phosphorylation was determined using Western blotsResultsBHK cells were found to endogenously express Sk1, SPP1 and the S1P1 and S1P2 receptors (n=8). S1P (1μM, n=6) significantly reduced iodide conductance by 43% (conventional) and 75% (real‐time). The negative effect of S1P on CFTR iodide conductance was enhanced following S1P2 overexpression but reduced following pretreatment with the S1P2 blocker JTE013 (5μM, n=6). The inhibitory effect of S1P was associated with AMPK phosphorylation and blocked by the AMPK inhibitor Compound C (80μM, n=5). Accordingly, AMPK activation by AICAR mimicked the effect of S1P on iodide conductance.ConclusionsThe S1P2‐ and AMPK‐dependent modulation of CFTR conductance describes a novel pathway through which S1P could potentially regulate its own degradation.

Pseudomonas or LPS exposure alters CFTR iodide efflux in 2WT2 epithelial cells with time and dose dependence

The most common heritable genetic disease in the United States, cystic fibrosis (CF), is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride channel that interacts with and regulates a number of other proteins. The bacteria Pseudomonas aeruginosa infects 80% of patients causing decreased pulmonary function and life expectancy. It is not known how malfunction of the chloride channel allows for preferential colonization of patients by a single pathogen. The hypothesis that CFTR interacts with toll-like receptor 4 (TLR4) to phagocytize bacteria was tested. A competitive antagonist of TLR4, MKLPS, was studied for its effect in gentamicin-protection-based bacterial invasion assays. Pre-incubation (15 min 50 μg/mL) with MKLPS did not alter the rate of phagocytosis of P. aeruginosa by cultured epithelia. However, further studies with GFP-transfected P. aeruginosa revealed prominent antibiotic resistant microcolonies were formed. If CFTR is involved in phagocytosis of the bacteria, then internalization was predicted to decrease in iodide efflux. Surprisingly, cultured epithelia exposed to P. aeruginosa for 15 min showed increased cAMP-activated iodide efflux through CFTR. In addition, 15-min exposure to bacterial cell wall component, LPS, purified from P. aeruginosa also increased CFTR iodide efflux in a dose-dependent manner (50, 100 and 200 μg/mL LPS had 25%, 37% and 47% increase). In a reversal of this phenomenon, shorter 5-min exposure to 100 μg/mL LPS resulted in a 25% decrease in forskolin-activated CFTR channel activity compared to controls. This data is consistent with a model in which CFTR is removed from the plasma membrane during phagocytosis of P. aeruginosa followed by recruitment of channels to the membrane to replace those removed during phagocytosis. More studies are needed to confirm this model, but this is the first report of a bacterial product causing a biphasic time-dependent and a dose-dependent alteration of CFTR channel activity.

Resveratrol Increases Iodide Trapping in the Rat Thyroid Cell Line FRTL-5

Resveratrol, a polyphenol found in grapes, exhibits several beneficial health effects by its antioxidant, antiinflammatory, and chemopreventive properties. The aim of the present study was to determine the effect of resveratrol on iodide trapping and efflux as well as its mode of action using FRTL-5 cells, having in mind the pivotal role of the natrium iodide symporter (NIS) in the treatment of differentiated thyroid cancers. Cells were treated with resveratrol for various times and doses, in the presence or absence of thyrotropin (TSH). Iodide trapping, iodide efflux, rat NIS (rNIS) protein expression, and cyclic AMP (cAMP) production were evaluated. Resveratrol increased iodide trapping in a time-dependent (optimal 6 hours) and dose-dependent (100 microM) way in the presence of TSH. It showed an additive effect when concomitantly added with an optimal dose of TSH. Resveratrol (50 microM) increased (threefold) rNIS protein expression. In TSH-deprived cells, resveratrol also provoked an increase in rNIS protein (>3-fold in 6 hours) with an optimum at 40 microM. Resveratrol did not inhibit iodide efflux from FRTL-5 cells. It neither increased intracellular cAMP nor induced the arborization of living cells, two TSH-induced effects. A non-cAMP mode of action is highly suspected. Resveratrol increases iodide trapping in FRTL-5 cells, increasing iodide influx and rNIS protein level even in the absence of TSH. It has an additive effect with TSH. Consequently, resveratrol could be a promising molecule for radioiodide therapy in follicular and papillary differentiated thyroid carcinoma in association with recombinant human TSH.

Identification of a novel water-soluble activator of wild-type and F508del CFTR: GPact-11a

One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.

Synthesis and Evaluation of Imidazo[2,1‐b]thiazoles as Iodide Efflux Inhibitors in Thyrocytes

The Na(+)/I(-) symporter (NIS) mediates iodide uptake in the thyroid gland as well as in other NIS-expressing cells. This transport is the basis for an emerging approach to selective cancer cell destruction by using radioiodide after targeted NIS gene transfer. Therapeutic efficacy requires that radioiodide retention be maximized in tumor cells. A first generation of forty imidazo[2,1-b]thiazole derivatives as iodide efflux inhibitors is described along with the evaluation of their biological properties. Structure-activity relationship studies by using radioiodide uptake in rat thyroid-derived cells (FRTL5) revealed that the 5,6-dihydroimidazo[2,1-b]thiazole heterocycle is required for activity. Introduction of electron-donor substituents on the 3-biphenyl moiety led to the discovery of novel potent compounds. A compound was identified with enhanced potency compared to reference 1. These molecules give the possibility to increase the cellular retention of radioiodide in NIS-expressing tumors, leading to higher absorbed doses and killing efficacy.

Clinical and molecular characterization of S1118F‐CFTR

Cystic fibrosis is a lethal autosomal recessive disorder usually associated with lung disease, pancreatic insufficiency and high sweat chloride levels. A patient admitted to Le Bonheur Children's Medical Center (LBCMC, Memphis, TN) showed symptoms of meconium ileus which required exploratory laparotomy, bowel resection and ileostomy. Genotyping showed DeltaF508/I1027T on one chromosome and S1118F on the other. Sweat testing on three different occasions gave negative and intermediate results (22.7, 24.6 mmol/L; 55.1, 58.6 mmol/L and 55.1, 58 mmol/L) and pancreatic elastase testing showed normal levels. To characterize S1118F-CFTR mutation at a molecular level to help understand the associated CF-phenotype. Molecular characterization of S1118F-CFTR mutant was studied in HEK-293 cells at 37 degrees C. Various biochemical methods such as Western blotting, real-time PCR, Pulse chase labeling and iodide efflux assay were employed. S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DeltaF508-CFTR exhibits atypical CF symptoms with intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency.

Rescue of Functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by Vasoactive Intestinal Peptide in the Human Nasal Epithelial Cell Line JME/CF15

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

Iodine assimilation by marine diatoms and other phytoplankton in nitrate‐replete conditions

Several marine phytoplankton species, primarily diatoms, were examined for the accumulation of iodide (10 species) and iodate (9 species) using radioactive iodine‐125 in f/2 artificial seawater, a nitrate‐enriched medium. Iodide accumulation (net uptake) rates were variable, and diatoms exhibited the highest rates. Emiliania huxleyi and Synechococcus sp. did not accumulate iodide. Accumulation rates ranged from 0 to 1.7 fmol cell−1 d−1. The diatom Porosira glacialis accumulated the greatest amount of iodide and was used to determine efflux rates of iodide. Iodide efflux was characterized by two distinct phases of iodide release: an initial rapid release rate of 10 amol cell−1 min−1 from the free space and a subsequent cellular release rate of 0.13 amol cell−1 min−1, which corresponds to a daily cellular release rate of 0.19 fmol cell−1 d−1. Accumulation of iodate ranged from 0 to 19 amol cell−1 d−1, with P. glacialis displaying the highest rate. Emiliania, Synechococcus, and Chaetoceros did not show significant iodate accumulation. Iodide is the preferred chemical species of iodine for uptake under nitrate‐replete conditions: iodide accumulation rates ranged from 3 to 90 times larger than those for iodate for the species studied. If the iodate accumulated is tightly coupled to its reduction to iodide, the accumulation rates suggest that phytoplankton‐mediated iodate reduction is not environmentally significant. If diatoms can reduce iodate to iodide, their overall contribution to surface‐water iodide, while living, would be further reduced because of their ability to reassimilate released iodide.

Bestrophin expression and function in the human pancreatic duct cell line, CFPAC‐1

Pancreatic duct epithelial cells (PDECs) have been shown to express calcium activated chloride channels (CaCCs) and there is evidence for their involvement in fluid secretion from these cells. The molecular identity of the CaCC in PDECs remains unknown. Recently, the bestrophin family of proteins have been proposed as a potential molecular candidate for CaCCs. Expression of bestrophins is strongly correlated with the function of CaCCs in a variety of tissues. In the present study, the expression of bestrophins has been investigated in the cystic fibrosis pancreatic duct cell line, CFPAC-1. Iodide efflux analysis was used to characterise native CaCCs in CFPAC-1 cell monolayers. Efflux was induced with the addition of UTP (100 microM, 10.2 +/- 1.5 nmol min(-1)), which was blocked by the chloride channel blockers niflumic acid (81%) and DIDS (90%). The UTP-stimulated iodide efflux was shown to be Ca(2+) dependent and cAMP independent. RT-PCR analysis of RNA isolated from CFPAC-1 cells demonstrated positive identification of all four human bestrophin mRNAs. Western blot of CFPAC-1 cell protein isolates with antibodies specific to human bestrophin 1 (hBest1) showed that hBest1 protein was expressed in this cell line. HBest1 was present on the cell surface, demonstrated using biotinylation and confocal imaging, as well as in the cytoplasm. SiRNA-mediated silencing of hBest1 in CFPAC-1 cells reduced the UTP-stimulated iodide efflux by around 40%. This study provides evidence that the bestrophins are expressed in pancreatic duct cells and, more specifically, that hBest1 plays a role in the CaCCs found in these cells.

A Soluble Sulfogalactosyl Ceramide Mimic Promotes ΔF508 CFTR Escape from Endoplasmic Reticulum Associated Degradation

AdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki approximately 10 microM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded Delta F508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased Delta F508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced Delta F508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with Delta F508CFTR/mock-transfected cells. Partial rescue of Delta F508CFTR by low-temperature glycerol increased SGC synthesis. AdaSGC also increased cellular endogenous SGC levels. SGC in the lung, liver, and kidney was severely depleted in Delta F508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis.

Chloride transport induced by the substitution of Asp555 with a Glu in the Na/HCO3 cotransporter SLC4A4 (NBCe1)

By structure/function analysis, we previously demonstrated that the substitution of Asp555 in NBCe1 with a Glu (D555E) causes the mutant transporter to produce currents by other anions. In this study, we further characterized the chloride transport of D555E by using two‐electrode voltage clamp and fluorescence measurements. In Xenopus oocytes expressing D555E, the application of a series of Cl concentrations without CO2/HCO3 produced outward currents in voltage‐clamp recordings. A similar application did not evoke outward currents in oocytes expressing wild type human NBCe1. The chloride currents induced by D555E were markedly diminished in CO2/HCO3 solution, consistent with the previous finding that D555E produces Cl currents in the absence of HCO3. To address the issue of whether the chloride conductance is not derived from endogenous oocyte proteins but is intrinsic to the mutant transporter, we expressed NBCe1 and D555E in HEK293 cells and measured chloride transport by using the fluorescence dye 6‐methoxy‐N‐(‐sulphopropyl) quinolinium SPQ. The expression of the transporters in cells was confirmed by immunoblot and immunocytochemistry with the antibody specific to the C‐terminal end of the protein. SPQ‐loaded cells were washed with a solution containing nitrate, and intracellular SPQ was then quenched by perfusion with iodide. After achieving basal fluorescence, iodide was replaced with nitrate to determine the iodide efflux. The fluorescence in D555E‐transfected cells significantly increased compared to that in NBCe1‐transfected cells. These data indicate that the chloride conductance associated with D555E is most likely to be intrinsic to the mutant transporter.

A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain

Aims Thyroid hormones (TH) play an important role in the development and functional maintenance of the central nervous system. The purpose of this study was to develop a radiotracer method for studying the in vivo efflux transport of iodide liberated by the TH metabolism in the brain. The rationale of our method is as follows: a radioiodinated compound can enter the brain and rapidly release iodide in situ; the iodide efflux rate can be estimated from the clearance of brain radioactivity after disappearance of the iodinated compound. Main methods 6-[ 125I]Iodo-9-pentylpurine ([ 125I]9Pe6IP) was designed to enter the brain and release 125I − by the reaction with glutathione and synthesized from the corresponding bromo derivative in a Br/ 125I exchange reaction. The brain kinetics of radioactivity and radioactive metabolites were investigated after intravenous injection of [ 125I]9Pe6IP into mice. The iodide efflux rate was estimated in mice pretreated with perchlorate, an inhibitor of iodide transport from the brain. Key findings High brain uptake (5.3% injected dose/g) was observed at 1 min, and almost complete conversion of [ 125I]9Pe6IP to 125I − occurred 10 min after injection. The 125I − uptake from the blood was negligible. 125I − was eliminated from the brain along a single-exponential curve with a half-life of 6.0 min. Furthermore, dose-dependent inhibition of 125I − efflux was observed in mice pretreated with perchlorate. Significance We conclude that 9Pe6IP labeled with 124I (positron emitter) or 123I (single-photon emitter) may be useful for studying the in vivo efflux transport of iodide in the brain using nuclear medicine imaging devices.

Release and transformations of inorganic iodine by marine macroalgae

A number of field and laboratory studies on the impact of marine macroalgae on dissolved inorganic iodine speciation are presented. Within tidally isolated rock pools, the brown macroalga Fucus serratus was found to both release stored iodide and to facilitate the reduction of iodate to iodide. In contrast, no discernible changes in iodine speciation were observed in rock pools containing green macroalgae of the genus Ulva. Incubation experiments confirmed that the macroalgae Laminaria digitata, F. serratus and Kallymenia antarctica release iodide, though the rate of release varied between species and between specimens of the same species. Application of oxidative stress by treatment with cell wall derived oligoguluronate elicitors increased the efflux of iodide by L. digitata approximately 20-fold. The release of iodide by macroalgae may impact upon the formation of volatile iodine species (molecular iodine and iodocarbons) that are of importance in the coastal atmosphere.

Angiotensin Ii Activates Calcium-Dependent Cl- Channels In Human Cardiac Fibroblasts

This study reports for the first time the presence of chloride channels on the plasma membrane of human cardiac fibroblasts in culture, by means of the iodide efflux and the patch clamp methods. The angiotensin II and the calcium ionophore A23187 activate a chloride conductance that shares pharmacological similarities with calcium-dependent chloride channels already described in other cell types. Using the iodide efflux technique it was shown that Ag II could induce an anionic efflux after binding to AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). Blockade of chloride efflux by calphostin C and KN 62 indicates that this activation is dependent on PKC and/or CaMKII. This calcium-dependent chloride current which is characterized in human cardiac fibroblasts is potentially involved in the secretion by cardiac fibroblasts of growth factors; collagen and pro-inflammatory mediators released in particular pathological conditions.

Synthesis and evaluation of photoreactive probes to elucidate iodide efflux in thyrocytes

Four photoreactive analogues of 3-biphenyl-4′-yl-5,6-dihydroimidazo[2,1- b]thiazole were prepared and evaluated as iodide sequestering agents in sodium iodide symporter-expressing cells. One of these new photoactivatable compounds retained biological activity and was further radiolabeled with tritium. This compound may provide a useful tool for labeling, purification, and identification of target protein responsible for iodide efflux in thyrocytes.

Pendrin Is a Novel Autoantigen Recognized by Patients with Autoimmune Thyroid Diseases

Pendrin is an apical protein of thyroid follicular cells, responsible for the efflux of iodide into the follicular lumen via an iodide-chloride transport mechanism. It is unknown whether pendrin is recognized by autoantibodies. Our objective was to examine the prevalence of pendrin antibodies in autoimmune thyroid diseases and compare with that of thyroglobulin, thyroperoxidase, TSH receptor, and sodium iodide symporter antibodies. In a prevalent case-control study, we analyzed the sera of 140 autoimmune thyroid disease cases (100 with Graves' disease and 40 with Hashimoto's thyroiditis) and 80 controls (50 healthy subjects, 10 patients with papillary thyroid cancer, 10 with systemic lupus erythematosus, and 10 with rheumatoid arthritis). Pendrin antibodies were measured by immunoblotting using extract of COS-7 cells transfected with pendrin and a rabbit polyclonal pendrin antibody. Pendrin antibodies were found in 81% of the cases and 9% of controls (odds ratio = 44; P < 0.0001). Among cases, pendrin antibodies were more frequent and of higher titers in Hashimoto's thyroiditis than in Graves' disease. Pendrin antibodies correlated significantly with thyroglobulin, thyroperoxidase, and sodium iodide symporter antibodies but not with TSH receptor antibodies. Pendrin antibodies were equally effective as thyroglobulin and thyroperoxidase antibodies in diagnosis of autoimmune thyroid diseases, especially Hashimoto's thyroiditis. The study identifies pendrin as a novel autoantigen recognized by patients with autoimmune thyroid diseases and proposes the use of pendrin antibodies as an accurate diagnostic tool.

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.

Combination Therapy and Noninvasive Imaging with a Dual Therapeutic Vector Expressing MDR1 Short Hairpin RNA and a Sodium Iodide Symporter

We investigated the feasibility of using combination gene therapy and noninvasive nuclear imaging after expression of the human sodium iodide symporter (hNIS) and inhibition of the multidrug resistance (MDR1) gene in colon cancer cells. HCT-15 cells were stably transfected with a dual expression vector, in which the hNIS gene, driven by a constitutive cytomegalovirus promoter, has been coupled to an MDR1 short hairpin RNA (shRNA) cassette. Cell lines stably expressing the hNIS gene and MDR1 shRNA (designated MN-61 and MN-62) were produced, and the expression of the NIS gene and MDR1 shRNA was examined by Western blotting, reverse transcription-polymerase chain reaction, and immunostaining. The functional activities of MDR1 shRNA were determined by paclitaxel uptake and sensitivity to doxorubicin. Functional NIS expression was confirmed by the uptake and efflux of (125)I and the cytotoxicity of (131)I. The effect of the combination of (131)I and doxorubicin was determined by an in vitro clonogenic assay. In vivo NIS expression was examined by small-animal PET with (124)I. The shMDR-NIS-expressing cells showed a significant decrease in the expression of MDR1 messenger RNA and its translated product, P-glycoprotein. The inhibition of P-glycoprotein expression by shRNA enhanced the intracellular accumulation of paclitaxel, the cellular retention of which is mediated by P-glycoprotein, thereby increasing sensitivity to the anticancer drug. The shMDR-NIS-expressing cells showed a significant increase of (125)I uptake, which was completely inhibited by KClO(4). Although the iodide efflux rate was rapid, the cell survival rate was markedly reduced by (131)I treatment. Interestingly, the combination of doxorubicin and a radioiodide ((131)I) displayed synergistic cytotoxicity that correlated with MDR1 inhibition and NIS expression in shMDR-NIS-expressing cells. Furthermore, in mice with shMDR-NIS-expressing tumor xenografts, small-animal PET with (124)I clearly visualized shMDR1-NIS-expressing tumors. We developed a dual expression vector with the NIS gene and MDR1 shRNA. This study represents a promising first step in investigations of the potential use of a combination of the NIS gene and MDR1 shRNA as a new therapeutic strategy allowing RNA interference-based gene therapy, NIS-based radioiodine therapy, and in vivo monitoring based on NIS imaging.

Effect of Sunitinib on Growth and Function of FRTL-5 Thyroid Cells

Sunitinib, a multitargeted vascular endothelial growth factor and receptor tyrosine kinase inhibitor, causes hypothyroidism in patients who take it for treatment of cancer. Although the pathophysiologic mechanism of the hypothyroidism is unclear, it has been claimed that it is due to inhibition of iodide uptake. To evaluate the pathologic mechanism of induction of the hypothyroidism, we studied the effect of sunitinib on FRTL-5 rat thyroid cells. We measured the effect of sunitinib on cell growth, (125)I-iodide uptake and efflux, TSH receptor (TSH-R), and sodium-iodide symporter (NIS) message. At 48 hours, sunitinib caused a dose-related inhibition of growth with LC(50) of 14.6 muM, but there was no apparent inhibition of growth at 24 hours at concentrations of 0.1-25 microM. Preincubation with sunitinib did not impair the response to TSH, indicating that it did not affect the TSH-R. Incubation with sunitinib for 24 hours caused a dose-related increase of (125)I-iodide uptake and did not reduce iodide efflux or NIS mRNA expression. The data indicate that sunitinib is unlikely to cause hypothyroidism by inhibition of iodide uptake.