Tamoxifen-induced, intestinal-specific deletion of Slc5a6 in adult mice leads to spontaneous inflammation: involvement of NF-κB, NLRP3, and gut microbiota.

The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-ß2.

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor ß2 (TGF-ß2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-ß2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-ß2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-ß2-mediated KCC2 trafficking and functional activation. TGF-ß2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-ß2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-ß2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.

Functional morphology of pituitary -thyroid and -adrenocortical axes in middle-aged male rats treated with Vitex agnus castus essential oil.

We previously reported that Vitex agnus-castus L. essential oil (VACEO), when administered to middle-aged males, exerts a bone-protective effect, induces silencing of locomotor activities and decreases pituitary prolactin immunopositivity. To further assess the putative endocrine effects of VACEO, we examined the pituitary-thyroid and -adrenocortical axes in our model. Sixteen-month-old Wistar rats were subcutaneously administered 60mg/kg of VACEO dissolved in sterile olive oil, while the control group received the same amount of vehicle alone for three weeks. Pituitaries, thyroids and adrenals were analyzed by qualitative and quantitative histological approaches. Concentration of thyroid stimulating hormone (TSH), total thyroxine and triiodothyronine (TH), adrenocorticotrophic hormone (ACTH), corticosterone in serum and in adrenal tissue were measured. In VACEO-treated rats, the relative volume density of pituitary thyrotrophs increased (p<0.001), while intensity of cytoplasmic TSHß immunostaining decreased (p<0.001), consistent with elevated TSH in serum (p<0.01). The thyroid tissue was characterized by a micro-follicular structure, increased relative volume of follicular epithelium (p<0.05), decreased volume of luminal colloid (p<0.001) and increased basolateral expression of sodium-iodide symporter-immunopositivity (p<0.05). Serum TH also increased (p<0.01). The relative volume density of pituitary corticotrophs decreased (p<0.05), compatible with decline in circulating ACTH (p<0.05). Neither tissue nor serum corticosterone levels were affected by VACEO treatment. In conclusion, the observed changes in TSH and ACTH strongly indicate central endocrine effects of prolonged VACEO treatment. In this respect, production of ACTH decreased without impact on corticosterone production. Increase in serum concentration of both TH and TSH are not compatible with a negative feedback loop and suggest a major change in set-point regulation of the hypothalamic-pituitary-thyroid axis.

Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake.

Functional complexes of the proton pump and chloride transporter in gastric acid secretion.

Proton(H+) of gastric acid(HCl) is actively secreted by gastric proton pump (H+, K+- ATPase) in the parietal cells. The proton pump is expressed in both tubulovesicles and apical membrane of the cells. In resting parietal cells, tubulovesicles are present in intracellular compartments underlying the apical membrane and forming a reticulated meshwork. Upon stimulation, tubulovesicles fuse each other and connect with the apical membrane, resulting in massive acid secretion. On the other hand, the mechanism of apical Cl- transport for HCl secretion is not fully understood, although several Cl- transporters and Cl- channels have been reported to be the candidate. Here, we summarized the function of Cl- transporters such as KCC4, a K+-Cl- cotransporter, and ClC-5, a Cl-/H+ exchanger, in gastric acid secretion.

S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na(+)/I(-) Symporter Expression.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

Enhanced antitumour drug delivery to cholangiocarcinoma through the apical sodium-dependent bile acid transporter (ASBT).

Novel antitumour drugs, such as cationic tyrosine kinase inhibitors, are useful in many types of cancer but not in others, such as cholangiocarcinoma (CCA), where their uptake through specific membrane transporters, such as OCT1, is very poor. Here we have investigated the usefulness of targeting cytostatic bile acid derivatives to enhance the delivery of chemotherapy to tumours expressing the bile acid transporter ASBT and whether this is the case for CCA. The analysis of paired samples of CCA and adjacent non-tumour tissue collected from human (n=15) and rat (n=29) CCA revealed that ASBT expression was preserved. Moreover, ASBT was expressed, although at different levels, in human and rat CCA cell lines. Both cells in vitro and rat tumours in vivo were able to carry out efficient uptake of bile acid derivatives. Using Bamet-UD2 (cisplatin-ursodeoxycholate conjugate) as a model ASBT-targeted drug, in vitro and in vivo antiproliferative activity was evaluated. ASBT expression enhanced the sensitivity to Bamet-UD2, but not to cisplatin, in vitro. In nude mice, Bamet-UD2 (more than cisplatin) inhibited the growth of human colon adenocarcinoma tumours with induced stable expression of ASBT. As compared with cisplatin, administration of Bamet-UD2 to rats with CCA resulted in an efficient liver and tumour uptake but low exposure of extrahepatic tissues to the drug. Consequently, signs of liver/renal toxicity were absent in animals treated with Bamet-UD2. In conclusion, endogenous or induced ASBT expression may be useful in pharmacological strategies to treat enterohepatic tumours based on the use of cytostatic bile acid derivatives.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Design and synthesis of a novel candidate compound NTI-007 targeting sodium taurocholate cotransporting polypeptide [NTCP]-APOA1-HBx-Beclin1-mediated autophagic pathway in HBV therapy.

Sodium taurocholate cotransporting polypeptide (NTCP) is a multiple transmembrane transporter predominantly expressed in the liver, functioning as a functional receptor for HBV. Through our continuous efforts to identify NTCP as a novel HBV target, we designed and synthesized a series of new compounds based on the structure of our previous compound NT-5. Molecular docking and MD simulation validated that a new compound named NTI-007 can tightly bind to NTCP, whose efficacy was also measured in vitro virological examination and cytotoxicity studies. Furthermore, autophagy was observed in NTI-007 incubated HepG2.2.15 cells, and results of q-PCR and Western blotting revealed that NTI-007 induced autophagy through NTCP-APOA1-HBx-Beclin1-mediated pathway. Taken together, considering crucial role of NTCP in HBV infection, NTCP-mediated autophagic pathway may provide a promising strategy of HBV therapy and given efficacy of NTI-007 triggering autophagy. Our study suggests pre-clinical potential of this compound as a novel anti-HBV drug candidate.

Advanced radioiodine-refractory differentiated thyroid cancer: the sodium iodide symporter and other emerging therapeutic targets.

Approximately 30% of patients with advanced, metastatic differentiated thyroid cancer have radioiodine-refractory disease, based on decreased expression of the sodium iodide symporter SLC5A5 (NIS), diminished membrane targeting of NIS, or both. Patients with radioiodine-refractory disease, therefore, are not amenable to (131)I therapy, which is the initial systemic treatment of choice for non-refractory metastatic thyroid cancer. Patients with radioiodine-refractory cancer have historically had poor outcomes, partly because these cancers often respond poorly to cytotoxic chemotherapy. In the past decade, however, considerable progress has been made in delineating the molecular pathogenesis of radioiodine-refractory thyroid cancer. As a result of the identification of key genetic and epigenetic alterations and dysregulated signalling pathways, multiple biologically targeted drugs, in particular tyrosine-kinase inhibitors, have been evaluated in clinical trials with promising results and have begun to meaningfully impact clinical practice. In this Review, we summarise the current knowledge of the molecular pathogenesis of advanced differentiated thyroid cancer and discuss findings from clinical trials of targeted drugs in patients with radioiodine-refractory disease. Additionally, we focus on the molecular basis of loss of NIS expression, function, or both in refractory disease, and discuss preclinical and clinical data on restoration of radioiodine uptake.

Macitentan does not interfere with hepatic bile salt transport.

Treatment of pulmonary arterial hypertension with the endothelin receptor antagonist bosentan has been associated with transient increases in liver transaminases. Mechanistically, bosentan inhibits the bile salt export pump (BSEP) leading to an intrahepatic accumulation of cytotoxic bile salts, which eventually results in hepatocellular damage. BSEP inhibition by bosentan is amplified by its accumulation in the liver as bosentan is a substrate of organic anion-transporting polypeptide (OATP) transport proteins. The novel endothelin receptor antagonist macitentan shows a superior liver safety profile. Introduction of the less acidic sulfamide moiety and increased lipophilicity yield a hepatic disposition profile different from other endothelin receptor antagonists. Passive diffusion rather than OATP-mediated uptake is the driving force for macitentan uptake into the liver. Interaction with the sodium taurocholate cotransporting polypeptide and BSEP transport proteins involved in hepatic bile salt homeostasis is therefore limited due to the low intrahepatic drug concentrations. Evidence for this conclusion is provided by in vitro experiments in drug transporter-expressing cell lines, acute and long-term studies in rats and dogs, absence of plasma bile salt changes in healthy human volunteers after multiple dosing, and finally the liver safety profile of macitentan in the completed phase III morbidity/mortality SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary Arterial Hypertension to Improve Clinical Outcome) trial.

Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice.

Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin+imipenem and cephalothin+neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin+imipenem but stimulated by cephalothin+neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism.

Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.

BACKGROUND: Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role in energy metabolism, a direct effect of AMPK modulation on thyroid function has only recently been reported, and much of its function in the thyroid is currently unknown. The aim of this study was to investigate the mechanism of AMPK modulation in iodide uptake. Furthermore, we wanted to investigate the potential of the AMPK inhibitor compound C as an enhancer of iodide uptake by thyrocytes. METHODS: The in vitro and in vivo effects of AMPK modulation on sodium-iodide symporter (NIS) protein levels and iodide uptake were examined in follicular rat thyroid cell-line cells and C57Bl6/J mice. Activation of AMPK by metformin resulted in a strong reduction of iodide uptake (up to sixfold with 5 mM metformin after 96 h) and NIS protein levels in vitro, whereas AMPK inhibition by compound C not only stimulated iodide uptake but also enhanced NIS protein levels both in vitro (up to sevenfold with 1 µM compound C after 96 h) and in vivo (1.5-fold after daily injections with 20 mg/kg for 4 days). We investigated the regulation of NIS expression by AMPK using a range of promoter constructs consisting of either the NIS promoter or isolated CRE (cAMP response element) and NF-κB elements, which are present within the NIS promoter. RESULTS: Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C stimulated its activity (3.4-fold) after 4 days. This largely coincides with CRE activation (0.6- and 3.0-fold). These experiments show that AMPK exerts its effects on iodide uptake, at least partly, through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1 knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds. These mice have a less active thyroid, as shown by reduced colloid volume and reduced responsiveness to thyrotropin. CONCLUSION: NIS expression and iodine uptake in thyrocytes can be modulated by metformin and compound C. These compounds exert their effect by modulation of AMPK, which, in turn, regulates the activation of the CRE element in the NIS promoter. Overall, this suggests that the use of AMPK modulating compounds may be useful for the enhancement of iodide uptake by thyrocytes, which could be useful for the treatment of thyroid cancer patients with radioactive iodine.

Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer.

BACKGROUND: Metastatic thyroid cancers that are refractory to radioiodine (iodine-131) are associated with a poor prognosis. In mouse models of thyroid cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists increase the expression of the sodium-iodide symporter and uptake of iodine. Their effects in humans are not known. METHODS: We conducted a study to determine whether the MAPK kinase (MEK) 1 and MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness to radioiodine in patients with metastatic thyroid cancer. After stimulation with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography (PET) was performed before and 4 weeks after treatment with selumetinib (75 mg twice daily). If the second iodine-124 PET study indicated that a dose of iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or lesions, therapeutic radioiodine was administered while the patient was receiving selumetinib. RESULTS: Of 24 patients screened for the study, 20 could be evaluated. The median age was 61 years (range, 44 to 77), and 11 patients were men. Nine patients had tumors with BRAF mutations, and 5 patients had tumors with mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the 20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS mutations). Eight of these 12 patients reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8 patients treated with radioiodine, 5 had confirmed partial responses and 3 had stable disease; all patients had decreases in serum thyroglobulin levels (mean reduction, 89%). No toxic effects of grade 3 or higher attributable by the investigators to selumetinib were observed. One patient received a diagnosis of myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with progression to acute leukemia. CONCLUSIONS: Selumetinib produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine; the effectiveness may be greater in patients with RAS-mutant disease. (Funded by the American Thyroid Association and others; ClinicalTrials.gov number, NCT00970359.).

α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4).

2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.

The sodium iodide symporter (NIS): regulation and approaches to targeting for cancer therapeutics.

Expression of the sodium iodide symporter (NIS) is required for efficient iodide uptake in thyroid and lactating breast. Since most differentiated thyroid cancer expresses NIS, ß-emitting radioactive iodide is routinely utilized to target remnant thyroid cancer and metastasis after total thyroidectomy. Stimulation of NIS expression by high levels of thyroid-stimulating hormone is necessary to achieve radioiodide uptake into thyroid cancer that is sufficient for therapy. The majority of breast cancer also expresses NIS, but at a low level insufficient for radioiodine therapy. Retinoic acid is a potent NIS inducer in some breast cancer cells. NIS is also modestly expressed in some non-thyroidal tissues, including salivary glands, lacrimal glands and stomach. Selective induction of iodide uptake is required to target tumors with radioiodide. Iodide uptake in mammalian cells is dependent on the level of NIS gene expression, but also successful translocation of NIS to the cell membrane and correct insertion. The regulatory mechanisms of NIS expression and membrane insertion are regulated by signal transduction pathways that differ by tissue. Differential regulation of NIS confers selective induction of functional NIS in thyroid cancer cells, as well as some breast cancer cells, leading to more efficient radioiodide therapy for thyroid cancer and a new strategy for breast cancer therapy. The potential for systemic radioiodide treatment of a range of other cancers, that do not express endogenous NIS, has been demonstrated in models with tumor-selective introduction of exogenous NIS.

Role of the putative N-glycosylation and PKC-phosphorylation sites of the human sodium-dependent multivitamin transporter (hSMVT) in function and regulation.

The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these sites significantly reduces the V(max) of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr(286) of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr(286) of hSMVT in the PKC-mediated regulation of biotin uptake.

Higher clearance of micafungin in neonates compared with adults: role of age-dependent micafungin serum binding.

Micafungin, a new echinocandin antifungal agent, has been used widely for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins. Micafungin body weight-adjusted clearance is higher in neonates than in adults, but the mechanisms underlying this difference are not understood. Previous work had revealed the roles of sinusoidal uptake (Na(+) -taurocholate co-transporting peptide, NTCP; organic anion transporting polypeptide, OATP) as well as canalicular efflux (bile salt export pump, BSEP; breast cancer resistance protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 were similar in neonates and in adults. However, the micafungin fraction unbound (f(u) ) in neonatal serum was about 8-fold higher than in the adult serum (0.033±0.012 versus 0.004±0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent serum protein binding of micafungin is responsible for its higher clearance in neonates compared with adults.

Increased densities of monocarboxylate transport protein MCT1 after chronic administration of nicotine in rat brain.

Chronic administration of nicotine is followed by a general stimulation of brain metabolism that results in a distinct increase of glucose transport protein densities for Glut1 and Glu3, and local cerebral glucose utilization (LCGU). This increase of LCGU might be paralleled by an enhanced production of lactate. Therefore, the question arose as to whether chronic nicotine infusion is accompanied by increased local densities of monocarboxylate transporter MCT1 in the brain. Secondly, we inquired whether LCGU might be correlated with local densities of MCT1 during normal conditions and after chronic nicotine infusion. Nicotine was given subcutaneously for 1 week by osmotic mini-pumps and local densities of MCT1 were measured by immunoautoradiographic methods in cryosections of rat brains. MCT1 density was significantly increased in 21 of 32 brain structures investigated (median increase 15.0+/-3.6%). Immunohistochemical stainings of these substructures revealed an over-expression of MCT1 within endothelial cells and astrocytes of treated animals. A comparison of 23 MCT1 densities with LCGU measured in the same structures in a previous study revealed a partial correlation between both parameters under control conditions and after chronic nicotine infusion. 10 out of 23 brain areas, which showed a significant increase of MCT1 density due to chronic nicotine infusion, also showed a significant increase of LCGU. In summary, our data show that chronic nicotine infusion induces a moderate increase of local and global density of MCT1 in defined brain structures. However, in terms of brain topologies and substructures this phenomenon did partially match with increased LCGU. It is concluded that MCT1 transporters were upregulated during chronic nicotine infusion at the level of brain substructures and, at least partially, independently of LCGU.