Screening for drugs potentially interfering with MCT8-mediated T3 transport in vitro identifies dexamethasone and some commonly used drugs as inhibitors of MCT8 activity.

BACKGROUND: Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity. AIM: This study aimed at investigating whether some common drugs having a structural similarity with TH and/or whose treatment is associated with thyroid function test abnormalities, or which behave as antagonists of TH action can inhibit MCT8-mediated T3 transport. METHODS: [125I]T3 uptake and efflux were measured in COS-7 cells transiently transfected with hMCT8 before and after exposure to increasing concentrations of hydrocortisone, dexamethasone, prednisone, prednisolone, amiodarone, desethylamiodarone, dronedarone, buspirone, carbamazepine, valproic acid, and L-carnitine. The mode of inhibition was also determined. RESULTS: Dexamethasone significantly inhibited T3 uptake at 10 µM; hydrocortisone reduced T3 uptake only at high concentrations, i.e. at 500 and 1000 µM; prednisone and prednisolone were devoid of inhibitory potential. Amiodarone caused a reduction of T3 uptake by MCT8 only at the highest concentrations used (44% at 50 µM and 68% at 100 µM), and this effect was weaker than that produced by desethylamiodarone and dronedarone; buspirone resulted a potent inhibitor, reducing T3 uptake at 0.1-10 µM. L-Carnitine inhibited T3 uptake only at 500 mM and 1 M. Kinetic experiments revealed a noncompetitive mode of inhibition for all compounds. All drugs inhibiting T3 uptake did not affect T3 release. CONCLUSION: This study shows a novel effect of some common drugs, which is inhibition of T3 transport mediated by MCT8. Specifically, dexamethasone, buspirone, desethylamiodarone, and dronedarone behave as potent inhibitors of MCT8.

pKa of Glu325 in LacY.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H+ across the cytoplasmic membrane of Escherichia coli (galactoside/H+ symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+ and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.

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.

Phosphoregulation of K+ -Cl- cotransporters during cell swelling: Novel insights.

The K+ -Cl- cotransporters (KCCs) belong to the cation-Cl- cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl- ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl- ] (Cli ), but the mechanisms at play are still ill-defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine-deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1-related proline/alanine-rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C-terminus of full-length KCC4 led to higher levels of heterologous K+ -Cl- cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric-sensitive kinase-dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.

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.

Depolarizing γ-aminobutyric acid contributes to glutamatergic network rewiring in epilepsy.

OBJECTIVE: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuits, an abnormal depolarizing γ-aminobutyric acidergic (GABAergic) drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post-status epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuits. METHODS: Osmotic minipumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody toward the pan-neurotrophin receptor p75 (p75NTR ). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2, and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric electroencephalographic and electrophysiological recordings were performed at day 5 and 2 months post-SE. RESULTS: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis, and significantly reduced the glutamatergic rMF sprouting within the dentate gyrus. This mechanism partially involves p75NTR signaling, as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3 days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. INTERPRETATION: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signaling fosters a long-lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. Ann Neurol 2017;81:251-265.

Neuroprotection by Propofol Post-Conditioning: Focus on PKMζ/KCC2 Pathway Activity.

Critical and major operations are often accompanied by brain ischemic complications. Previous studies found that propofol post-conditioning provided neuroprotective functions through upregulating the expression of potassium chloride cotransporter 2 (KCC2) in gamma-aminobutyric acid (GABA) interneurons. Membrane expression and phosphorylation represents KCC2 activity, which were modulated by a protein kinase C (PKC)-dependent mechanism. However, the role of propofol in increasing KCC2 phosphorylation and the involvement of protein kinase Mζ (PKMζ), a major subtype of PKC, in the KCC2 pathway remained unclear. In this study, we established middle cerebral artery occlusion model in rats to evaluate the long-term recovery of brain functions using behavioral experiments. KCC2 and PKMζ were assessed via western blot. We used the selective inhibitor, zeta inhibitory peptide (ZIP), to investigate the relationship between KCC2 and PKMζ. Intracellular chloride concentration in the hippocampal CA1 area was measured to determine KCC2 activity. We found that propofol, infused at a speed of 20 mg kg-1 h-1 for 2 h at the onset of reperfusion, improved neurological deficits and cognitive dysfunction following ischemia/reperfusion injury. PKMζ expression was significantly upregulated, which improved KCC2 membrane expression and phosphorylation in the ischemic hippocampal CA1 area, and these effects could last up to 28 days. But ZIP inhibited this process. Ultimately, we showed that propofol increased KCC2 phosphorylation and PKMζ was the upstream of KCC2. Propofol led to long-term recovery of brain functions by upregulating the activity of the PKMζ/KCC2 pathway.

K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

BACKGROUND: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). METHODS: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. RESULTS: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. CONCLUSIONS: The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

Increase of KCC2 in hippocampal synaptic plasticity disturbances after perinatal ethanol exposure.

Low to moderate perinatal ethanol exposure (PEE) may have disastrous consequences for the central nervous system resulting notably in permanent cognitive deficits. Learning and memory are mediated in the hippocampus by long-term potentiation (LTP) and long term depression (LTD), two forms of synaptic plasticity. PEE decreases LTP but also abnormally facilitates LTD (Kervern et al. ) through a presently unknown mechanism. We studied in rat hippocampus slice, the involvement of the chloride co-transporters NKCC1 and KCC2, in the role of GABAA inhibitions in facilitated LTD after moderate PEE. After PEE and in contrast to control slices, facilitated LTD in CA1 field was reduced by the GABAA receptor antagonist bicuculline with no changes in sensitivity to bicuculline and in GABA and benzodiazepine binding sites. Also, sensitivity to diazepam was unaltered, whereas aberrant LTD was blocked. Immunohistochemistry and protein analysis demonstrated an increase in KCC2 protein level at cell membrane in CA1 after PEE with no change in NKCC1 expression. Specifically, both monomeric and dimeric forms of KCC2 were increased in CA1. Bumetanide (10-100 µM), a dose-dependent blocker of NKCC1 and KCC2, or VU0240551 (10 µM) a specific antagonist of KCC2, corrected the enhanced LTD and interestingly bumetanide also restored the lower LTP after PEE. These results demonstrate for the first time an upregulation of the KCC2 co-transporter expression after moderate PEE associated with disturbances in GABAergic neurotransmission modulating bidirectional synaptic plasticity in the hippocampus. Importantly, bumetanide compensated deficits in both LTP and LTD, revealing its potential therapeutic properties.

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.).

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.

In vitro evidence that phosphatidylcholine protects against indomethacin/bile acid-induced injury to cells.

Indomethacin is a powerful analgesic nonsteroidal anti-inflammatory drug (NSAID), but is limited in use by its primary side effect to cause gastrointestinal bleeding and serious injury. One factor important for exacerbating NSAID injury is the presence of bile acids, which may interact with indomethacin to form toxic mixed micelles in the gut. The development of a safer gastrointestinal formulation of indomethacin that is chemically complexed with phosphatidylcholine (PC-indomethacin) may offer an improved therapeutic agent, particularly in the presence of bile acid, but its potential protective mechanism is incompletely understood. Intestinal epithelial cells (IEC-6) were tested for injury with indomethacin (alone and plus various bile acids) compared with PC-indomethacin (alone and plus bile acids). To explore a role for bile acid uptake into cells as a requirement for NSAID injury, studies were performed using Madin-Darby canine kidney cells transfected with the apical sodium-dependent bile acid transporter (ASBT). Indomethacin, but not PC-indomethacin, was directly and dose-dependently injurious to IEC-6 cells. Similarly, the combination of any bile acid plus indomethacin, but not PC-indomethacin, induced cell injury. The expression of ASBT had a modest effect on the acute cytotoxicity of indomethacin in the presence of some conjugated bile acids. Complexing PC with indomethacin protected against the acute intestinal epithelial injury caused by indomethacin regardless of the presence of bile acids. The presence of luminal bile acid, but not its carrier-mediated uptake into the enterocyte, is required for acute indomethacin-induced cell injury. It is likely that initial cell damage induced by indomethacin occurs at or near the cell membrane, an effect exacerbated by bile acids and attenuated by PC.

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.

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process.

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 µM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS.

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.

Pharmacotherapeutic targeting of cation-chloride cotransporters in neonatal seizures.

Seizures are a common manifestation of acute neurologic insults in neonates and are often resistant to the standard antiepileptic drugs that are efficacious in children and adults. The paucity of evidence-based treatment guidelines, coupled with a rudimentary understanding of disease pathogenesis, has made the current treatment of neonatal seizures empiric and often ineffective, highlighting the need for novel therapies. Key developmental differences in γ-aminobutyric acid (GABA)ergic neurotransmission between the immature and mature brain, and trauma-induced alterations in the function of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2, probably contribute to the poor efficacy of standard antiepileptic drugs used in the treatment of neonatal seizures. Although CCCs are attractive drug targets, bumetanide and other existing CCC inhibitors are suboptimal because of pharmacokinetic constraints and lack of target specificity. Newer approaches including isoform-specific NKCC1 inhibitors with increased central nervous system penetration, and direct and indirect strategies to enhance KCC2-mediated neuronal chloride extrusion, might allow therapeutic modulation of the GABAergic system for neonatal seizure treatment. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Disrupted Cl(-) homeostasis contributes to reductions in the inhibitory efficacy of diazepam during hyperexcited states.

The K(+) -Cl(-) cotransporter type 2 is the major Cl(-) extrusion mechanism in most adult neurons. This process in turn leads to Cl(-) influx upon activation of γ-aminobutyric acid type A (GABAA ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABAA receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABAA signaling and become less sensitive to therapeutic drugs that target GABAA receptors. To date, it is unknown if alterations in the neuronal Cl(-) gradient affect the efficacies of diazepam and propofol. We therefore used the in vitro model of glutamate-induced hyperexcitability to test if alterations in the Cl(-) gradient affect the efficacy of GABAA modulators. We exclusively utilised the gramicidin perforated-patch-clamp configuration to preserve the endogenous Cl(-) gradient in rat neurons. Brief exposure to glutamate reduced the inhibitory efficacy of diazepam within 5 min, which was caused by the collapse of the Cl(-) gradient, and not due to reductions in GABAA receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate-induced appearance of depolarising GABAA -mediated currents. Similarly, pharmacological inhibition of K(+) -Cl(-) cotransporter type 2 by furosemide disrupted Cl(-) homeostasis and reduced the efficacy of diazepam but not propofol. Collectively our results suggest that pathological hyperexcitable conditions could cause the rapid accumulation of intracellular Cl(-) and the appearance of depolarising GABAA -mediated currents that would decrease the efficacy of diazepam.

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.