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Apical Membrane (apical + membrane)

Distribution by Scientific Domains

Life Sciences	46%
Medical Sciences	37%
Chemistry	15%

Distribution within Life Sciences

Anatomy & Physiology	26%
Cell & Molecular Biology	13%

Selected Abstracts

Localization and functional characterization of the human NKCC2 isoforms

ACTA PHYSIOLOGICA, Issue 3 2010
I. Carota
Abstract Aim:, Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na+/K+/2Cl, cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. Methods:, RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. Results:, All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl, affinity as determined by 86Rb+ uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. Conclusion:, The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function. [source]

Upregulation of Brain Expression of P-Glycoprotein in MRP2-deficient TR - Rats Resembles Seizure-induced Up-regulation of This Drug Efflux Transporter in Normal Rats

EPILEPSIA, Issue 4 2007
Katrin Hoffmann
Summary:,Purpose: The multidrug resistance protein 2 (MRP2) is a drug efflux transporter that is expressed predominantly at the apical domain of hepatocytes but seems also to be expressed at the apical membrane of brain capillary endothelial cells that form the blood,brain barrier (BBB). MRP2 is absent in the transport-deficient (TR,) Wistar rat mutant, so that this rat strain was very helpful in defining substrates of MRP2 by comparing tissue concentrations or functional activities of compounds in MRP2-deficient rats with those in transport-competent Wistar rats. By using this strategy to study the involvement of MRP2 in brain access of antiepileptic drugs (AEDs), we recently reported that phenytoin is a substrate for MRP2 in the BBB. However, one drawback of such studies in genetically deficient rats is the fact that compensatory changes with upregulation of other transporters can occur. This prompted us to study the brain expression of P-glycoprotein (Pgp), a major drug efflux transporter in many tissues, including the BBB, in TR, rats compared with nonmutant (wild-type) Wistar rats. Methods: The expression of MRP2 and Pgp in brain and liver sections of TR, rats and normal Wistar rats was determined with immunohistochemistry, by using a novel, highly selective monoclonal MRP2 antibody and the monoclonal Pgp antibody C219, respectively. Results: Immunofluorescence staining with the MRP2 antibody was found to label a high number of microvessels throughout the brain in normal Wistar rats, whereas such labeling was absent in TR, rats. TR, rats exhibited a significant up-regulation of Pgp in brain capillary endothelial cells compared with wild-type controls. No such obvious upregulation of Pgp was observed in liver sections. A comparable overexpression of Pgp in the BBB was obtained after pilocarpine-induced seizures in wild-type Wistar rats. Experiments with systemic administration of the Pgp substrate phenobarbital and the selective Pgp inhibitor tariquidar in TR, rats substantiated that Pgp is functional and compensates for the lack of MRP2 in the BBB. Conclusions: The data on TR, rats indicate that Pgp plays an important role in the compensation of MRP2 deficiency in the BBB. Because such a compensatory mechanism most likely occurs to reduce injury to the brain from cytotoxic compounds, the present data substantiate the concept that MRP2 performs a protective role in the BBB. Furthermore, our data suggest that TR, rats are an interesting tool to study consequences of overexpression of Pgp in the BBB on access of drugs in the brain, without the need of inducing seizures or other Pgp-enhancing events for this purpose. [source]

Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)

FEBS JOURNAL, Issue 7 2002
Anne T. Nies
The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin,Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP,MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP,MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells. [source]

Fibroblast growth factor 23 reduces expression of type IIa Na+/Pi co-transporter by signaling through a receptor functionally distinct from the known FGFRs in opossum kidney cells

GENES TO CELLS, Issue 5 2005
Xiaomei Yan
Fibroblast growth factor (FGF) 23 is an important phosphaturic factor that inhibits inorganic phosphate (Pi) reabsorption from the renal proximal tubule. Its overproduction and proteolysis-resistant mutation such as R179Q cause tumor-induced osteomalacia and autosomal dominant hypophosphatemic rickets, respectively. To clarify the signaling mechanisms of FGF23 that mediate the reduction of Pi reabsorption, we inhibited the function of the known FGFRs in opossum kidney (OK-E) cells by expressing a dominant-negative (DN) form of FGFR. OK-E cells, which represent the renal proximal tubular cells, expressed all four known FGFRs. FGF23(R179Q) bound to and activated FGFR2, a prominent FGFR expressed in OK-E cells. The activated receptor transmitted a signal to increase the expression of type IIa Na+/Pi co-transporter and the Pi uptake. Expression of FGFR2(DN), which suppresses the major FGFR-mediated signal through the FRS2,-ERK pathway, reversed the function of FGF23(R179Q). When FGF23(R179Q) was applied to the basolateral side of polarized OK-E cells, regardless of the FGFR2(DN) expression, the apical Pi uptake decreased significantly. The apical application of FGF23(R179Q) in the polarized cells did not show such decrease but increase. The exogenously expressed FGFR2 was detectable only at the apical membrane. These results suggest that an FGF23 receptor, which is functionally distinct from the known FGFRs, is expressed at the basolateral membrane of OK-E cells. [source]

Secretin activation of the apical Na+ -dependent bile acid transporter is associated with cholehepatic shunting in rats,

HEPATOLOGY, Issue 5 2005
Gianfranco Alpini
The role of the cholangiocyte apical Na+ -dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct,ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate-dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine-sensitive ASBT translocation to the cholangiocyte plasma membrane and 3H-TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater-than-expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment-dependent bile acid,induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo. (HEPATOLOGY 2005.) [source]

p.R254Q mutation in the aquaporin-2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin-induced phosphorylation,

HUMAN MUTATION, Issue 10 2009
Paul JM Savelkoul
Abstract Vasopressin regulates human water homeostasis by re-distributing homotetrameric aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2-p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2-p.S256A, which mimics non-phosphorylated AQP2. In polarized MDCK cells, AQP2-p.R254Q was retained and was distributed similarly to that of unstimulated wt-AQP2 or AQP2-p.S256A. Upon co-expression, AQP2-p.R254Q interacted with, and retained wt-AQP2 in intracellular vesicles. In contrast to wild-type AQP2, forskolin did not increase AQP2-p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2-p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley-Liss, Inc. [source]

Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1

INSECT MOLECULAR BIOLOGY, Issue 6 2002
Michael Gill
Abstract The bacterium Bacillus thuringiensis synthesizes toxins (,-endotoxins) that are highly specific for insects. Once ingested, the activated form of the toxin binds to a specific receptor(s) located on the midgut epithelial cells, inserts into the membrane causing the formation of leakage pores and eventual death of the susceptible insect larvae. Manduca sexta larvae are highly susceptible to Cry1Ac1, a toxin that is believed to bind M. sexta Aminopeptidase N, a glycoprotein located on the apical membrane. However, the binding data obtained to date only support the interaction of Cry1Ac1 with APN in vitro. To explore the in vivo role of APN, we have utilized the GAL4 enhancer trap technique to drive the expression of M. sexta APN in both midgut and mesodermal tissues of Cry1Ac1 insensitive Drosophila larvae. Transgenic Drosophila fed the toxin were now killed, demonstrating that APN can function as a receptor for Cry1Ac1 in vivo. [source]

Chromium nanoparticle exhibits higher absorption efficiency than chromium picolinate and chromium chloride in Caco-2 cell monolayers

JOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 2 2008
L.-Y. Zha
Summary This study was conducted to determine whether chromium nanoparticle (CrNano) exhibited higher absorption efficiency and possessed unique absorption mechanism in comparison to chromium picolinate (CrPic) and chromium chloride (CrCl3), as was postulated by previous reports. Twenty-one-day-old Caco-2 cell monolayers grown on semipermeable membranes in Snapwell tissue culture bichambers were incubated with CrNano, CrPic or CrCl3 to examine their transport and uptake respectively. In the concentration range of 0.2,20 ,mol/l, transport of CrNano, CrPic and CrCl3 across Caco-2 monolayers both in apical-to-basolateral and basolateral-to-apical direction was concentration-, and time-dependent, and temperature independent. The apparent permeability coefficient (Papp) of CrNano was between 5.89 and 7.92 × 10,6 cm/s and that of CrPic and CrCl3 was between 3.52 and 5.31 × 10,6 cm/s and between 0.97 and 1.37 × 10,6 cm/s respectively. Uptake of CrNano, CrPic and CrCl3 by both apical and basolateral membranes was concentration- and time-dependent. Uptake of CrNano by apical membrane was significantly (p < 0.05) decreased when the incubation temperature was reduced from 37 °C to 4 °C. The transport efficiency of CrNano, CrPic and CrCl3 after incubation for 120 min at 37 °C was 15.83% ± 0.76%, 9.08% ± 0.25% and 2.11% ± 0.53% respectively. The uptake efficiency of CrNano, CrPic and CrCl3 was 10.08% ± 0.76%, 4.73% ± 0.60% and 0.88% ± 0.08% respectively. It was concluded that the epithelial transport of CrNano, CrPic and CrCl3 across the Caco-2 cell monolayers was mainly via passive transport pathways. In addition, CrNano exhibited considerably higher absorption efficiency than both CrPic and CrCl3 in Caco-2 cell monolayers. [source]

Expression of GLUT8 in mouse intestine: Identification of alternative spliced variants

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009
Amparo Romero
Abstract GLUT8 is a facilitative glucose transporter composed of 10 exons coding for a 477 amino acids protein. It is mainly expressed in the testis, but it has also been studied in a number of tissues such as brain, adipose tissue, and liver. In this work, we have characterized the expression of GLUT8 in the small and large intestine under normal physiological conditions. Protein assay revealed low GLUT8 protein levels in the intestine compared to the testis, with higher levels in the colon than in the small intestine. Immunohistochemistry studies showed an intracellular localization of GLUT8 in enterocytes and colonocytes with a supranuclear distribution next to the apical membrane. GLUT8 immunoreactivity was also detected in the crypt cells. Interestingly, we have identified three additional transcriptional variants in mouse intestine (mGLUT-SP1, mGLUT8-SP2, and mGLUT8-SP3) produced by the deletion of one, two, and four exons, respectively, whereas only the entire mRNA was detected in the testis. Expression of these alternative variants did not have an effect on glucose consumption in 3T3-L1 cells. Although the specific function of GLUT8 in intestine remains unclear, the alternative splicing of GLUT8 could reflect a mechanism for the regulation of the gene expression in a tissue-specific manner by targeting GLUT8 mRNA for nonsense-mediated decay. J. Cell. Biochem. 106: 1068,1078, 2009. © 2009 Wiley-Liss, Inc. [source]

OCTN3: A Na+ -independent L -carnitine transporter in enterocytes basolateral membrane

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005
J.M. Durán
L -carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+ -free conditions chicken enterocytes take up L -carnitine until the cell to medium L -carnitine ratio is 1. This uptake was inhibited by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L - 3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+ -independent, (ii) trans-stimulated by intravesicular L -carnitine, and (iii) cis-inhibited by TEA and cold L -carnitine. L - 3H-carnitine efflux from L - 3H-carnitine preloaded enterocytes was also Na+ -independent, and trans-stimulated by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L -carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl- L -carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L -carnitine transport activity measured in the three experimental conditions. © 2004 Wiley-Liss, Inc. [source]

ASSESSING ABSORBABILITY OF BIOACTIVE COMPONENTS IN ALOE USING IN VITRO DIGESTION MODEL WITH HUMAN INTESTINAL CELL

JOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2010
SOON-MI SHIM
ABSTRACT This study used a simulated in vitro digestion model coupled with caco-2 cell to assess the digestive stability and absorption of aloin, aloe-emodin and aloenin A. Aloenin A and aloe-emodin were stable and entirely recovered during simulated digestion, but 50% of aloin was lost. Approximately 53.2, 7.3 and 28.7% of aloe-emodin, aloenin A and aloin, respectively, was transported into both apical and basolateral compartments after 1 h incubation in caco-2 cell. The involvement of several transporter proteins for aloin and aloenin A was examined. An inhibitor of SGLT1 on apical surface (phloridzin) or that of GLUT2 on basolateral membrane (cytochalasin B) reduced the absorption of aloin by 40 or 60%, respectively, indicating that aloin is likely to be a partial substrate of SGLT1. In the presence of an efflux transporter inhibitor (verapamil), the transport of aloenin A through an intentinal apical membrane increased up to 2.1 times compared with the control (without verapamil). PRACTICAL APPLICATIONS Our results on both digestive stability and intestinal absorption characteristics of bioactive components in aloe could be of helpful information for promoting its bioavailability. The in vitro technique described in this study provides a rapid and cost-effective alternative for predicting bioavailability of biomarkers in aloe functional food. [source]

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood,brain barrier

JOURNAL OF NEUROCHEMISTRY, Issue 1 2008
Dorota Miecz
Abstract Carnitine ,-hydroxy-,-(trimethylammonio)butyrate , a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low ,-oxidation in this organ. In order to enter the brain, carnitine has to cross the blood,brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters , organic cation/carnitine transporter 2 (OCTN2) and B0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood,brain barrier by real-time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house-keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood,brain barrier with use of anti-OCTN2 antibodies. Z -axis analysis of the confocal microscope pictures of endothelial cells, with anti-P-glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters. [source]

Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

PLANT BIOTECHNOLOGY JOURNAL, Issue 2 2010
Abdoreza Davoodi-Semiromi
Summary Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10+ T cell but not Foxp3+ regulatory T cells, suppression of interferon-, and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity. [source]

Taurine uptake across the human intestinal brush-border membrane is via two transporters: H+ -coupled PAT1 (SLC36A1) and Na+ - and Cl, -dependent TauT (SLC6A6)

THE JOURNAL OF PHYSIOLOGY, Issue 4 2009
Catriona M. H. Anderson
Taurine is an essential amino acid in some mammals and is conditionally essential in humans. Taurine is an abundant component of meat and fish-based foods and has been used as an oral supplement in the treatment of disorders such as cystic fibrosis and hypertension. The purpose of this investigation was to identity the relative contributions of the solute transporters involved in taurine uptake across the luminal membrane of human enterocytes. Distinct transport characteristics were revealed following expression of the candidate solute transporters in Xenopus laevis oocytes: PAT1 (SLC36A1) is a H+ -coupled, pH-dependent, Na+ - and Cl, -independent, low-affinity, high-capacity transporter for taurine and ,-alanine; TauT (SLC6A6) is a Na+ - and Cl, -dependent, high-affinity, low-capacity transporter of taurine and ,-alanine; ATB0,+ (SLC6A14) is a Na+ - and Cl, -dependent, high-affinity, low-capacity transporter which accepts ,-alanine but not taurine. Taurine uptake across the brush-border membrane of human intestinal Caco-2 cell monolayers showed characteristics of both PAT1- and TauT-mediated transport. Under physiological conditions, Cl, -dependent TauT-mediated uptake predominates at low taurine concentrations, whereas at higher concentrations typical of diet, Cl, -independent PAT1-mediated uptake is the major absorptive mechanism. Real-time PCR analysis of human duodenal and ileal biopsy samples demonstrates that PAT1, TauT and ATB0,+ mRNA are expressed in each tissue but to varying degrees. In conclusion, this study is the first to demonstrate both taurine uptake via PAT1 and functional coexpression of PAT1 and TauT at the apical membrane of the human intestinal epithelium. PAT1 may be responsible for bulk taurine uptake during a meal whereas TauT may be important for taurine supply to the intestinal epithelium and for taurine capture between meals. [source]

Apical SK potassium channels and Ca2+ -dependent anion secretion in endometrial epithelial cells

THE JOURNAL OF PHYSIOLOGY, Issue 3 2008
Melissa L. Palmer
Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (Isc) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K+ dependent. The current,voltage relationship was slightly inwardly rectifying with a reversal potential of ,52 ± 2 mV, and the PK/PNa ratio was 14, indicating high selectivity for K+. Concentration,response relationships for apamin and dequalinium had IC50 values of 0.5 nm and 1.8 ,m, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 ,m BAPTA-AM completely blocked the effects of UTP on K+ channel activation, indicating that the apamin-sensitive current was also Ca2+ dependent. Moreover, channel activation was blocked by calmidazolium (IC50= 5 ,m), suggesting a role for calmodulin in Ca2+ -dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17, produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K+ channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca2+ near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation. [source]

Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 9 2006
G. Einecke
One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis. [source]

V-ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009
Martin Voss
Abstract The activity of vacuolar H+ -ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc. [source]

Wingless can't fly so it hitches a ride with dynein

BIOESSAYS, Issue 10 2001
Steven H. Myster
Asymmetric RNA localization is required for many developmental processes in a wide range of organisms. For example, wingless and pair-rule transcripts are localized to the apical membrane of polarized cells. It has been unclear, however, if this localization is important for biological activity and, in addition, how the transcripts are transported. Two recent studies(1,2) have identified cis -elements and trans -acting factors that are required for the asymmetric localization of mRNAs. Correct localization is shown to be required for biological activity, and a mechanism of RNA transport involving the microtubule motor dynein has been revealed. BioEssays 23:869,872, 2001. © 2001 John Wiley & Sons, Inc. [source]

Der Epitheliale Natrium Kanal.

BIOLOGIE IN UNSERER ZEIT (BIUZ), Issue 5 2009
15 Jahre Kanalarbeiten
Abstract Epitheliale Natrium Kanäle (ENaCs) sind Natrium leitende Ionenkanäle, die in der apikalen Membran vieler Epithelien vorkommen. Sie sind der zentrale Faktor zum transepithelialen Transport von Natrium , und damit einhergehend von Wasser. ENaCs tragen zu einer Vielzahl physiologischer Prozesse bei: Regulation des Salz- und Wasserhaushalts, des Blutdrucks oder des Wassergehaltes der Lunge. Auch Erkrankungen wie Bluthochdruck oder Lungenödeme lassen sich auf gestörte Regulationen des ENaC zurückführen. Dieser Artikel beschreibt die Funktion und Physiologie Epithelialer Natrium Kanäle, sowie zelluläre Mechanismen, die die Aktivität dieser Ionenkanäle bestimmen. Epithelial sodium channels (ENaCs) are sodium-conducting ion channels that are located in the apical membrane of various epithelia. They represent the rate-limiting step for transeptihelial sodium- and thereby water-transport. ENaCs contribute to a variety of physiological processes including the regulation of salt and water homeostasis, blood pressure or the water content of the lung. Further, malfunctions in ENaC regulation contribute to the pathogenesis of diseases like hypertension or pulmonary oedema. This article describes the function and physiology of epithelial sodium channels as well as cellular mechanisms that determine the activity of these ion channels. [source]

Transport characteristics of L -citrulline in renal apical membrane of proximal tubular cells

BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 3 2009
Keisuke Mitsuoka
Abstract L -Citrulline has diagnostic potential for renal function, because its plasma concentration increases with the progression of renal failure. Although L -citrulline extracted by glomerular filtration in kidney is mostly reabsorbed, the mechanism involved is not clearly understood. The present study was designed to characterize L -citrulline transport across the apical membranes of renal epithelial tubular cells, using primary-cultured rat renal proximal tubular cells, as well as the human kidney proximal tubular cell line HK-2. L -Citrulline was transported in a Na+ -dependent manner from the apical side of both cell types cultured on permeable supports with a microporous membrane. Kinetic analysis indicated that the transport involves two distinct Na+ -dependent saturable systems and one Na+ -independent saturable system in HK-2 cells. The uptake was competitively inhibited by neutral and cationic, but not anionic amino acids. Relatively large cationic and anionic compounds inhibited the uptake, but smaller ones did not. In HK-2 cells, mRNA expression of SLC6A19 and SLC7A9, which encode B0AT1 and b0,+AT, respectively, was detected by RT-PCR. In addition, L -citrulline transport was significantly decreased in HK-2 cells in which either SLC6A19 or SLC7A9 was silenced. Hence, these results suggest that amino acid transporters B0AT1 and b0,+AT are involved in the reabsorption of L -citrulline in the kidney, at least in part, by mediating the apical membrane transport of L -citrulline in renal tubule cells. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Lysosomal trapping of amodiaquine: impact on transport across intestinal epithelia models

BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 6 2008
Rose Hayeshi
Abstract The lipophilic weak base amodiaquine is an antimalarial drug that has been in use for over 40 years. Little is known of amodiaquine's mechanism of transport across membranes. Transport experiments of amodiaquine in Caco-2 cells showed a low recovery of 30% and rapid disappearance from the apical chamber. Compounds structurally similar to amodiaquine, and those affecting non-specific binding of amodiaquine or the pH of the system, were tested to unravel the mechanism behind these observations. Chloroquine and ammonium chloride increased the transmonolayer permeability of amodiaquine and decreased its accumulation in Caco-2 cells, whereas BSA had no effect. Chloroquine and BSA decreased plastic binding whereas ammonium chloride had no effect. This suggests that amodiaquine is trapped in acidic cell compartments such as lysosomes. Amodiaquine was also trapped in rat intestinal tissue. In addition, permeability from the apical to basolateral direction was significantly higher, suggesting an active uptake over the apical membrane of the rat tissue. It can be concluded that amodiaquine is trapped in acidic cell compartments due to its base properties and recovery may be improved by the use of ammonium chloride rather than BSA in transport experiments. Further studies are required to confirm whether amodiaquine is actively absorbed in the intestine. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Transcellular transport of genistein, a soybean-derived isoflavone, across human colon carcinoma cell line (Caco-2)

BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2001
Masataka Oitate
Abstract Genistein, a soybean-derived isoflavone, is thought to have an anticarcinogenic action, but little is known about the cellular mechanisms of its intestinal absorption. This study was designed to investigate the absorption mechanisms of genistein using human colon carcinoma cell line, Caco-2 cells. The apical-to-basolateral transcellular transport of genistein across a Caco-2 cell monolayer was significantly greater than that in the opposite direction. An uptake experiment revealed that cellular uptake of genistein by Caco-2 cells was concentrative. The transcellular transport of genistein was saturable and temperature-dependent, and was inhibited by other flavonoids such as rutin, quercetin, (+)-catechin and (,)-epicatechin. These results suggest that genistein is transported across Caco-2 cells by a carrier-mediated system, located on the apical membrane. Copyright © 2001 John Wiley & Sons, Ltd. [source]

Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

BRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2000
H L McAlroy
Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (ISC, a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca2+]i) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y2, P2Y4 and P2Y6 receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase,polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which ISC and [Ca2+]i were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in ISC was accompanied by a [Ca2+]i -signal. Ionomycin consistently increased [Ca2+]i in such polarized cells but caused no discernible change in ISC. However, subsequent application of apical ATP or UTP evoked a small rise in ISC but no rise in [Ca2+]i. UDP evoked no such response. As well as evoking increases in [Ca2+]i, the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca2+]i. British Journal of Pharmacology (2000) 131, 1651,1658; doi:10.1038/sj.bjp.0703743 [source]

Purinergic regulation of the epithelial Na+ channel

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2009
Lauren M O'Mullane
Summary 1. The epithelial Na+ channel (ENaC) is a major conductive pathway that transports Na+ across the apical membrane of the distal nephron, the respiratory tract, the distal colon and the ducts of exocrine glands. The ENaC is regulated by hormonal and humoral factors, including extracellular nucleotides that are available from the epithelial cells themselves. 2. Extracellular nucleotides, via the P2Y2 receptors (P2Y2Rs) at the basolateral and apical membrane of the epithelia, trigger signalling systems that inhibit the activity of the ENaC and activate Ca2+ -dependent Cl, secretion. 3. Recent data from our laboratory suggest that stimulation of the P2Y2Rs at the basolateral membrane inhibits ENaC activity by a signalling mechanism that involves G,, subunits freed from a pertussis toxin (PTX)-sensitive G-protein and phospholipase C (PLC) ,4. A similar signalling mechanism is also partially responsible for inhibition of the ENaC during activation of apical P2Y2Rs. 4. Stimulation of apical P2Y2Rs also activates an additional signalling mechanism that inhibits the ENaC and involves the activated G, subunit of a PTX-insensitive G-protein and activation of an unidentified PLC. The effect of this PTX-insensitive system requires the activity of the basolateral Na+/K+/2Cl, cotransporter. [source]

DIFFERENTIAL REGULATION OF ANGIOTENSIN II RECEPTORS DURING RENAL INJURY AND COMPENSATORY HYPERTROPHY IN THE RAT

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2005
Emma Joly
SUMMARY 1.,The renin-angiotensin system may be involved in the compensatory adaptations occurring after the reduction of renal mass and during the consecutive changes leading to chronic renal failure. We therefore investigated the regulation of angiotensin II receptors in two models of renal hypertrophy in the rat: hypertrophy following uninephrectomy (UNx) or subtotal nephrectomy (STNx). The level of angiotensin type 1 (AT1A -R and AT1B -R) and type 2 (AT2 -R) receptor mRNA was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) in specific renal zones and the intrarenal distribution of angiotensin II receptors was analysed by immunohistochemistry. 2.,In the UNx rats, AT1 -R mRNA expression was not modified in the cortex or in the inner stripe of the outer medulla of the residual kidney at any time after the surgery (1, 4 and 12 weeks). In contrast, AT1 -R mRNA expression was significantly reduced in these zones in STNx rats (,33% and ,40%, respectively). This downregulation was organ-specific, as AT1 -R mRNA levels were not modified in the liver. The proportions of AT1 -R subtype (AT1A and AT1B) mRNA were unchanged by UNx or STNx. Very low levels of AT2 -R mRNA were found in the cortex of all groups. Immunostaining revealed a similar localization of AT1 -R in mesangial cells, proximal tubule, basolateral membrane of thick ascending limb, in both models of hypertrophy. AT1 -R labelling was also detected in the apical membrane of intercalated cells of cortical collecting ducts. 3.,This differential mRNA expression of angiotensin II receptors during compensatory hypertrophy and renal injury suggests that the development of renal hypertrophy is independent of AT1 -R and AT2 -R gene expression levels. [source]

Dietary pectin up-regulates monocaboxylate transporter 1 in the rat gastrointestinal tract

EXPERIMENTAL PHYSIOLOGY, Issue 4 2009
Doaa Kirat
This work was undertaken to study the effect of pectin feeding on the expression level, cellular localization and functional activity of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of rats. The results indicated that MCT1 protein level was significantly increased along the entire length of the gastrointestinal tract of pectin-fed rats in comparison with control animals. Immunohistochemical analysis revealed an increase in MCT1 in the stratified squamous epithelia of the forestomach as well as in the basolateral membranes of the cells lining the gastric pit of the glandular stomach of pectin-fed rats when compared with control animals. The parietal cells, which showed barely any or no detectable MCT1 in the control group, exhibited a strong intensity of MCT1 on the basolateral membranes in pectin-fed rats. In the small intestine of pectin-fed rats, strong immunopositivity for MCT1 was detected in the brush border and basolateral membranes of the absorptive enterocytes lining the entire villi, while in control rats, weak reactivity was detected on the brush border membrane in a few absorptive enterocytes in the villus tip. In the large intestine of control animals, MCT1 was detected on the basolateral membranes of the epithelia lining the caecum and colon. This staining intensity was markedly increased in pectin-fed rats, along with the appearance of strong reactivity for MCT1 on the apical membranes of the surface and crypt epithelia of caecum and colon. Our results also showed that MCT1 co-localizes with its chaperone, basigin (CD147), in the rat gastrointestinal tract, and that the pectin feeding increased the expression of CD147. In vivo functional studies revealed an enhanced acetate absorption in the colon of pectin-fed in comparison with control animals. We conclude that MCT1 is up-regulated along the gastrointestinal tract of pectin-fed rats, which might represent an adaptive response to the increased availability of its substrates. [source]

Lipid formulation strategies for enhancing intestinal transport and absorption of P-glycoprotein (P-gp) substrate drugs: In vitro/In vivo case studies

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2007
Panayiotis P. Constantinides
Abstract The intestinal efflux pump, P-glycoprotein (P-gp), located in the apical membranes of intestinal absorptive cells, can reduce the bioavailability of a wide range of drugs which are substrates for this membrane transporter. In addition to anticancer and anti-HIV drugs, NCEs for other disease indications are P-gp substrates and there is considerable interest in inhibiting P-gp and thus increasing the bioavailability of these molecules. In this review article, an overview of P-gp and its role in drug transport and absorption will be presented first and then formulation strategies to effectively inhibit P-gp will be discussed and compared. These strategies independently and in combination, are: (a) coadministration of another P-gp substrate/specific inhibitor, and (b) incorporation of a nonspecific lipid and/or polymer excipient in the formulation. The first approach, although very effective in inhibiting P-gp, utilizes a second active compound in the formulation and thus imposes regulatory constraints and long development timelines on such combination products. Excipient inhibitors appear to have minimal nonspecific pharmacological activity and thus potential side effects of specific active compound inhibitors can be avoided. Case studies will be presented where specific active compounds, surfactants, polymers, and formulations incorporating these molecules are shown to significantly improve the intestinal absorption of poorly soluble and absorbed drugs as a result of P-gp inhibition and enhanced drug transport in vitro. ©2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:235,248, 2007 [source]

Structure, function, and regulation of renal organic anion transporters

MEDICINAL RESEARCH REVIEWS, Issue 6 2002
Guofeng You
Abstract Renal elimination of anionic drugs, xenobiotics, and toxins is necessary for the survival of mammalian species. This process is mediated by vectorial transport from blood to urine through the cooperative functions of specific transporters in the basolateral and apical membranes of the proximal tubule epithelium. The first step of this process is the extraction of organic anions from the peritubular blood plasma into proximal tubule cells largely through the organic anion transporter (OAT) pathway. Therefore, the OAT pathway is one of the major sites for body drug clearance/detoxification. As a result, it is also the site for drug,drug interaction and drug-induced nephrotoxicity. To maximize therapeutic efficacy and minimize toxicity, the structure-function relationships of OATs and their regulation must be defined. The recent cloning and identification of OATs have paved the way for such investigations. This review summarizes the available data on the general properties of OATs, focusing in particular on the recent progress made from the author's laboratory as well as from other's, on the molecular characterization of the structure-function relationships of OATs and their regulatory mechanisms. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 6, 602,616, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10019 [source]

Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells

THE JOURNAL OF PHYSIOLOGY, Issue 2 2007
Santanu Bose
Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B12) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T1/2) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM. [source]