RGMb protects against acute kidney injury by inhibiting tubular cell necroptosis via an MLKL-dependent mechanism.

PubMed

Liu, Wenjing; Chen, Binbin; Wang, Yang; Meng, Chenling; Huang, Huihui; Huang, Xiao-Ru; Qin, Jinzhong; Mulay, Shrikant R; Anders, Hans-Joachim; Qiu, Andong; Yang, Baoxue; Freeman, Gordon J; Lu, Hua Jenny; Lin, Herbert Y; Zheng, Zhi-Hua; Lan, Hui-Yao; Huang, Yu; Xia, Yin

2018-02-13

Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.

Localization of the Calcium Regulated Citrate Transport Process in Proximal Tubule Cells

PubMed Central

Hering-Smith, Kathleen S.; Mao, Weibo; Schiro, Faith R.; Coleman-Barnett, Joycelynn; Pajor, Ana M.; Hamm, L. Lee

2014-01-01

Urinary citrate is an important inhibitor of calcium stone formation. Most of citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical > basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles. PMID:24652587

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

PubMed

Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

2006-03-01

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action. Copyright 2006 Wiley-Liss, Inc.

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

PubMed

Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

2018-04-01

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.

PubMed

Shirasawa, Madoka; Fujiwara, Naoyuki; Hirabayashi, Susumu; Ohno, Hideki; Iida, Junko; Makita, Koshi; Hata, Yutaka

2004-02-01

Lung alveolar epithelial cells are comprised of type I (ATI) and type II (ATII) cells. ATI cells are polarized, although they have very flat morphology. The identification of marker proteins for apical and basolateral membranes of ATI cells is important to investigate into the differentiation of ATI cells. In this paper, we characterized receptor for advanced glycation end-products (RAGE) as a marker for ATI cells. RAGE was localized on basolateral membranes of ATI cells in the immunoelectron microscopy and its expression was enhanced in a parallel manner to the differentiation of ATI cells in vivo and in primary cultures of ATII cells. RAGE and T1 alpha, a well-known ATI marker protein, were targeted to basolateral and apical membranes, respectively, when expressed in polarized Madine Darby canine kidney cells. Moreover, RAGE was expressed in ATI cells after T1 alpha in vivo and in ex in vivo organ cultures. In conclusion, RAGE is a marker for basolateral membranes of well-differentiated ATI cells. ATI cells require some signal provided by the in vivo environment to express RAGE.

Differential subcellular membrane recruitment of Src may specify its downstream signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah

2008-04-15

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 andmore » flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.« less

Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish

USGS Publications Warehouse

Hiroi, J.; Yasumasu, S.; McCormick, S.D.; Hwang, P.-P.; Kaneko, T.

2008-01-01

Cation-chloride cotransporters, such as the Na+/K +/2Cl- cotransporter (NKCC) and Na+/Cl - cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify 'freshwater-type' and 'seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na+/K+-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/H+ exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in freshwater that incorporates apically located NCC. We also reevaluate a traditional ion-uptake model incorporating NHE3; the mRNA was highly expressed in freshwater, and the immunoreactivity was found at the apical membrane of other freshwater-specific MRCs.

Altered expression of junctional adhesion molecule 4 in injured podocytes.

PubMed

Harita, Yutaka; Miyauchi, Naoko; Karasawa, Tamaki; Suzuki, Koichi; Han, Gi Dong; Koike, Hiroko; Igarashi, Takashi; Shimizu, Fujio; Kawachi, Hiroshi

2006-02-01

Recent investigations have revealed the importance of glomerular podocytes with its diaphragm as the major filtration barrier. Junctional adhesion molecule 4 (JAM4) has been identified as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 and is reported to be expressed on podocytes. To elucidate the role of JAM4 on podocytes, we examined the expression of JAM4 and MAGI-1 in normal and two different proteinuric rat models: puromycin aminonucleoside (PAN) nephropathy and anti-nephrin antibody-induced (ANA) nephropathy, one model with and one without effacement of podocyte foot processes. JAM4 was detected by immunomicroscopy at the apical membrane of normal podocytes. JAM4 immunostaining was focally increased in the podocytes in PAN nephropathy but not in ANA nephropathy. In proteinuric podocytes, the expression of JAM4 was distinct from that of MAGI-1 or other slit diaphragm molecules such as nephrin and ZO-1. Close colocalization of JAM4 and ezrin was maintained in PAN nephropathy. By immunoelectron microscopy, the signals for JAM4 were detected at the free apical membrane of the podocytes with effaced foot processes. Studies with selective detergent extract revealed that the subcellular localization of JAM4 was altered in PAN nephropathy. Thus the altered expression of JAM4 appears to be associated with morphological changes in podocytes and can be a useful marker of injured podocytes. JAM4 may have a different role at the apical membrane besides the role as a junctional molecule and is likely associated with the unique structure of this epithelium.

Carbonic anhydrases in chick extra-embryonic structures: a role for CA in bicarbonate reabsorption through the chorioallantoic membrane.

PubMed

Gabrielli, M Gabriella

2004-06-01

The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model.

PubMed

Sasamoto, Kouhei; Marunaka, Rie; Niisato, Naomi; Sun, Hongxin; Taruno, Akiyuki; Pezzotti, Giuseppe; Yamamoto, Toshiro; Kanamura, Narisato; Zhu, Wenliang; Nishio, Kyosuke; Inui, Toshio; Eaton, Douglas C; Marunaka, Yoshinori

2017-01-01

Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC. © 2017 The Author(s). Published by S. Karger AG, Basel.

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

PubMed Central

Zacchi, Paola; Stenmark, Harald; Parton, Robert G.; Orioli, Donata; Lim, Filip; Giner, Angelika; Mellman, Ira; Zerial, Marino; Murphy, Carol

1998-01-01

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells. PMID:9490718

Developmental expression of SLC26A4 (Pendrin) during amelogenesis in developing rodent teeth

PubMed Central

Bronckers, Antonius LJJ; Guo, Jing; Zandieh-Doulabi, Behrouz; Bervoets, Theodore J; Lyaruu, Donacian M.; Li, Xiangming; Wangemann, Philine; DenBesten, Pamela

2012-01-01

Ameloblasts need to regulate pH during formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as pH regulator. SLC26A4 does not appear critical for ameloblast functioning and is likely compensated by other pH regulators. PMID:22243245

Inhibitory effect of calcium on non-heme iron absorption may be related to translocation of DMT-1 at the apical membrane of enterocytes.

PubMed

Thompson, Ben A V; Sharp, Paul A; Elliott, Ruan; Fairweather-Tait, Susan J

2010-07-28

Many studies show that calcium reduces iron absorption from single meals, but the underlying mechanism is not known. We tested the hypothesis that calcium alters the expression and/or functionality of iron transport proteins. Differentiated Caco-2 cells were treated with ferric ammonium citrate and calcium chloride, and ferritin, DMT-1, and ferroportin were quantified in whole-cell lysate and cell-membrane fractions. Calcium attenuated the iron-induced increase in cell ferritin levels in a dose-dependent manner; a significant decrease was seen at calcium concentrations of 1.25 and 2.5 mM but was only evident after a 16-24 h incubation period. Calcium and iron treatments decreased DMT-1 protein in Caco-2 cell membranes, although total DMT-1 in whole cell lysates was unchanged by either iron or calcium. No change was seen in ferroportin expression. Our data suggest that calcium reduces iron bioavailability by decreasing DMT-1 expression at the apical cell membrane, thereby downregulating iron transport into the cell.

Distinct lipid rafts in subdomains from human placental apical syncytiotrophoblast membranes.

PubMed

Godoy, Valeria; Riquelme, Gloria

2008-01-01

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

PubMed Central

Yu, Ling; Helms, My N.; Yue, Qiang; Eaton, Douglas C.

2008-01-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane. PMID:18784262

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.

PubMed

Yu, Ling; Helms, My N; Yue, Qiang; Eaton, Douglas C

2008-11-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

Localization of aPKC lambda/iota and its interacting protein, Lgl2, is significantly associated with lung adenocarcinoma progression.

PubMed

Imamura, Naoko; Horikoshi, Yosuke; Matsuzaki, Tomohiko; Toriumi, Kentaro; Kitatani, Kanae; Ogura, Go; Masuda, Ryota; Nakamura, Naoya; Takekoshi, Susumu; Iwazaki, Masayuki

2013-12-20

Atypical protein kinase C lambda/iota (aPKC λ/ι) is expressed in several human cancers; however, the correlation between aPKC λ/ι localization and cancer progression in human lung adenocarcinoma (LAC) remains to be clarified. We found that patients with a high level of aPKC λ/ι expression in LAC had significantly shorter overall survival than those with a low level of aPKC λ/ι expression. In addition, localization of aPKC λ/ι in the apical membrane or at the cell-cell contact was associated with both lymphatic invasion and metastasis. The intercellular adhesion molecule, E-cadherin, was decreased in LACs with highly expressed aPKC λ/ι at the invasion site of tumor cells. This result suggested that the expression levels of aPKC λ/ι and E-cadherin reflect the progression of LAC. On double-immunohistochemical analysis, aPKC λ/ι and Lgl2, a protein that interacts with aPKC λ/ι, were co-localized within LACs. Furthermore, we found that Lgl2 bound the aPKC λ/ι-Par6 complex in tumor tissue by immune-cosedimentation analysis. Apical membrane localization of Lgl2 was correlated with lymphatic invasion and lymph node metastasis. These results thus indicate that aPKC λ/ι expression is altered upon the progression of LAC. This is also the first evidence to show aPKC λ/ι overexpression in LAC and demonstrates that aPKC λ/ι localization at the apical membrane or cell-cell contact is associated with lymphatic invasion and metastasis of the tumor.

Constitutive apical membrane recycling in Aplysia enterocytes.

PubMed

Keeton, Robert Aaron; Runge, Steven William; Moran, William Michael

2004-11-01

In Aplysia californica enterocytes, alanine-stimulated Na+ absorption increases both apical membrane exocytosis and fractional capacitance (fCa; a measure of relative apical membrane surface area). These increases are thought to reduce membrane tension during periods of nutrient absorption that cause the enterocytes to swell osmotically. In the absence of alanine, exocytosis and fCa are constant. These findings imply equal rates of constitutive endocytosis and exocytosis and constitutive recycling of the apical plasma membrane. Thus, the purpose of this study was to confirm and determine the relative extent of constitutive apical membrane recycling in Aplysia enterocytes. Biotinylated lectins are commonly used to label plasma membranes and to investigate plasma membrane recycling. Of fourteen biotinylated lectins tested, biotinylated wheat germ agglutinin (bWGA) bound preferentially to the enterocytes apical surface. Therefore, we used bWGA, avidin D (which binds tightly to biotin), and the UV fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated avidin D to assess the extent of constitutive apical membrane recycling. A temperature-dependent (20 vs. 4 degrees C) experimental protocol employed the use of two tissues from each of five snails and resulted in a approximately 60% difference in apical surface fluorescence intensity. Because the extent of membrane recycling is proportional to the difference in surface fluorescence intensity, this difference reveals a relatively high rate of constitutive apical membrane recycling in Aplysia enterocytes.

Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line

PubMed Central

Brice, Nicola L; Dolphin, Annette C

1999-01-01

Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. We expressed the VDCC α1A, α1B or α1C subunits either alone or in combination with accessory subunits α2-δ and the different β subunits, and examined their localization immunocytochemically. An α1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. The combination α1C/α2-δ and all β subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. In contrast, the α1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. The α1A subunit was consistently observed at the apical membrane in the combinations α1A/α2-δ/β1b or β4. In contrast, when co-expressed with α2-δ/β2a, α1A was clearly targeted to the basolateral membrane. In conclusion, the VDCC α1 subunit appears to be the primary determinant for targeting the VDCC complex, but the β subunit can modify this destination, particularly for α1A. PMID:10066897

Expression of a novel isoform of Na+/H+ exchanger 3 in the kidney and intestine of banded houndshark, Triakis scyllium

PubMed Central

Li, Shanshan; Takabe, Souichirou; Chen, An-Ping; Romero, Michael F.; Umezawa, Takahiro; Nakada, Tsutomu; Hyodo, Susumu; Hirose, Shigehisa

2013-01-01

Na+/H+ exchanger 3 (NHE3) provides one of the major Na+ absorptive pathways of the intestine and kidney in mammals, and recent studies of aquatic vertebrates (teleosts and elasmobranchs) have demonstrated that NHE3 is expressed in the gill and plays important roles in ion and acid-base regulation. To understand the role of NHE3 in elasmobranch osmoregulatory organs, we analyzed renal and intestinal expressions and localizations of NHE3 in a marine elasmobranch, Japanese banded houndshark (Triakis scyllium). mRNA for Triakis NHE3 was most highly expressed in the gill, kidney, spiral intestine, and rectum. The kidney and intestine expressed a transcriptional isoform of NHE3 (NHE3k/i), which has a different amino terminus compared with that of NHE3 isolated from the gill (NHE3g), suggesting that NHE3k/i and NHE3g arise from a single gene by alternative promoter usage. Immunohistochemical analyses of the Triakis kidney demonstrated that NHE3k/i is expressed in the apical membrane of a part of the proximal and late distal tubules in the sinus zone. In the bundle zone of the kidney, NHE3k/i was expressed in the apical membrane of the early distal tubules known as the diluting segment. In the spiral intestine and rectum, NHE3k/i was localized toward the apical membrane of the epithelial cells. The transcriptional levels of NHE3k/i were increased in the kidney when Triakis was acclimated in 130% seawater, whereas those in the spiral intestine were increased in fish acclimated in diluted seawater. These results suggest that NHE3 is involved in renal Na+ reabsorption, urine acidification, and intestinal Na+ absorption in elasmobranchs. PMID:23485868

The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

PubMed

Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

2015-12-01

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. Copyright © 2015 the American Physiological Society.

Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth.

PubMed

Bronckers, Antonius L J J; Guo, Jing; Zandieh-Doulabi, Behrouz; Bervoets, Theodore J; Lyaruu, Donacian M; Li, Xiangming; Wangemann, Philine; DenBesten, Pamela

2011-12-01

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators. © 2011 Eur J Oral Sci.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar

USGS Publications Warehouse

Sundh, Henrik; Nilsen, Tom O.; Lindström, Jenny; Hasselberg-Frank, Linda; Stefansson, Sigurd O.; McCormick, Stephen D.; Sundell, K.

2014-01-01

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmonSalmo salar. Emphasis was placed on Na+, K+-ATPase (NKA) and Na+, K+, Cl− co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na+, Cl−co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Molecular Mechanisms of Antidiuretic Effect of Oxytocin

PubMed Central

Li, Chunling; Wang, Weidong; Summer, Sandra N.; Westfall, Timothy D.; Brooks, David P.; Falk, Sandor; Schrier, Robert W.

2008-01-01

Oxytocin is known to have an antidiuretic effect, but the mechanisms underlying this effect are not completely understood. We infused oxytocin by osmotic minipump into vasopressin-deficient Brattleboro rats for five days and observed marked antidiuresis, increased urine osmolality, and increased solute-free water reabsorption. Administration of oxytocin also significantly increased the protein levels of aquaporin-2 (AQP2), phosphorylated AQP2 (p-AQP2), and AQP3 in the inner medulla and in the outer medulla plus cortex. Immunohistochemistry demonstrated increased AQP2 and p-AQP2 expression and trafficking to the apical plasma membrane of principal cells in the collecting duct, and increased AQP3 expression in the basolateral membrane. These oxytocin-induced effects were blocked by treatment with the vasopressin V2 receptor antagonist SR121463B, but not by treatment with the oxytocin receptor antagonist GW796679X. We conclude that vasopressin V2 receptors mediate the antidiuretic effects of oxytocin, including increased expression and apical trafficking of AQP2, p-AQP2, and increased AQP3 protein expression. PMID:18057218

Apical targeting of the formin Diaphanous in Drosophila tubular epithelia

PubMed Central

Rousso, Tal; Shewan, Annette M; Mostov, Keith E; Schejter, Eyal D; Shilo, Ben-Zion

2013-01-01

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane. DOI: http://dx.doi.org/10.7554/eLife.00666.001 PMID:23853710

Plasma membrane localization of multidrug resistance-associated protein homologs in brain capillary endothelial cells.

PubMed

Zhang, Yan; Schuetz, John D; Elmquist, William F; Miller, Donald W

2004-11-01

Several multidrug resistance-associated protein (MRP) homologs are expressed in brain microvessel endothelial cells forming the blood-brain barrier (BBB). The influence of these MRP transporters on BBB permeability will be dependent on their localization within the brain microvessel endothelial cells. Using two different and complementary approaches, the localization of various MPR homologs (MRP1, MRP4, and MRP5) was examined in primary cultured bovine brain microvessel endothelial cells (BBMECs). The first approach involved centrifugal separation of apical and basolateral plasma membranes of cultured BBMECs. The membrane fractions were then subjected to Western blot analysis for MRPs. The second approach used confocal laser scanning microscopy to determine membrane localization of MRPs in BBMECs. Results show a predominantly apical plasma membrane distribution for MRP1 and MRP5, and an almost equal distribution of MRP4 on the apical and basolateral plasma membrane of BBMECs. These studies provide the first demonstration of the localization of MRP1, MRP4, and MRP5 homologs in brain microvessel endothelial cells. The present studies also indicate that the localization of MRPs in the endothelial cells forming the BBB is different from that observed in polarized epithelial cells and thus may contribute to the reduced entry and enhanced elimination of organic anions and nucleotides in the brain.

Differential Expression of Renal Outer Medullary K+ Channel and Voltage-gated K+ Channel 7.1 in Bladder Urothelium of Patients With Interstitial Cystitis/Painful Bladder Syndrome.

PubMed

Lee, Jane-Dar; Lee, Ming-Huei; Yang, Wen-Kai; Wang, Kuan-Lin; Lee, Tsung-Han

2017-03-01

To investigate the changes including expression and localization of 2 potassium channels, renal outer medullary K + channel (ROMK) and voltage-gated K + channel 7.1 (KCNQ1), after increased urinary potassium leakage in patients with interstitial cystitis/painful bladder syndrome (IC/PBS). The study group included 24 patients with IC/PBS and a control group consisting of 12 volunteers without any IC/PBS symptoms. Bladder biopsies were taken from both groups. We determined the protein expression and distribution of potassium channels using immunoblotting, immunohistochemistry, and immunofluorescent staining under confocal laser microscopy. The results revealed that ROMK was predominantly expressed in apical cells of the bladder urothelium at significantly higher levels (3.3-fold) in the study group than in the control group. In contrast, KCNQ1 was expressed in the basolateral membrane according to confocal microscopy results and did not significantly differ between groups. Our data showed that the abundance of ROMK protein in apical cells was increased in the IC/PBS group, whereas KCNQ1, which was distributed in the basolateral membrane of the bladder urothelium, showed similar abundance between groups. These results suggest that upregulation of the ROMK channel in apical cells might permit avid potassium flux into the bladder lumen to maintain intracellular K + homeostasis in the dysfunctional urothelium. Copyright © 2016 Elsevier Inc. All rights reserved.

Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis

PubMed Central

Luo, Jun; Wang, Heng; Kang, Di; Guo, Xuan; Wan, Ping; Wang, Dou; Chen, Jiong

2016-01-01

Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. Here we show that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, we demonstrate that the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components could rescue epithelial polarity defects resulted from loss of Dlg5. Furthermore, we performed a structure-function analysis of Dlg5 and found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5’s functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif could be individually targeted to the apical membrane, while the central linker region could be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK could be individually targeted to apical, AJ and basolateral membranes. PMID:27211898

Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium

PubMed Central

Cario, Elke; Brown, Dennis; McKee, Mary; Lynch-Devaney, Kathryn; Gerken, Guido; Podolsky, Daniel K.

2002-01-01

Commensal-associated molecular patterns, the major products of nonpathogenic bacteria, are present at high concentrations at the apical surface of the intestinal epithelium. However, the nature of the interaction of commensal-associated molecular patterns with the lumenal surface of the epithelium has not been defined. We have recently demonstrated that intestinal epithelial cells constitutively express several Toll-like receptors (TLRs) in vitro and in vivo that seem to be the key receptors responsible for immune cell activation in response to various bacterial products. In this study we characterize the subcellular distribution of two major TLRs, TLR2 and TLR4, and their ligand-specific dynamic regulation in the model human intestinal epithelial cell line T84. Immunocytochemical studies indicate that TLR2 and TLR4 are constitutively expressed at the apical pole of differentiated T84 cells. After stimulation with lipopolysaccharide or peptidoglycan, TLRs selectively traffic to cytoplasmic compartments near the basolateral membrane. Thus, we demonstrate that TLRs are positioned at the apical pole where they are poised to monitor the sensitive balance of the lumenal microbial array. The results of this dynamic epithelial surveillance can then be conveyed to the underlying cell populations of the lamina propria via these innate immune pattern recognition receptors. PMID:11786410

Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

PubMed Central

Attardo, Alessio; Calegari, Federico; Haubensak, Wulf; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2008-01-01

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors. PMID:18545663

The urothelium of a hibernator: the American black bear

PubMed Central

Spector, David A; Deng, Jie; Coleman, Richard; Wade, James B

2015-01-01

The American black bear undergoes a 3–5 month winter hibernation during which time bears do not eat, drink, defecate, or urinate. During hibernation renal function (GFR) is 16–50% of normal but urine is reabsorbed across the urinary bladder (UB) urothelium thus enabling metabolic recycling of all urinary constituents. To elucidate the mechanism(s) whereby urine is reabsorbed, we examined the UBs of five nonhibernating wild bears using light, electron (EM), and confocal immunofluorescent (IF) microscopy–concentrating on two components of the urothelial permeability barrier – the umbrella cell apical membranes and tight junctions (TJ). Bear UB has the same tissue layers (serosa, muscularis, lamina propria, urothelia) and its urothelia has the same cell layers (basal, intermediate, umbrella cells) as other mammalians. By EM, the bear apical membrane demonstrated a typical mammalian scalloped appearance with hinge and plaque regions – the latter containing an asymmetric trilaminar membrane and, on IF, uroplakins Ia, IIIa, and IIIb. The umbrella cell TJs appeared similar to those in other mammals and also contained TJ proteins occludin and claudin - 4, and not claudin –2. Thus, we were unable to demonstrate urothelial apical membrane or TJ differences between active black bears and other mammals. Expression and localization of UT-B, AQP-1 and -3, and Na+, K+-ATPase on bear urothelial membranes was similar to that of other mammals. Similar studies of urothelia of hibernating bears, including evaluation of the apical membrane lipid bilayer and GAGs layer are warranted to elucidate the mechanism(s) whereby hibernating bears reabsorb their daily urine output and thus ensure successful hibernation. PMID:26109187

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1.

PubMed

Nagamori, Shushi; Wiriyasermkul, Pattama; Guarch, Meritxell Espino; Okuyama, Hirohisa; Nakagomi, Saya; Tadagaki, Kenjiro; Nishinaka, Yumiko; Bodoy, Susanna; Takafuji, Kazuaki; Okuda, Suguru; Kurokawa, Junko; Ohgaki, Ryuichi; Nunes, Virginia; Palacín, Manuel; Kanai, Yoshikatsu

2016-01-19

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.

PubMed

Howard, M; Jiang, X; Stolz, D B; Hill, W G; Johnson, J A; Watkins, S C; Frizzell, R A; Bruton, C M; Robbins, P D; Weisz, O A

2000-08-01

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

PubMed

Hirt, B; Penkova, Z H; Eckhard, A; Liu, W; Rask-Andersen, H; Müller, M; Löwenheim, H

2010-07-28

Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Meniere's disease) or sensorineural hearing loss (i.e. in Sjögren's syndrome). Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Signaling-Dependent Control of Apical Membrane Size and Self-Renewal in Rosette-Stage Human Neuroepithelial Stem Cells.

PubMed

Medelnik, Jan-Philip; Roensch, Kathleen; Okawa, Satoshi; Del Sol, Antonio; Chara, Osvaldo; Mchedlishvili, Levan; Tanaka, Elly M

2018-06-05

In the developing nervous system, neural stem cells are polarized and maintain an apical domain facing a central lumen. The presence of apical membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling apical membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls apical membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and apical membrane components. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

PubMed

Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

2000-03-01

Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

Influenza A Virus Hemagglutinin and Neuraminidase Mutually Accelerate Their Apical Targeting through Clustering of Lipid Rafts

PubMed Central

Ohkura, Takashi; Momose, Fumitaka; Ichikawa, Reiko; Takeuchi, Kaoru

2014-01-01

ABSTRACT In polarized epithelial cells, influenza A virus hemagglutinin (HA) and neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical plasma membrane for viral assembly and budding. Previous studies have indicated that the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA are required for association with lipid rafts, but the raft dependencies of their apical targeting are controversial. Here, we show that coexpression of HA with NA accelerated their apical targeting through accumulation in lipid rafts. HA was targeted to the apical plasma membrane even when expressed alone, but the kinetics was much slower than that of HA in infected cells. Coexpression experiments revealed that apical targeting of HA and NA was accelerated by their coexpression. The apical targeting of HA was also accelerated by coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and the deletion of the CT in HA and NA that reduced their association with lipid rafts abolished the acceleration of their apical transport, indicating that the lipid raft association is essential for efficient apical trafficking of HA and NA. An in situ proximity ligation assay (PLA) revealed that HA and NA were accumulated and clustered in the cytoplasmic compartments only when both were associated with lipid rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings. We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism of the accelerated apical transport of HA and NA via clustering of lipid rafts. IMPORTANCE Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase (NA) of influenza virus, key players for particle assembly, contain determinants for apical sorting and lipid raft association. However, it remains to be elucidated how lipid rafts contribute to the apical trafficking and budding. We investigated the relation of lipid raft association of HA and NA to the efficiency of apical trafficking. We show that coexpression of HA and NA induces their accumulation in lipid rafts and accelerates their apical targeting, and we suggest that the accelerated apical transport likely occurs by clustering of lipid rafts at the TGN. This finding provides the first evidence that two different raft-associated viral proteins induce lipid raft clustering, thereby accelerating apical trafficking of the viral proteins. PMID:24965459

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

PubMed

Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

2018-01-01

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells*

PubMed Central

Striz, Anneliese C.; Tuma, Pamela L.

2016-01-01

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5′ nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface. PMID:26957544

Bicarbonate, NBCe1, NHE, and Carbonic Anhydrase Activity Enhance Lactate-H+ Transport in Bovine Corneal Endothelium

PubMed Central

Nguyen, Tracy T.

2011-01-01

Purpose. To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO3−, sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux. Methods. MCT1–4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pHi were measured in BCEC in HCO3−-free or HCO3−-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na+/H+ exchange blocker), disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells. Results. MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pHi measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpHi/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO3− but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO3−, was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA. Conclusions. MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H+ transport. Lactate dependent proton flux can activate Na+/H+ exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO3−, HCO3− transport, NHE and CA activity. PMID:21896839

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

PubMed Central

Ruch, Travis R.; Bryant, David M.; Mostov, Keith E.; Engel, Joanne N.

2017-01-01

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens. PMID:27881661

Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium.

PubMed

Joseph, D P; Miller, S S

1991-04-01

1. Intracellular voltage recordings using conventional and double-barrelled chloride-selective microelectrodes have been used to identify several transport mechanisms at the apical and basolateral membranes of the isolated bovine retinal pigment epithelium (RPE)-choroid preparation. Intracellular recordings were obtained from two cell populations, melanotic (pigmented) and amelanotic (non-pigmented). The electrical properties of these two populations are practically identical. For melanotic cells the average apical resting membrane potential (VA) is -61 +/- 2 mV (mean +/- S.E.M., n = 49 cells, thirty-three eyes). For these cells the ratio of apical to basolateral membrane resistance (a) was 0.22 +/- 0.02. The mean transepithelial voltage and resistance were 6 +/- 1 mV and 138 +/- 7 omega cm2, respectively. 2. The apical membrane, which faces the distal retina, contains a Ba(2+)-inhibitable K+ conductance and a ouabain-inhibitable, electrogenic Na(+)-K+ pump. In addition it contains a bumetanide-sensitive mechanism, the putative Na(+)-K(+)-Cl- cotransporter. The basolateral membrane contains a DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid)-inhibitable chloride channel. The relative conductances of the apical and basolateral membranes to K+ and Cl- are TK approximately 0.9 and TCl approximately 0.7, respectively. 3. The ouabain-induced fast phase of apical membrane depolarization (0-30 s) was used to calculate the equivalent resistances of the apical (RA) and basolateral (RB) cell membranes, as well as the paracellular or shunt resistance (RS). They are: 3190 +/- 400, 17920 +/- 2730 and 2550 +/- 200 omega (mean +/- S.E.M., n = 9 tissues), respectively. From these data the equivalent electromotive forces (EMF) at the apical (EA) and basolateral (EB) membranes were also calculated. They are: -69 +/- 5.0 and -24 +/- 5.0 mV, respectively. 4. Intracellular Cl- activity (aiCl) was measured using double-barreled ion-selective microelectrodes. In the steady state aiCl = 61 +/- 4.0 mM and the Nernst potential ECl = -13.5 +/- 1.5 mV (mean +/- S.E.M., n = 4). 5. In the intact eye or in retina, RPE-choroid preparations it has been shown that the transition between light and dark alters the K+ concentration in the extracellular (or subretinal) space between the photoreceptors and the apical membrane of the RPE. These light-induced changes in subretinal [K+]o were qualitatively simulated in vitro by altering apical K+ between 5 and 2 mM. This produced a sequence of voltage changes at the apical and basolateral membranes that had three operationally distinct phases. Phase 1 is generated by the combination of an apical membrane K+ diffusion potential and inhibition of the electrogenic Na(+)-K+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

PubMed Central

Zihni, Ceniz; Munro, Peter M.G.; Elbediwy, Ahmed; Keep, Nicholas H.; Terry, Stephen J.; Harris, John

2014-01-01

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation. PMID:24379416

The barley MATE gene, HvAACT1, increases citrate efflux and Al(3+) tolerance when expressed in wheat and barley.

PubMed

Zhou, Gaofeng; Delhaize, Emmanuel; Zhou, Meixue; Ryan, Peter R

2013-08-01

Aluminium is toxic in acid soils because the soluble Al(3+) inhibits root growth. A mechanism of Al(3+) tolerance discovered in many plant species involves the release of organic anions from root apices. The Al(3+)-activated release of citrate from the root apices of Al(3+)-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al(3+) tolerance of these important cereal species. HvAACT1 was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al(3+) tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil. Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al(3+) tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al(3+) tolerance of important crop plants.

Role of the BK channel (KCa1.1) during activation of electrogenic K+ secretion in guinea pig distal colon

PubMed Central

Zhang, Jin; Halm, Susan T.

2012-01-01

Secretagogues acting at a variety of receptor types activate electrogenic K+ secretion in guinea pig distal colon, often accompanied by Cl− secretion. Distinct blockers of KCa1.1 (BK, Kcnma1), iberiotoxin (IbTx), and paxilline inhibited the negative short-circuit current (Isc) associated with K+ secretion. Mucosal addition of IbTx inhibited epinephrine-activated Isc (epiIsc) and transepithelial conductance (epiGt) consistent with K+ secretion occurring via apical membrane KCa1.1. The concentration dependence of IbTx inhibition of epiIsc yielded an IC50 of 193 nM, with a maximal inhibition of 51%. Similarly, IbTx inhibited epiGt with an IC50 of 220 nM and maximal inhibition of 48%. Mucosally added paxilline (10 μM) inhibited epiIsc and epiGt by ∼50%. IbTx and paxilline also inhibited Isc activated by mucosal ATP, supporting apical KCa1.1 as a requirement for this K+ secretagogue. Responses to IbTx and paxilline indicated that a component of K+ secretion occurred during activation of Cl− secretion by prostaglandin-E2 and cholinergic stimulation. Analysis of KCa1.1α mRNA expression in distal colonic epithelial cells indicated the presence of the ZERO splice variant and three splice variants for the COOH terminus. The presence of the regulatory β-subunits KCaβ1 and KCaβ4 also was demonstrated. Immunolocalization supported the presence of KCa1.1α in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K+ secretion involving apical membrane KCa1.1 during activation by several secretagogue types, but the observed K+ secretion likely required the activity of additional K+ channel types in the apical membrane. PMID:23064759

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

PubMed Central

Bravo-Zehnder, Marcela; Orio, Patricio; Norambuena, Andrés; Wallner, Martin; Meera, Pratap; Toro, Ligia; Latorre, Ramón; González, Alfonso

2000-01-01

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells. PMID:11069304

Debris buster is a Drosophila scavenger receptor essential for airway physiology.

PubMed

Wingen, Almut; Carrera, Pilar; Ekaterini Psathaki, Olympia; Voelzmann, André; Paululat, Achim; Hoch, Michael

2017-10-01

Scavenger receptors class B (SR-B) are multifunctional transmembrane proteins, which in vertebrates participate in lipid transport, pathogen clearance, lysosomal delivery and intracellular sorting. Drosophila has 14 SR-B members whose functions are still largely unknown. Here, we reveal a novel role for the SR-B family member Debris buster (Dsb) in Drosophila airway physiology. Larvae lacking dsb show yeast avoidance behavior, hypoxia, and severe growth defects associated with impaired elongation and integrity along the airways. Furthermore, in dsb mutant embryos, the barrier function of the posterior spiracles, which are critical for gas exchange, is not properly established and liquid clearance is locally impaired at the spiracular lumen. We found that Dsb is specifically expressed in a group of distal epithelial cells of the posterior spiracle organ and not throughout the entire airways. Furthermore, tissue-specific knockdown and rescue experiments demonstrate that Dsb function in the airways is only required in the posterior spiracles. Dsb localizes in intracellular vesicles, and a subset of these associate with lysosomes. However, we found that depletion of proteins involved in vesicular transport to the apical membrane, but not in lysosomal function, causes dsb-like airway elongation defects. We propose a model in which Dsb sorts components of the apical extracellular matrix which are essential for airway physiology. Since SR-B LIMP2-deficient mice show reduced expression of several apical plasma membrane proteins, sorting of proteins to the apical membrane is likely an evolutionary conserved function of Dsb and LIMP2. Our data provide insights into a spatially confined function of the SR-B Dsb in intracellular trafficking critical for the physiology of the whole tubular airway network. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters*

PubMed Central

Giral, Hector; Lanzano, Luca; Caldas, Yupanqui; Blaine, Judith; Verlander, Jill W.; Lei, Tim; Gratton, Enrico; Levi, Moshe

2011-01-01

The sodium-dependent phosphate (Na/Pi) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of Pi. The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in Pi reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/Pi transporters. Pdzk1−/− mice adapted to chronic low Pi diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/Pi transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/Pi transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low Pi concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/Pi transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c. PMID:21388960

Isolation of plasma membrane fractions from the intestinal epithelial model T84.

PubMed

Kaoutzani, P; Parkos, C A; Delp-Archer, C; Madara, J L

1993-05-01

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

NHE8 plays important roles in gastric mucosal protection

PubMed Central

Xu, Hua; Li, Jing; Chen, Huacong; Wang, Chunhui

2013-01-01

Sodium/hydrogen exchanger (NHE) 8 is an apically expressed membrane protein in the intestinal epithelial cells. It plays important roles in sodium absorption and bicarbonate secretion in the intestine. Although NHE8 mRNA has been detected in the stomach, the precise location and physiological role of NHE8 in the gastric glands remain unclear. In the current study, we successfully detected the expression of NHE8 in the glandular region of the stomach by Western blotting and located NHE8 protein at the apical membrane in the surface mucous cells by a confocal microscopic method. We also identified the expression of downregulated-in-adenoma (DRA) in the surface mucous cells in the stomach. Using NHE8−/− mice, we found that NHE8 plays little or no role in basal gastric acid production, yet NHE8−/− mice have reduced gastric mucosal surface pH and higher incidence of developing gastric ulcer. DRA expression was reduced significantly in the stomach in NHE8−/− mice. The propensity for gastric ulcer, reduced mucosal surface pH, and low DRA expression suggest that NHE8 is indirectly involved in gastric bicarbonate secretion and gastric mucosal protection. PMID:23220221

Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells.

PubMed

Procino, G; Barbieri, C; Carmosino, M; Rizzo, F; Valenti, G; Svelto, M

2010-02-01

Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.

Role of the Intestinal Bile Acid Transporters in Bile Acid and Drug Disposition

PubMed Central

Dawson, Paul A.

2011-01-01

Membrane transporters expressed by the hepatocyte and enterocyte play critical roles in maintaining the enterohepatic circulation of bile acids, an effective recycling and conservation mechanism that largely restricts these potentially cytotoxic detergents to the intestinal and hepatobiliary compartments. In doing so, the hepatic and enterocyte transport systems ensure a continuous supply of bile acids to be used repeatedly during the digestion of multiple meals throughout the day. Absorption of bile acids from the intestinal lumen and export into the portal circulation is mediated by a series of transporters expressed on the enterocyte apical and basolateral membranes. The ileal apical sodium-dependent bile acid cotransporter (abbreviated ASBT; gene symbol, SLC10A2) is responsible for the initial uptake of bile acids across the enterocyte brush border membrane. The bile acids are then efficiently shuttled across the cell and exported across the basolateral membrane by the heteromeric Organic Solute Transporter, OSTα-OSTβ. This chapter briefly reviews the tissue expression, physiology, genetics, pathophysiology, and transport properties of the ASBT and OSTα-OSTα. In addition, the chapter discusses the relationship between the intestinal bile acid transporters and drug metabolism, including development of ASBT inhibitors as novel hypocholesterolemic or hepatoprotective agents, prodrug targeting of the ASBT to increase oral bioavailability, and involvement of the intestinal bile acid transporters in drug absorption and drug-drug interactions. PMID:21103970

Balance between apical membrane growth and luminal matrix resistance determines epithelial tubule shape.

PubMed

Dong, Bo; Hannezo, Edouard; Hayashi, Shigeo

2014-05-22

The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cong, Yingying; Li, Xiaoxue; Bai, Yunyun

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus releasedmore » into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.« less

Trafficking Ion Transporters to the Apical Membrane of Polarized Intestinal Enterocytes.

PubMed

Engevik, Amy Christine; Goldenring, James R

2018-01-02

Epithelial cells lining the gastrointestinal tract require distinct apical and basolateral domains to function properly. Trafficking and insertion of enzymes and transporters into the apical brush border of intestinal epithelial cells is essential for effective digestion and absorption of nutrients. Specific critical ion transporters are delivered to the apical brush border to facilitate fluid and electrolyte uptake. Maintenance of these apical transporters requires both targeted delivery and regulated membrane recycling. Examination of altered apical trafficking in patients with Microvillus Inclusion disease caused by inactivating mutations in MYO5B has led to insights into the regulation of apical trafficking by elements of the apical recycling system. Modeling of MYO5B loss in cell culture and animal models has led to recognition of Rab11a and Rab8a as critical regulators of apical brush border function. All of these studies show the importance of apical membrane trafficking dynamics in maintenance of polarized epithelial cell function. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

PubMed

Dang, Viet D; Jella, Kishore Kumar; Ragheb, Ragy R T; Denslow, Nancy D; Alli, Abdel A

2017-12-01

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells. © FASEB.

Regulation of Dab2 expression in intestinal and renal epithelia by development.

PubMed

Vázquez-Carretero, María D; García-Miranda, Pablo; Calonge, María L; Peral, María J; Ilundáin, Anunciación A

2011-01-01

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

PubMed Central

Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

2016-01-01

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

PubMed Central

Walsh, Dustin R.; Nolin, Thomas D.

2015-01-01

Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

The barley MATE gene, HvAACT1, increases citrate efflux and Al3+ tolerance when expressed in wheat and barley

PubMed Central

Zhou, Gaofeng; Delhaize, Emmanuel; Zhou, Meixue; Ryan, Peter R.

2013-01-01

Background and Aims Aluminium is toxic in acid soils because the soluble Al3+ inhibits root growth. A mechanism of Al3+ tolerance discovered in many plant species involves the release of organic anions from root apices. The Al3+-activated release of citrate from the root apices of Al3+-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al3+ tolerance of these important cereal species. Methods HvAACT1 was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al3+ tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil. Key Results and Conclusions Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al3+ tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al3+ tolerance of important crop plants. PMID:23798600

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

PubMed

Mitsuoka, Keisuke; Shirasaka, Yoshiyuki; Fukushi, Akimasa; Sato, Masanobu; Nakamura, Toshimichi; Nakanishi, Takeo; Tamai, Ikumi

2009-04-01

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 B(0)AT1 and b(0,+)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 B(0)AT1 and b(0,+)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.

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions

PubMed Central

Ip, Yuen K.; Hiong, Kum C.; Goh, Enan J. K.; Boo, Mel V.; Choo, Celine Y. L.; Ching, Biyun; Wong, Wai P.; Chew, Shit F.

2017-01-01

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam. PMID:29066980

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions.

PubMed

Ip, Yuen K; Hiong, Kum C; Goh, Enan J K; Boo, Mel V; Choo, Celine Y L; Ching, Biyun; Wong, Wai P; Chew, Shit F

2017-01-01

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca 2+ to, and simultaneously an augmented removal of H + from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca 2+ -ATPase ( PMCA ) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa . The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca 2+ in exchange for H + . More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA /PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane.

PubMed

Röper, K; Corbeil, D; Huttner, W B

2000-09-01

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

PubMed

Shimura, M; Yuan, Y; Chang, J T; Zhang, S; Campochiaro, P A; Zack, D J; Hughes, B A

2001-03-01

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) > K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) > K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

PubMed Central

Hu, Ping; Lacruz, Rodrigo S.; Smith, Charles E.; Smith, Susan M.; Kurtz, Ira; Paine, Michael L.

2012-01-01

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na+/Ca2+) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na+/Ca2+-K+ ) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca2+ requirements vastly increase but exactly how Ca2+ traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca2+ membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1–6). NCKX are bidirectional electrogenic transporters regulating Ca2+ transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca2+ transport during amelogenesis. PMID:22677781

Serine 269 phosphorylated aquaporin-2 is targeted to the apical membrane of collecting duct principal cells

PubMed Central

Moeller, Hanne B.; Knepper, Mark A.; Fenton, Robert A.

2012-01-01

Trafficking of the water channel aquaporin-2 to the apical plasma membrane of the collecting duct is mediated by arginine vasopressin, rendering the cell permeable to water. We recently identified a novel form of aquaporin-2 that is phosphorylated at serine-269 (pS269-AQP2). Using antibodies specific for this form of the water channel, we detected rat and mouse pS269-AQP2 in the connecting tubule and throughout the collecting duct system. Using confocal immunofluorescence microscopy with organelle-specific markers and immunogold electron microscopy, we found that pS269-AQP2 was found only on the apical plasma membrane of principal cells. In vasopressin-deficient Brattleboro rats, pS269-AQP2 was undetectable but dramatically increased in abundance after these rats were treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP). This increase occurred only at the apical plasma membrane, even after long-term dDAVP treatment. Following dDAVP there was a time-dependent redistribution of total aquaporin-2 from predominantly intracellular vesicles to the apical plasma membrane, clathrin-coated vesicles, early endosomal compartments, and lysosomes. However, pS269-AQP2 was found only on the apical plasma membrane at any time. Our results show that S269 phosphorylated aquaporin-2 is exclusively associated with the apical plasma membrane, where it escapes endocytosis to remain at the cell surface. PMID:18843259

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments.

PubMed

Stoops, Emily H; Hull, Michael; Caplan, Michael J

2016-12-01

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans-Golgi network is a well-established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post-endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post-endocytic pools of this protein are subjected to distinct sorting processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

PubMed Central

1987-01-01

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates. PMID:3654750

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

PubMed

Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

2011-01-01

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

PubMed

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

PubMed Central

Moyer, Bryan D.; Denton, Jerod; Karlson, Katherine H.; Reynolds, Donna; Wang, Shusheng; Mickle, John E.; Milewski, Michal; Cutting, Garry R.; Guggino, William B.; Li, Min; Stanton, Bruce A.

1999-01-01

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells. J. Clin. Invest. 104:1353–1361 (1999). PMID:10562297

Gene expression and localization of two types of AQP5 in Xenopus tropicalis under hydration and dehydration.

PubMed

Shibata, Yuki; Sano, Takahiro; Tsuchiya, Nobuhito; Okada, Reiko; Mochida, Hiroshi; Tanaka, Shigeyasu; Suzuki, Masakazu

2014-07-01

Two types of aquaporin 5 (AQP5) genes (aqp-xt5a and aqp-xt5b) were identified in the genome of Xenopus tropicalis by synteny comparison and molecular phylogenetic analysis. When the frogs were in water, AQP-xt5a mRNA was expressed in the skin and urinary bladder. The expression of AQP-xt5a mRNA was significantly increased in dehydrated frogs. AQP-xt5b mRNA was also detected in the skin and increased in response to dehydration. Additionally, AQP-xt5b mRNA began to be slightly expressed in the lung and stomach after dehydration. For the pelvic skin of hydrated frogs, immunofluorescence staining localized AQP-xt5a and AQP-xt5b to the cytoplasm of secretory cells of the granular glands and the apical plasma membrane of secretory cells of the small granular glands, respectively. After dehydration, the locations of both AQPs in their respective glands did not change, but AQP-xt5a was visualized in the cytoplasm of secretory cells of the small granular glands. For the urinary bladder, AQP-xt5a was observed in the apical plasma membrane and cytoplasm of a number of granular cells under normal hydration. After dehydration, AQP-xt5a was found in the apical membrane and cytoplasm of most granular cells. Injection of vasotocin into hydrated frogs did not induce these changes in the localization of AQP-xt5a in the small granular glands and urinary bladder, however. The results suggest that AQP-xt5a might be involved in water reabsorption from the urinary bladder during dehydration, whereas AQP-xt5b might play a role in water secretion from the small granular gland. Copyright © 2014 the American Physiological Society.

Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium.

PubMed

Wade, James B; Fang, Liang; Coleman, Richard A; Liu, Jie; Grimm, P Richard; Wang, Tong; Welling, Paul A

2011-06-01

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.

The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

USDA-ARS?s Scientific Manuscript database

Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II

PubMed Central

Wang, Lin; Soroka, Carol J.; Boyer, James L.

2002-01-01

PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II–associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep. PMID:12370274

Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

PubMed Central

Butterworth, Michael B.; Edinger, Robert S.; Johnson, John P.; Frizzell, Raymond A.

2005-01-01

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation. PMID:15623897

Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.

PubMed

Butterworth, Michael B; Edinger, Robert S; Johnson, John P; Frizzell, Raymond A

2005-01-01

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na(+) transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, (MPK)CCD(14)) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (I(SC)), membrane capacitance (C(T)), and conductance (G(T)) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 +/- 6% (n = 31) increase in I(SC) and a 15.3 +/- 1.5% (n = 15) increase in C(T). Selective membrane permeabilization demonstrated that the C(T) increase was due to an increase in apical membrane capacitance. I(SC) and C(T) declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (approximately 1 h each cycle) resulted in response fatigue; DeltaI(SC) decreased approximately 10% per stimulation-recovery cycle. When channel production was blocked by cycloheximide, DeltaI(SC) decreased approximately 15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated I(SC), and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the I(SC) responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins

PubMed Central

Bernadskaya, Yelena Y.; Patel, Falshruti B.; Hsu, Hsiao-Ting; Soto, Martha C.

2011-01-01

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin–radixin–moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes. PMID:21697505

Transmembrane mucins as novel therapeutic targets.

PubMed

Constantinou, Pamela E; Danysh, Brian P; Dharmaraj, Neeraja; Carson, Daniel D

2011-11-01

Membrane-tethered mucin glycoproteins are abundantly expressed at the apical surfaces of simple epithelia, where they play important roles in lubricating and protecting tissues from pathogens and enzymatic attack. Notable examples of these mucins are MUC1, MUC4 and MUC16 (also known as cancer antigen 125). In adenocarcinomas, apical mucin restriction is lost and overall expression is often highly increased. High-level mucin expression protects tumors from killing by the host immune system, as well as by chemotherapeutic agents, and affords protection from apoptosis. Mucin expression can increase as the result of gene duplication and/or in response to hormones, cytokines and growth factors prevalent in the tumor milieu. Rises in the normally low levels of mucin fragments in serum have been used as markers of disease, such as tumor burden, for many years. Currently, several approaches are being examined that target mucins for immunization or nanomedicine using mucin-specific antibodies.

Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.

PubMed

Castillon, Guillaume Alain; Michon, Laetitia; Watanabe, Reika

2013-06-01

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

Changes in membrane conductances and areas associated with bicarbonate secretion in turtle bladder.

PubMed

Rich, A; Dixon, T E; Clausen, C

1990-02-01

Transepithelial impedance-analysis studies were performed in turtle bladder epithelium in order to measure changes in the different epithelial membranes resulting from stimulation of electrogenic bicarbonate secretion. Changes in membrane conductance relate to changes in ionic permeability, whereas changes in membrane capacitance relate to changes in membrane area, since most biological membranes exhibit a specific capacitance of approximately 1 muF/cm2. The results of this investigation are summarized as follows: (i) cAMP and carbachol, agents which have been shown previously to stimulate electrogenic bicarbonate secretion, result in increases in apical-membrane conductance and capacitance; (ii) these changes occur concomitantly with the observed change in transport (measured using the short-circuit-current technique), thereby suggesting that bicarbonate secretion may be regulated in part by changes in the chloride conductance of the apical membrane; (iii) the increase in conductance does not reflect an increase in the membrane's specific conductance, thereby indicating that it results from the addition of membrane possessing similar ionic permeability as the existing apical membrane; (iv) the magnitude of the changes in capacitance indicate that a minor cell population (beta-type carbonic-anhydrase-rich cells) increase their apical-membrane area by several-fold; (v) a lack of transport-associated changes in the basolateral-membrane parameters suggest that transport is not regulated by alterations in basolateral-membrane ionic conductance or area; (vi) a lack of colchicine sensitivity, coupled with the magnitude of the changes in apical-membrane capacitance, indicate that the membrane remodeling processes are different from those involved in the regulation of proton secretion in a different cell population (alpha-type carbonic-anhydrase-rich cells).

Hyaluronan and Stone Disease

NASA Astrophysics Data System (ADS)

Asselman, Marino

2008-09-01

Kidney stones cannot be formed as long as crystals are passed in the urine. However, when crystals are retained it becomes possible for them to aggregate and form a stone. Crystals are expected to be formed not earlier than the distal tubules and collecting ducts. Studies both in vitro and in vivo demonstrate that calcium oxalate monohydrate crystals do not adhere to intact distal epithelium, but only when the epithelium is proliferating or regenerating, so that it possesses dedifferentiated cells expressing hyaluronan, osteopontin (OPN) and their mutual receptor CD44 at the apical cell membrane. The polysaccharide hyaluronan is an excellent crystal binding molecule because of its negative ionic charge. We hypothesized that the risk for crystal retention in the human kidney would be increased when tubular cells express hyaluronan at their apical cell membrane. Two different patient categories in which nephrocalcinosis frequently occurs were studied to test this hypothesis (preterm neonates and kidney transplant patients). Hyaluronan (and OPN) expression at the luminal membrane of tubular cells indeed was observed, which preceded subsequent retention of crystals in the distal tubules. Tubular nephrocalcinosis has been reported to be associated with decline of renal function and thus further studies to extend our knowledge of the mechanisms of retention and accumulation of crystals in the kidney are warranted. Ultimately, this may allow the design of new strategies for the prevention and treatment of both nephrocalcinosis and nephrolithiasis in patients.

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harvey, B.; Lacoste, I.; Ehrenfeld, J.

1991-04-01

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride, indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes ofmore » principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+).« less

Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

PubMed Central

Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

2012-01-01

SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

PGE(2) activation of apical membrane Cl(-) channels in A6 epithelia: impedance analysis.

PubMed Central

Păunescu, T G; Helman, S I

2001-01-01

Measurements of transepithelial electrical impedance of continuously short-circuited A6 epithelia were made at audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E(2) (PGE(2)) modulates Cl(-) transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral membrane resistances were determined by nonlinear curve-fitting of the impedance vectors at relatively low frequencies (<50 Hz) to equations (Păunescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838--851) where depressed Nyquist impedance semicircles were characteristic of the membrane impedances under control Na(+)-transporting and amiloride-inhibited conditions. In all tissues (control, amiloride-blocked, and amiloride-blocked and furosemide-pretreated), PGE(2) caused relatively small (< approximately 3 microA/cm(2)) and rapid (<60 s) maximal increase of chloride current due to activation of a rather large increase of apical membrane conductance that preceded significant activation of Na(+) transport through amiloride-sensitive epithelial Na(+) channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion whose relaxation frequency was near 150 Hz. Analysis of the time-dependent changes of the complex frequency-dependent equivalent capacitance of the cells at frequencies >1.5 kHz revealed that the mean 9.8% increase of capacitance caused by PGE(2) was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical membrane Na(+) transport. PMID:11463630

Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

PubMed Central

Youker, Robert T.; Bruns, Jennifer R.; Costa, Simone A.; Rbaibi, Youssef; Lanni, Frederick; Kashlan, Ossama B.; Teng, Haibing; Weisz, Ora A.

2013-01-01

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery. PMID:23637462

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney.

PubMed

Guggino, W B; Oberleithner, H; Giebisch, G

1985-07-01

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

PubMed Central

1985-01-01

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway. PMID:2411847

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

PubMed Central

Smith, Laura; Litman, Paul; Kohli, Ekta; Amick, Joseph; Page, Richard C.; Misra, Saurav

2013-01-01

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+ exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization. PMID:23636454

Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

PubMed Central

Lipardi, Concetta; Mora, Rosalia; Colomer, Veronica; Paladino, Simona; Nitsch, Lucio; Rodriguez-Boulan, Enrique; Zurzolo, Chiara

1998-01-01

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes. PMID:9456321

Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes.

PubMed

Pelaseyed, Thaher; Gustafsson, Jenny K; Gustafsson, Ida J; Ermund, Anna; Hansson, Gunnar C

2013-08-15

We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the enterocyte apical glycocalyx. Carbachol (CCh) stimulation of the small intestine results in gel-forming mucin secretion from goblet cells, something that requires adjacent enterocytes to secrete chloride and bicarbonate for proper mucin formation. Surface labeling and confocal imaging demonstrated that apically expressed MUC17 in Caco-2 cells and Muc3(17) in murine enterocytes were endocytosed upon stimulation with CCh. Relocation of MUC17 in response to CCh was specific as MUC3 and MUC12 did not relocate following CCh stimulation. MUC17 colocalized with PDZK1 under basal conditions, while MUC17 relocated to the terminal web and into early endosomes after CCh stimulation. CCh stimulation concomitantly internalized the Na(+/)H(+) exchanger 3 (NHE3) and recruited cystic fibrosis transmembrane conductance regulator (CFTR) to the apical membranes, a process that was important for CFTR-mediated bicarbonate secretion necessary for proper gel-forming mucin unfolding. The reason for the specific internalization of MUC17 is not understood, but it could limit the diffusion barrier for ion secretion caused by the apical enterocyte glycocalyx or alternatively act to sample luminal bacteria. Our results reveal well-orchestrated mucus secretion and trafficking of ion channels and the MUC17 mucin.

Effects of cevimeline on the immunolocalization of aquaporin-5 and the ultrastructure of salivary glands in Sjögren's syndrome model mice.

PubMed

Nishimura, Hidehiko; Yakeishi, Akira; Saga, Tsuyoshi; Yamaki, Koh-Ichi

2009-01-01

Sjögren's syndrome (SS) is an autoimmune disorder whose main symptoms include xerostomia and dry eyes. It has been demonstrated that abnormal expression of aquaporin (AQP)-5 in the parotid and submandibular glands in SS model mice was corrected by cevimeline. In the present study, we orally administered cevimeline hydrochloride (cevimeline) to female MRL/l mice, which are widely used as a model for SS, to immunohistochemically investigate the localization of AQP-5 in the salivary glands. We also assessed the ultrastructure of acinar cells in the submandibular glands. AQP-5 was expressed in the apical and lateral cell membranes of acinar cells in the parotid and submandibular glands of normal mice, but not in the sublingual glands. In contrast, AQP-5 was expressed not only in the cell membranes in the apical domains but also in the cytoplasm in the SS model mice, indicating that the localization of AQP-5 was disordered in the SS model mice. After administration of cevimeline, AQP-5 was predominantly localized in the cellular apical domains of the acinar cells. Electron microscopy revealed that administration of cevimeline to the SS model mice and normal mice markedly reduced the number of secretory granules, increased the area of the rough endoplasmic reticulum, and expanded the intercellular gaps in the cells of the submandibular acini. Condensed vacuoles were also observed in the Golgi apparatuses, indicating that secondary enhancement of secretion and production of saliva had occurred. Consequently, the results of the present study demonstrate that the administration of cevimeline to the SS model mice increased salivary secretion in the submandibular glands. Furthermore, cevimeline transiently normalized the localization of AQP-5 expression in the parotid and submandibular glands.

Expression Analysis of the Transmembrane Mucin MUC20 in Human Corneal and Conjunctival Epithelia

PubMed Central

Woodward, Ashley M.; Argüeso, Pablo

2014-01-01

Purpose. Cell surface mucins are a group of highly O-glycosylated transmembrane glycoproteins responsible for the protection of epithelial cells on mucosal surfaces. The aim of this study was to investigate the localization and regulation of mucin 20 (MUC20) at the ocular surface. Methods. Localization of MUC20 in human corneal and conjunctival epithelia was evaluated by immunofluorescence microscopy. Immortalized corneal (HCLE) and conjunctival (HCjE) cell lines were grown at different stages of differentiation and subjected to quantitative PCR and Western blot analyses. Cell surface proteins on apical cell membranes were biotinylated and isolated by neutravidin chromatography. Results. The MUC20 was detected throughout the entire human ocular surface epithelia, predominantly in cell membranes within intermediate cell layers. In conjunctiva, MUC20 also was observed in the cytoplasm of apical cells within the stratified squamous epithelium, but not in goblet cells. Quantitative PCR and immunoblotting demonstrated expression of MUC20 in HCLE and HCjE cells. Induction of differentiation with serum-containing medium resulted in upregulation of MUC20 mRNA and protein. Biotin labeling of the surface of stratified cultures revealed low levels of MUC20 protein on apical glycocalyces. Further, MUC20 was not detected in the cell culture media or in human tears, suggesting that the extracellular domain of MUC20 is not released from the ocular surface as described previously for other cell surface mucins. Conclusions. Our results indicate that MUC20 is a novel transmembrane mucin expressed by the human corneal and conjunctival epithelia, and suggest that differential expression of MUC20 during differentiation has a role in maintaining ocular surface homeostasis. PMID:25168902

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

PubMed

Krey, Jocelyn F; Dumont, Rachel A; Wilmarth, Philip A; David, Larry L; Johnson, Kenneth R; Barr-Gillespie, Peter G

2018-01-24

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout ( rda )]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle. SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia. Copyright © 2018 the authors 0270-6474/18/380843-15$15.00/0.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia.

PubMed

Blaug, Sasha; Rymer, Jodi; Jalickee, Stephen; Miller, Sheldon S

2003-04-01

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

PubMed Central

Dubreuil, Ronald R.; Maddux, Pratumtip Boontrakulpoontawee; Grushko, Tanya A.; Macvicar, Gary R.

1997-01-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells. PMID:9348534

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

PubMed

Dubreuil, R R; Maddux, P B; Grushko, T A; MacVicar, G R

1997-10-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul

2010-04-15

N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mousemore » proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.« less

Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia.

PubMed

Khanal, Ichha; Elbediwy, Ahmed; Diaz de la Loza, Maria Del Carmen; Fletcher, Georgina C; Thompson, Barry J

2016-07-01

In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli. © 2016. Published by The Company of Biologists Ltd.

Purinergic P2Y receptors in airway epithelia: from ion transport to immune functions.

PubMed

Hao, Yuan; Ko, Wing-hung

2014-02-25

The regulated transport of salt and water is essential to the integrated function of many organ systems, including the respiratory, reproductive, and digestive tracts. Airway epithelial fluid secretion is a passive process that is driven by osmotic forces, which are generated by ion transport. The main determinant of a luminally-directed osmotic gradient is the mucosal transport of chloride ions (Cl(-)) into the lumen. As with many epithelial cells, a number of classic signal transduction cascades are involved in the regulation of ion transport. There are two well-known intracellular signaling systems: an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) and an increase in the rate of synthesis of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP). Therefore, Cl(-) secretion is primarily activated via the opening of apical Ca(2+)- or cAMP-dependent Cl(-) channels at the apical membrane. The opening of basolateral Ca(2+)- or cAMP-activated K(+) channels, which hyperpolarizes the cell to maintain the driving force for Cl(-) exit through apical Cl(-) channels that are constitutively open, is also important in regulating transepithelial ion transport. P2Y receptors are expressed in the apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Human airway epithelial cells express multiple nucleotide receptors. Extracellular nucleotides, such as UTP and ATP, are calcium-mobilizing secretagogues. They are released into the extracellular space from airway epithelial cells and act on the same cell in an autocrine fashion to stimulate transepithelial ion transport. In addition, recent data support the role of P2Y receptors in releasing inflammatory cytokines in the bronchial epithelium and other immune cells.

Role of NH3 and NH4+ transporters in renal acid-base transport.

PubMed

Weiner, I David; Verlander, Jill W

2011-01-01

Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.

Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix

PubMed Central

Ayala-Figueroa, Jesus; Parry, Jean M.; Pu, Pu; Hall, David H.

2016-01-01

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity. PMID:27482894

Control of apical membrane chloride permeability in the renal A6 cell line by nucleotides

PubMed Central

Banderali, U; Brochiero, E; Lindenthal, S; Raschi, C; Bogliolo, S; Ehrenfeld, J

1999-01-01

The effect of extracellular nucleotides applied on the apical side of polarised A6 cells grown on permeant filters was investigated by measuring the changes in (i) the 36Cl efflux through the apical membranes, (ii) the intracellular chloride concentrations (aCli, measured with N-(6-methoxyquinolyl) acetoethyl ester, MQAE), (iii) ICl, the short-circuit current in the absence of Na+ transport and (iv) the characteristics of the apical chloride channels using a patch-clamp approach. ATP or UTP (0.1-500 μm) transiently stimulated ICl. The sequence of purinergic agonist potencies was UTP = ATP > ADP >> the P2X-selective agonist β,γ-methylene ATP = the P2Y-selective agonist 2-methylthioATP. Suramin (100 μm) as the P2Y antagonist Reactive Blue 2 (10 μm) had no effect on the UTP (or ATP)-stimulated current. These findings are consistent with the presence of P2Y2-like receptors located on the apical membranes of A6 cells. Apical application of adenosine also transiently increased ICl. This effect was blocked by theophylline while the UTP-stimulated ICl was not. The existence of a second receptor, of the P1 type is proposed. ATP (or UTP)-stimulated ICl was blocked by apical application of 200 μmN-phenylanthranilic acid (DPC) or 100 μm niflumic acid while 100 μm glibenclamide was ineffective. Ionomycin and thapsigargin both transiently stimulated ICl; the nucleotide stimulation of ICl was not suppressed by pre-treatment with these agents. Chlorpromazin (50 μm), a Ca2+-calmodulin inhibitor strongly inhibited the stimulation of ICl induced either by apical UTP or by ionomycin application. BAPTA-AM pre-treatment of A6 cells blocked the UTP-stimulated ICl. Niflumic acid also blocked the ionomycin stimulated ICl. A fourfold increase in 36Cl effluxes through the apical membranes was observed after ATP or UTP application. These increases of the apical chloride permeability could also be observed when following aCli changes. Apical application of DPC (1 mm) or 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB; 500 μm) produced an incomplete inhibition of 36Cl effluxes through the apical membranes in ATP-stimulated and in untreated monolayers. In single channel patch-clamp experiments, an apical chloride channel with a unitary single channel conductance of 7.3 ± 0.6 pS (n = 12) was usually observed. ATP application induced the activation of one or more of these channels within a few minutes. These results indicate that multiple purinergic receptor subtypes are present in the apical membranes of A6 cells and that nucleotides can act as modulators of Cl− secretion in renal cells. PMID:10457087

An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

PubMed Central

Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Pereira, Lenore

1999-01-01

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway. PMID:10482621

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

PubMed

Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

2016-04-01

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

Differential localization of lipid phosphate phosphatases 1 and 3 to cell surface subdomains in polarized MDCK cells.

PubMed

Jia, Yan-Jun; Kai, Masahiro; Wada, Ikuo; Sakane, Fumio; Kanoh, Hideo

2003-09-25

Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, we found that LPP1 was located exclusively at the apical surface whereas LPP3 was distributed mostly in the basolateral subdomain. We identified a novel apical sorting signal at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. Our work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

PubMed Central

Stoops, Emily H.; Hull, Michael; Olesen, Christina; Mistry, Kavita; Harder, Jennifer L.; Rivera-Molina, Felix; Toomre, Derek

2015-01-01

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins. PMID:26504168

Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells

PubMed Central

Verkade, Paul; Harder, Thomas; Lafont, Frank; Simons, Kai

2000-01-01

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929–942), only small (∼100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly (∼10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins. PMID:10684254

Ion transport in goby intestine: cellular mechanism of urotensin II stimulation.

PubMed

Loretz, C A; Howard, M E; Siegel, A J

1985-08-01

The Na- and Cl-absorbing goby posterior intestinal epithelium is composed predominantly of mitochondria-rich, tall columnar cells. Glass intracellular microelectrode recording technique was applied to absorptive cells of this relatively leaky epithelium to measure apical cell membrane potential difference (psi mc) and apical membrane fractional resistance. As determined by ion-substitution studies, absorptive cells are characterized by a large, Ba2+-inhibitable apical K conductance, which is a major factor determining psi mc and smaller Cl and Na conductances. Inhibition of the apical Na-Cl-coupled influx directly by furosemide or indirectly by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine produced hyperpolarization of psi mc, consistent with the greater apical membrane conductance to Cl than Na. The urophysial neurosecretory peptide urotensin II, which stimulates Na-Cl-coupled absorption, markedly depolarized psi mc in posterior intestinal tissues from 5% seawater-adapted gobies. This response is consistent with a stimulatory effect of urotensin II at the apical membrane carrier rather than at the basolateral Na-K-ATPase. Urotensin II is without effect on psi mc in tissues from seawater-adapted fish and somatostatin, a natural analogue of urotensin II, is without effect on tissues from fish adapted to either salinity. This specificity parallels that determined using radiotracer fluxes.

Transport of volatile solutes through AQP1

PubMed Central

Cooper, Gordon J; Zhou, Yuehan; Bouyer, Patrice; Grichtchenko, Irina I; Boron, Walter F

2002-01-01

For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3. Additionally, the water channel protein aquaporin 1 (AQP1), expressed at high levels in erythrocytes, can increase membrane CO2 permeability when expressed in Xenopus oocytes. Similarly, nodulin-26, which is closely related to AQP1, can act as a conduit for NH3. A key question is whether aquaporins, which are abundant in virtually every tissue that transports O2 and CO2 at high levels, ever play a physiologically significant role in the transport of small volatile molecules. Preliminary data are consistent with the hypothesis that AQP1 enhances the reabsorption of HCO3− by the renal proximal tubule by increasing the CO2 permeability of the apical membrane. Other preliminary data on Xenopus oocytes heterologously expressing the electrogenic Na+-HCO3− cotransporter (NBC), AQP1 and carbonic anhydrases are consistent with the hypothesis that the macroscopic cotransport of Na+ plus two HCO3− occurs as NBC transports Na+ plus CO32- and AQP1 transports CO2 and H2O. Although data – obtained on AQP1 reconstituted into liposomes or on materials from AQP1 knockout mice – appear inconsistent with the model that AQP1 mediates substantial CO2 transport in certain preparations, the existence of unstirred layers or perfusion-limited conditions may have masked the contribution of AQP1 to CO2 permeability. PMID:12096045

Melanocortin 3 Receptor Has a 5′ Exon That Directs Translation of Apically Localized Protein From the Second In-Frame ATG

PubMed Central

Park, Jeenah; Sharma, Neeraj

2014-01-01

Melanocortin-3 receptor (MC3R) is a canonical MSH receptor that plays an essential role in energy homeostasis. Variants in MC3R have been implicated in obesity in humans and mice. However, interpretation of the functional consequences of these variants is challenging because the translational start site of MC3R is unclear. Using 5′ rapid amplification of cDNA ends, we discovered a novel upstream exon that extends the length of the 5′ untranslated region (UTR) in MC3R without changing the open-reading frame. The full-length 5′ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin-Darby canine kidney cells, consistent with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes relocalization of melanocortin receptor accessory protein 2, an accessory factor for melanocortin-2 receptor, to the apical membrane, coincident with the location of MC3R. In contrast, protein synthesized from MC3R cDNAs lacking the 5′ UTR displayed diffuse cytosolic distribution and has no effect on the distribution of melanocortin receptor accessory protein 2. Our findings demonstrate that a previously unannotated 5′ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells. Together, our work provides insight on the structure of human MC3R and reveals a new pathway for regulation of energy metabolism. PMID:25051171

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism.

PubMed

Wani, Nissar Ahmad; Kaur, Jyotdeep

2011-03-01

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. Copyright © 2010 Wiley-Liss, Inc.

Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

PubMed

Yang, Wen-Kai; Wu, Yu-Ching; Tang, Cheng-Hao; Lee, Tsung-Han

2016-08-01

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

KCNQ and KCNE Potassium Channel Subunit Expression in Bovine Retinal Pigment Epithelium

PubMed Central

Zhang, Xiaoming; Hughes, Bret A.

2013-01-01

Human, monkey, and bovine retinal pigment epithelial (RPE) cells exhibit an M-type K+ current, which in many other cell types is mediated by channels composed of KCNQ α-subunits and KCNE auxiliary subunits. Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 in the monkey RPE. Here, we investigated the expression of KCNQ and KCNE subunits in native bovine RPE. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE, but, in Western blot analysis of RPE plasma membranes, only KCNQ5 was detected. Among the five members of the KCNE gene family, transcripts for KCNE1, KCNE2, KCNE3, and KCNE4 were detected in bovine RPE, but only KCNE1 and KCNE2 proteins were detected. Immunohistochemistry of frozen bovine retinal sections revealed KCNE1 expression near the apical and basal membranes of the RPE, in cone outer segments, in the outer nuclear layer, and throughout the inner retina. The localization of KCNE1 in the RPE basal membrane, where KCNQ5 was previously found to be present, suggests that this β-subunit may contribute to M-type K+ channels in this membrane. PMID:24416770

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

PubMed Central

1996-01-01

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

Elastic Properties of Pore-Spanning Apical Cell Membranes Derived from MDCK II Cells.

PubMed

Nehls, Stefan; Janshoff, Andreas

2017-10-17

The mechanical response of adherent, polarized cells to indentation is frequently attributed to the presence of an endogenous actin cortex attached to the inner leaflet of the plasma membrane. Here, we scrutinized the elastic properties of apical membranes separated from living cells and attached to a porous mesh in the absence of intracellular factors originating from the cytosol, organelles, the substrate, neighbors, and the nucleus. We found that a tension-based model describes the data very well providing essentially the prestress of the shell generated by adhesion of the apical membrane patches to the pore rim and the apparent area compressibility modulus, an intrinsic elastic modulus modulated by the surface excess stored in membrane reservoirs. Removal of membrane-associated proteins by proteases decreases the area compressibility modulus, whereas fixation and cross-linking of proteins with glutaraldehyde increases it. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions.

PubMed

Sharma, Shiwani; Ang, Sharyn L; Shaw, Marie; Mackey, David A; Gécz, Jozef; McAvoy, John W; Craig, Jamie E

2006-06-15

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.

Golgi sorting regulates organization and activity of GPI-proteins at apical membranes

PubMed Central

Tivodar, Simona; Formiggini, Fabio; Ossato, Giulia; Gratton, Enrico; Tramier, Marc; Coppey-Moisan, Maïté; Zurzolo, Chiara

2014-01-01

Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface. PMID:24681536

Expression and role of the genes involved in the transport of bile acids in the liver and kidneys in mice.

PubMed

Attakpa, Eugène S; Djibril, Naguibou M; Baba-Moussa, Farid; Yessoufou, Ganiou; Sezan, Alphonse

2013-01-01

Bile acids are synthesized in the liver from cholesterol. This study investigated the impact and expression of different carriers of bile acid in the liver and kidneys. Eight-week-old male mice were used, which were fed for 15 days and divided into two groups: 15 mice fed with standard diet (control group) and another 15 mice fed with a rich diet of 5% cholesterol (second group). Bile acid dosage was based on their oxidation by 7α hydroxyl-steroid dehydrogenize. The mRNA expression was quantitatively analyzed by the real time of polymerase chain reaction (RT-PCR), and the expression of the renal carrier bile acid protein was analyzed by Western blot. The expression of bile salt export pump involved in the uptake of bile acids in the basolateral membrane of hepatocytes revealed no differences between the two groups of mice. However, the expression of multidrug resistance-associated protein 2 was reduced in mice of the second group. Moreover, the expressions of organic anion transporting polypeptide 4, organic anion transporting polypeptide 1, and sodium taurocholate co-transporting polypeptide (Ntcp) involved in the uptake of bile acids in the apical pole of hepatocytes are suppressed in mice of the second group. The expression of multidrug resistance-associated protein 3 involved in the secretion of bile acids in the apical membrane of hepatocytes revealed no significant differences between the two groups. In mice of the second group, blood concentration of bile acids on the last day was increased. In those mice, the expression of intestinal bile acid transporter was reduced in the kidneys compared with the control mice.

H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

PubMed

Thwaites, D T; Ford, D; Glanville, M; Simmons, N L

1999-09-01

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane.

PubMed

Pedersen, Gitte A; Jensen, Helene H; Schelde, Anne-Sofie B; Toft, Charlotte; Pedersen, Hans N; Ulrichsen, Maj; Login, Frédéric H; Amieva, Manuel R; Nejsum, Lene N

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane

PubMed Central

Pedersen, Gitte A.; Jensen, Helene H.; Schelde, Anne-Sofie B.; Toft, Charlotte; Pedersen, Hans N.; Ulrichsen, Maj; Login, Frédéric H.; Amieva, Manuel R.

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth. PMID:28636623

Does apical membrane GLUT2 have a role in intestinal glucose uptake?

PubMed

Naftalin, Richard J

2014-01-01

It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border. The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route. As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface. These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations.

Does apical membrane GLUT2 have a role in intestinal glucose uptake?

PubMed Central

Naftalin, Richard J

2014-01-01

It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border. The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route. As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface. These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations. PMID:25671087

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

KIFC3, a microtubule minus end–directed motor for the apical transport of annexin XIIIb–associated Triton-insoluble membranes

PubMed Central

Noda, Yasuko; Okada, Yasushi; Saito, Nobuhito; Setou, Mitsutoshi; Xu, Ying; Zhang, Zheizeng; Hirokawa, Nobutaka

2001-01-01

We have identified and characterized a COOH-terminal motor domain–type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end–directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100–insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413–1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb–associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells. PMID:11581287

Iodide handling by the thyroid epithelial cell.

PubMed

Nilsson, M

2001-01-01

Iodination of thyroglobulin, the key event in the synthesis of thyroid hormone, is an extracellular process that takes place inside the thyroid follicles at the apical membrane surface that faces the follicular lumen. The supply of iodide involves two steps of TSH-regulated transport, basolateral uptake and apical efflux, that imprint the polarized phenotype of the thyroid cell. Iodide uptake is generated by the sodium/iodide symporter present in the basolateral plasma membrane. A candidate for the apical iodide-permeating mechanism is pendrin, a chloride/iodide transporting protein recently identified in the apical membrane. In physiological conditions, transepithelial iodide transport occurs without intracellular iodination, despite the presence of large amounts of thyroglobulin and thyroperoxidase inside the cells. The reason is that hydrogen peroxide, serving as electron acceptor in iodide-protein binding and normally produced at the apical cell surface, is rapidly degraded by cytosolic glutathione peroxidase once it enters the cells. Iodinated thyroglobulin in the lumen stores not only thyroid hormone but iodine incorporated in iodotyrosine residues as well. After endocytic uptake and degradation of thyroglobulin, intracellular deiodination provides a mechanism for recycling of iodide to participate in the synthesis of new thyroid hormone at the apical cell surface.

[Ultrastructure and cytochemistry of the pellicle and apical complexes of the kinete of Babesia bigemina and Babesia ovis in the hemolymph and oavry of the tick].

PubMed

Weber, G

1980-02-01

The term kinete is used in this paper for the cigar-shaped, motile development stages (VERMICULE") OF Babesia occurring intra- and extracellularly in hemolymph and overy (including oocytes) of vectors, hard ticks (Ixodoidea). The structure of, and cytochemical activities of hydrolases (acid phosphatase, nonspecific esterase) in the pellicle and the apical complex was studied at the fine-structural level in kinetes of Babesia bigemina Smith & Kilborne, in hemolympho of female Boophilus microplus Canestrini. The cytochemistry of acid hydrolases was studied also in kinetes of Babesia ovis (Babès) Starcovici, in hemolymph and ovary of Rhipicephalus bursa Canestrini & Fanzago. The pellicle of the B. bigemina kinetes is composted of 3 membranes (pellicular complex): an outer membrane, approximately 8 nm thick (the plasmalemma) and 2 innder ones, each approximately nm thick, lying closely together. The outer membrane appears to be covered by a structureless coat, 3 nm thick. The space between the inner double membrane and the plasmalemma is 7.5 nm. The whole pellicular complex is 30 nm in diameter. The 2 inner pellicular membranes appear to be derived from the endoplasmic reticulum (ER) for the following reasons: (a) a layer of hydrolase-active material is enclosed by these membranes; (b) in the spheroid parasite stages which transform from kinetes inside hemocytes, the inner double membrane is apparently replaced by an ER cisterna; (c) the thickness of each of the inner pellicular membranes is approximately the same as that of the ER membrane. There are circular openings in the pellicular double membrane with average diameters of 100 nm; despite some similarity to micropores, they have a specific structure. The term Intrapellikularfenster (IPF) (intrapellicular windows) or pseudomicropores is proposed for these pellicular differentiations. The margin of an IPF is formed by the 2 inner membranes folding into each other; cytoplasmic, electron-dense material is accumulated alongside this edge. Unlike that of micropores, the plasmalemma of the IPF is not invaginated. The IPF appears as a single, dark ring in tangential sections. At times, rhoptry-like bodies are associated with the openings. The function of the IPF is not known. An intrapellicular opening similar to the IPF, although wider, is present at the apex of the parasite. Its margin coincides with the inners edge of the apical ring. Typical subpellicular microtubuli were not observed in the Babesia kinetes. The apical complex of the B. bigemina kinetes consists of an Apikalschirm (apical umbrella), a crown of microtubuli beneath it, and rhoptries: micronemes are also present in large numbers. The Apikalschirm is located beneath the pellicle of the apical pole of the parasite. It is a wheel-like structure composed of spokes radiating from a wide, hub=like central ring (apical ring). It should be stressed that the apical ring is not identical with the polar ring described as an integral part of the pellicular complex in other Apicomplexa...

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PubMed

Nickel, Sabrina; Selo, Mohammed Ali; Fallack, Juliane; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

2017-12-01

Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

The Balance of HCO3- Secretion vs. Reabsorption in the Endometrial Epithelium Regulates Uterine Fluid pH

PubMed Central

Xie, Zhang-Dong; Guo, Yi-Min; Ren, Mei-Juan; Yang, Jichun; Wang, Shao-Fang; Xu, Tong-Hui; Chen, Li-Ming; Liu, Ying

2018-01-01

Uterine fluid contains a high concentration of HCO3- which plays an essential role in sperm capacitation and fertilization. In addition, the HCO3- concentration in uterine fluid changes periodically during the estrous cycle. It is well-known that the endometrial epithelium contains machineries involving the apical SLC26 family anion exchangers for secreting HCO3- into the uterine fluid. In the present study, we find for the first time that the electroneutral Na+/HCO3- cotransporter NBCn1 is expressed at the apical membrane of the endometrial epithelium. The protein abundance of the apical NBCn1 and that of the apical SLC26A4 and SLC26A6 are reciprocally regulated during the estrous cycle in the uterus. NBCn1 is most abundant at diestrus, whereas SLC26A4/A6 are most abundant at proestrus/estrus. In the ovariectomized mice, the expression of uterine NBCn1 is inhibited by β-estradiol, but stimulated by progesterone, whereas that of uterine SLC26A4/A6 is stimulated by β-estradiol. In vivo perfusion studies show that the endometrial epithelium is capable of both secreting and reabsorbing HCO3-. Moreover, the activity for HCO3- secretion by the endometrial epithelium is significantly higher at estrus than it is at diestrus. The opposite is true for HCO3- reabsorption. We conclude that the endometrial epithelium simultaneously contains the activity for HCO3- secretion involving the apical SLC26A4/A6 and the activity for HCO3- reabsorption involving the apical NBCn1, and that the acid-base homeostasis in the uterine fluid is regulated by the finely-tuned balance of the two activities. PMID:29422866

Membrane recycling at the infranuclear pole of the outer hair cell

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.

2015-12-01

Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues.

PubMed

Greenwalt, D E; Mather, I H

1985-02-01

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

PubMed

Bortfeld, M; Rius, M; König, J; Herold-Mende, C; Nies, A T; Keppler, D

2006-01-01

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells

PubMed Central

Perez Bay, Andres E.; Schreiner, Ryan; Benedicto, Ignacio; Paz Marzolo, Maria; Banfelder, Jason; Weinstein, Alan M.; Rodriguez-Boulan, Enrique J.

2016-01-01

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station. PMID:27180806

Immunization with apical membrane antigen 1 confers sterile infection-blocking immunity against Plasmodium sporozoite challenge in a rodent model.

PubMed

Schussek, Sophie; Trieu, Angela; Apte, Simon H; Sidney, John; Sette, Alessandro; Doolan, Denise L

2013-10-01

Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8(+) and CD4(+) T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity.

Renal Na+-K+-Cl− cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent

PubMed Central

Welker, Pia; Böhlick, Alexandra; Mutig, Kerim; Salanova, Michele; Kahl, Thomas; Schlüter, Hartmut; Blottner, Dieter; Ponce-Coria, Jose; Gamba, Gerardo; Bachmann, Sebastian

2008-01-01

Apical bumetanide-sensitive Na+-K+-2Cl− cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40–70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na+-Cl− cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-β-cyclodextrin (MβCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MβCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport. PMID:18579701

Rapid Turnover of Stereocilia Membrane Proteins: Evidence from the Trafficking and Mobility of Plasma Membrane Ca2+-ATPase 2

PubMed Central

Grati, M'hamed; Schneider, Mark E.; Lipkow, Karen; Strehler, Emanuel E.; Wenthold, Robert J.; Kachar, Bechara

2007-01-01

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of ∼5–7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01– 0.005 μm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that ∼60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1– 0.2 μm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal. PMID:16763047

Visualization of CD44 and CD133 in Normal Pancreas and Pancreatic Ductal Adenocarcinomas

PubMed Central

Immervoll, Heike; Hoem, Dag; Steffensen, Ole Johnny; Miletic, Hrvoje; Molven, Anders

2011-01-01

Tumor-initiating cells of pancreatic ductal adenocarcinoma (PDAC) have been isolated based on expression of either CD133 or CD44. The authors aimed to visualize pancreatic cells simultaneously expressing both these cell surface markers by employing the same antibodies commonly used in cell-sorting studies. Normal and diseased pancreatic tissue, including 51 PDAC cases, were analyzed. CD44 and CD133 expression was determined by immunohistochemical double staining on formalin-fixed material and subcellular protein distribution evaluated by immunofluorescence/confocal microscopy. In the normal pancreas, CD44 and CD133 were coexpressed in the centroacinar regions but in non-overlapping subcellular compartments. As expected, CD44 was found mainly basolaterally, whereas CD133 was present on the apical/endoluminal membrane. This was also the case in chronically inflamed/atrophic pancreatic tissue and in PDAC. In some malignant ducts, CD44 was found at the apical cell membrane adjacent to but never overlapping with CD133 expression. CD44 level was significantly associated with the patient’s lymph node status. In conclusion, a CD44+/CD133+ cell population does exist in the normal and neoplastic pancreas. The preferentially centroacinar localization of the doubly positive cells in the normal parenchyma suggests that this population could be of particular interest in attempts to identify tumor-initiating cells in PDAC. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:21411814

Fructose-induced increases in expression of intestinal fructolytic and gluconeogenic genes are regulated by GLUT5 and KHK

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Tharabenjasin, Phuntila; Gao, Nan

2015-01-01

Marked increases in fructose consumption have been tightly linked to metabolic diseases. One-third of ingested fructose is metabolized in the small intestine, but the underlying mechanisms regulating expression of fructose-metabolizing enzymes are not known. We used genetic mouse models to test the hypothesis that fructose absorption via glucose transporter protein, member 5 (GLUT5), metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein in brain 11a (Rab11a)-dependent endosomes are required for the regulation of intestinal fructolytic and gluconeogenic enzymes. Fructose feeding increased the intestinal mRNA and protein expression of these enzymes in the small intestine of adult wild-type (WT) mice compared with those gavage fed with lysine or glucose. Fructose did not increase expression of these enzymes in the GLUT5 knockout (KO) mice. Blocking intracellular fructose metabolism by KHK ablation also prevented fructose-induced upregulation. Glycolytic hexokinase I expression was similar between WT and GLUT5- or KHK-KO mice and did not vary with feeding solution. Gavage feeding with the fructose-specific metabolite glyceraldehyde did not increase enzyme expression, suggesting that signaling occurs before the hydrolysis of fructose to three-carbon compounds. Impeding GLUT5 trafficking to the apical membrane using intestinal epithelial cell-specific Rab11a-KO mice impaired fructose-induced upregulation. KHK expression was uniformly distributed along the villus but was localized mainly in the basal region of the cytosol of enterocytes. The feedforward upregulation of fructolytic and gluconeogenic enzymes specifically requires GLUT5 and KHK and may proactively enhance the intestine's ability to process anticipated increases in dietary fructose concentrations. PMID:26084694

Altered Regulation of Airway Epithelial Cell Chloride Channels in Cystic Fibrosis

NASA Astrophysics Data System (ADS)

Frizzell, Raymond A.; Rechkemmer, Gerhard; Shoemaker, Richard L.

1986-08-01

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that β -adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.

PubMed

Slimane, Tounsia Aït; Trugnan, Germain; Van IJzendoorn, Sven C D; Hoekstra, Dick

2003-02-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

PubMed Central

Slimane, Tounsia Aït; Trugnan, Germain; van IJzendoorn, Sven C.D.; Hoekstra, Dick

2003-01-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains (“rafts”) are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway. PMID:12589058

Relationship between aquaporin-5 expression and saliva flow in streptozotocin-induced diabetic mice?

PubMed

Soyfoo, M S; Bolaky, N; Depoortere, I; Delporte, C

2012-07-01

To investigate the expression and distribution of AQP5 in submandibular acinar cells from sham- and streptozotocin (STZ)-treated mice in relation to the salivary flow. Mice were sham or STZ injected. Distribution of AQP5 subcellular expression in submandibular glands was determined by immunohistochemistry. AQP5 labelling indices (LI), reflecting AQP5 subcellular distribution, were determined in acinar cells. Western blotting was performed to determine the expression of AQP5 in submandibular glands. Blood glycaemia and osmolality and saliva flow rates were also determined. AQP5 immunoreactivity was primarily located at the apical and apical-basolateral membranes of submandibular gland acinar cells from sham- and STZ-treated mice. No significant differences in AQP5 protein levels were observed between sham- and STZ-treated mice. Compared to sham-treated mice, STZ-treated mice had significant increased glycaemia, while no significant differences in blood osmolality were observed. Saliva flow rate was significantly decreased in STZ-treated mice as compared to sham-treated mice. In STZ-treated mice, significant reduction in salivary flow rate was observed without any concomitant modification in AQP5 expression and localization. © 2011 John Wiley & Sons A/S.

GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes

PubMed Central

Ait-Omar, Amal; Monteiro-Sepulveda, Milena; Poitou, Christine; Le Gall, Maude; Cotillard, Aurélie; Gilet, Jules; Garbin, Kevin; Houllier, Anne; Château, Danièle; Lacombe, Amélie; Veyrie, Nicolas; Hugol, Danielle; Tordjman, Joan; Magnan, Christophe; Serradas, Patricia; Clément, Karine; Leturque, Armelle; Brot-Laroche, Edith

2011-01-01

OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-14C]-3-O-methyl glucose into lumen. RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology. PMID:21852673

Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype

PubMed Central

Gilbert, Merle L.; Yang, Linghai; Su, Thomas

2015-01-01

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus. PMID:25587115

Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier.

PubMed

Arana, Maite R; Tocchetti, Guillermo N; Rigalli, Juan P; Mottino, Aldo D; Villanueva, Silvina S M

2016-07-01

The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

PubMed Central

Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K.

2016-01-01

ABSTRACT Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/− Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus. PMID:27923925

A dynamic model of saliva secretion

PubMed Central

Palk, Laurence; Sneyd, James; Shuttleworth, Trevor J.; Yule, David I.; Crampin, Edmund J.

2010-01-01

We construct a mathematical model of the parotid acinar cell with the aim of investigating how the distribution of K+ and Cl− channels affects saliva production. Secretion of fluid is initiated by Ca2+ signals acting the Ca2+ dependent K+ and Cl− channels. The opening of these channels facilitates the movement of Cl− ions into the lumen which water follows by osmosis. We use recent results into both the release of Ca2+ from internal stores via the inositol (1,4,5)-trisphosphate receptor (IP3R) and IP3 dynamics to create a physiologically realistic Ca2+ model which is able to recreate important experimentally observed behaviours seen in parotid acinar cells. We formulate an equivalent electrical circuit diagram for the movement of ions responsible for water flow which enables us to calculate and include distinct apical and basal membrane potentials to the model. We show that maximum saliva production occurs when a small amount of K+ conductance is located at the apical membrane, with the majority in the basal membrane. The maximum fluid output is found to coincide with a minimum in the apical membrane potential. The traditional model whereby all Cl− channels are located in the apical membrane is shown to be the most efficient Cl− channel distribution. PMID:20600135

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

PubMed

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

2011-09-18

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis

PubMed Central

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A.; Hall, David H.; Fleming, John T.; Gobel, Verena

2011-01-01

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single postmitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity via sphingolipid synthesis, and reveal ceramideglucosyltransferases (CGTs) as endpoint biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids (GSLs), CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and suggest they sort new components to the expanding apical membrane. PMID:21926990

Recycling Endosomes of Polarized Epithelial Cells Actively Sort Apical and Basolateral Cargos into Separate Subdomains

PubMed Central

Thompson, Anthony; Nessler, Randy; Wisco, Dolora; Anderson, Eric; Winckler, Bettina

2007-01-01

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B–dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity. PMID:17494872

Repulsion between Lys258 and upstream arginines explains the missorting of the AQP2 mutant p.Glu258Lys in nephrogenic diabetes insipidus.

PubMed

Kamsteeg, Erik-Jan; Stoffels, Monique; Tamma, Grazia; Konings, Irene B M; Deen, Peter M T

2009-10-01

Regulation of body water homeostasis occurs by the vasopressin-dependent sorting of aquaporin-2 (AQP2) water channels to and from the apical membrane of renal principal cells. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease that renders the kidney unresponsive to vasopressin, resulting in polyuria and polydipsia. The AQP2 mutant c.772G>A; p.Glu258Lys (AQP2-E258K) causes dominant NDI by oligomerizing with wild-type AQP2 and missorting of this AQP2 complex to multivesicular bodies instead of the apical membrane. The motif causing this missorting of AQP2-E258K was identified here. Functional analyses and plasma membrane expression studies of truncation mutants in oocytes revealed that AQP2-E258K shortened to Leu259 is still intracellular retained. Alanine scanning and glutamic acid to arginine exchanges revealed increased function and plasma membrane expression for AQP2-E258K mutants with the following additional changes: Leu259Ala, Arg252Glu, Arg253Glu, or Arg252Ala-Arg254Ala, or for the AQP2 mutant p.Glu258Ala, indicating that the motif RRRxxxK(258)L confers AQP2-E258K retention. Fusion of this motif to aquaporin-1 also resulted in missorting of that water channel, indicating that this retention motif is transferable. In conclusion, our data reveal that the RRRxxxKL motif and repulsion between K258 and the arginine-triplet within this motif are the primary cause of missorting of AQP2-E258K in NDI.

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

PubMed Central

Liégeois, Samuel; Benedetto, Alexandre; Garnier, Jean-Marie; Schwab, Yannick; Labouesse, Michel

2006-01-01

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals. PMID:16785323

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins.

PubMed

Revenu, Céline; Ubelmann, Florent; Hurbain, Ilse; El-Marjou, Fatima; Dingli, Florent; Loew, Damarys; Delacour, Delphine; Gilet, Jules; Brot-Laroche, Edith; Rivero, Francisco; Louvard, Daniel; Robine, Sylvie

2012-01-01

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.

Mechanisms of bicarbonate secretion: lessons from the airways.

PubMed

Bridges, Robert J

2012-08-01

Early studies showed that airway cells secrete HCO(3)(-) in response to cAMP-mediated agonists and HCO(3)(-) secretion was impaired in cystic fibrosis (CF). Studies with Calu-3 cells, an airway serous model with high expression of CFTR, also show the secretion of HCO(3)(-) when cells are stimulated with cAMP-mediated agonists. Activation of basolateral membrane hIK-1 K(+) channels inhibits HCO(3)(-) secretion and stimulates Cl(-) secretion. CFTR mediates the exit of both HCO(3)(-) and Cl(-) across the apical membrane. Entry of HCO(3)(-) on a basolateral membrane NBC or Cl(-) on the NKCC determines which anion is secreted. Switching between these two secreted anions is determined by the activity of hIK-1 K(+) channels.

cAmp activation of apical membrane Cl(-) channels: theoretical considerations for impedance analysis.

PubMed Central

Păunescu, T G; Helman, S I

2001-01-01

Transepithelial electrical impedance analysis provides a sensitive method to evaluate the conductances and capacitances of apical and basolateral plasma membranes of epithelial cells. Impedance analysis is complicated, due not only to the anatomical arrangement of the cells and their paracellular shunt pathways, but also in particular to the existence of audio frequency-dependent capacitances or dispersions. In this paper we explore implications and consequences of anatomically related Maxwell-Wagner and Cole-Cole dielectric dispersions that impose limitations, approximations, and pitfalls of impedance analysis when tissues are studied under widely ranging spontaneous rates of transport, and in particular when apical membrane sodium and chloride channels are activated by adenosine 3',5'-cyclic monophosphate (cAMP) in A6 epithelia. We develop the thesis that capacitive relaxation processes of any origin lead not only to dependence on frequency of the impedance locus, but also to the appearance of depressed semicircles in Nyquist transepithelial impedance plots, regardless of the tightness or leakiness of the paracellular shunt pathways. Frequency dependence of capacitance precludes analysis of data in traditional ways, where capacitance is assumed constant, and is especially important when apical and/or basolateral membranes exhibit one or more dielectric dispersions. PMID:11463629

SHIP2 Regulates Lumen Generation, Cell Division, and Ciliogenesis through the Control of Basolateral to Apical Lumen Localization of Aurora A and HEF 1.

PubMed

Hamze-Komaiha, Ola; Sarr, Sokavuth; Arlot-Bonnemains, Yannick; Samuel, Didier; Gassama-Diagne, Ama

2016-12-06

Lumen formation during epithelial morphogenesis requires the creation of a luminal space at cell interfaces named apical membrane-initiation sites (AMISs). This is dependent upon integrated signaling from mechanical and biochemical cues, vesicle trafficking, cell division, and processes tightly coupled to ciliogenesis. Deciphering relationships between polarity determinants and lumen or cilia generation remains a fundamental issue. Here, we report that Src homology 2 domain-containing inositol 5-phosphatase 2 (SHIP2), a basolateral determinant of polarity, regulates RhoA-dependent actin contractility and cell division to form AMISs. SHIP2 regulates mitotic spindle alignment. SHIP2 is expressed in G1 phase, whereas Aurora A kinase is enriched in mitosis. SHIP2 binds Aurora A kinase and the scaffolding protein HEF1 and promotes their basolateral localization at the expense of their luminal expression connected with cilia resorption. Furthermore, SHIP2 expression increases cilia length. Thus, our findings offer new insight into the relationships among basolateral proteins, lumen generation, and ciliogenesis. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3.

PubMed

Søndergaard, Helle Bach; Brodin, Birger; Nielsen, Carsten Uhd

2008-06-01

The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis-Menten kinetics with a Km value of 1.3 +/- 0.3 mM and a Vmax value of 0.60 +/- 0.06 nmol cm(-2) min(-1). Transepithelial apical to basolateral transport of 50 microM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of delta-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (approximately 80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PubMed

Ban, Yuriko; Cooper, Leanne J; Fullwood, Nigel J; Nakamura, Takahiro; Tsuzuki, Masakatsu; Koizumi, Noriko; Dota, Atsuyoshi; Mochida, Chikako; Kinoshita, Shigeru

2003-06-01

To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

PubMed

Le Maout, S; Sewing, S; Coudrier, E; Elalouf, J M; Pongs, O; Merot, J

1996-01-01

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

Electron tomographic characterization of a vacuolar reticulum and of six vesicle types that occupy different cytoplasmic domains in the apex of tip-growing Chara rhizoids.

PubMed

Limbach, Christoph; Staehelin, L Andrew; Sievers, Andreas; Braun, Markus

2008-04-01

We provide a 3D ultrastructural analysis of the membrane systems involved in tip growth of rhizoids of the green alga Chara. Electron tomography of cells preserved by high-pressure freeze fixation has enabled us to distinguish six different types of vesicles in the apical cytoplasm where the tip growth machinery is accommodated. The vesicle types are: dark and light secretory vesicles, plasma membrane-associated clathrin-coated vesicles (PM-CCVs), Spitzenkoerper-associated clathrin-coated vesicles (Sp-CCVs) and coated vesicles (Sp-CVs), and microvesicles. Each of these vesicle types exhibits a distinct distribution pattern, which provides insights into their possible function for tip growth. The PM-CCVs are confined to the cytoplasm adjacent to the apical plasma membrane. Within this space they are arranged in clusters often surrounding tubular plasma membrane invaginations from which CCVs bud. This suggests that endocytosis and membrane recycling are locally confined to specialized apical endocytosis sites. In contrast, exocytosis of secretory vesicles occurs over the entire membrane area of the apical dome. The Sp-CCVs and the Sp-CVs are associated with the aggregate of endoplasmic reticulum membranes in the center of the growth-organizing Spitzenkoerper complex. Here, Sp-CCVs are seen to bud from undefined tubular membranes. The subapical region of rhizoids contains a vacuolar reticulum that extends along the longitudinal cell axis and consists of large, vesicle-like segments interconnected by thin tubular domains. The tubular domains are encompassed by thin filamentous structures resembling dynamin spirals which could drive peristaltic movements of the vacuolar reticulum similar to those observed in fungal hyphae. The vacuolar reticulum appears to serve as a lytic compartment into which multivesicular bodies deliver their internal vesicles for molecular recycling and degradation.

Polarity and transport properties of rabbit kidney proximal tubule cells on collagen IV-coated porous membranes.

PubMed

Genestie, I; Morin, J P; Vannier, B; Lorenzon, G

1995-07-01

A high degree of functional polarity has been obtained in primary cultures of rabbit kidney proximal tubule cells grown on collagen IV-coated porous membranes. Tight confluency was attained 6 days after seeding and maintained for at least 6 more days, as shown by analysis of paracellular inulin diffusion. From day 6 onward, L-lactate, ammonia, and D-glucose concentration gradient and a pH difference of approximately 1 unit developed between the two nutrient medium compartments. Confluent monolayers expressed organic ion transport properties higher than those formerly reported for other cell models. Transcellular transport of 20 microM tetraethylammonium was directed from basal to apical compartment and was specifically inhibited by mepiperphenidol (1 mM). Unidirectional transport of 2.4 microM p-aminohippurate also occurred from basal to apical compartment, was saturable, and specifically inhibited by probenecid (1 mM). These results suggest that rabbit kidney proximal tubule cells, cultured under the experimental conditions described here, may be a useful model for the in vitro study of highly polarized renal transport processes.

Epithelial Membrane Protein 2 and β1 integrin signaling regulate APC-mediated processes.

PubMed

Lesko, Alyssa C; Prosperi, Jenifer R

2017-01-01

Adenomatous Polyposis Coli (APC) plays a critical role in cell motility, maintenance of apical-basal polarity, and epithelial morphogenesis. We previously demonstrated that APC loss in Madin Darby Canine Kidney (MDCK) cells increases cyst size and inverts polarity independent of Wnt signaling, and upregulates the tetraspan protein, Epithelial Membrane Protein 2 (EMP2). Herein, we show that APC loss increases β1 integrin expression and migration of MDCK cells. Through 3D in vitro model systems and 2D migration analysis, we have depicted the molecular mechanism(s) by which APC influences polarity and cell motility. EMP2 knockdown in APC shRNA cells revealed that APC regulates apical-basal polarity and cyst size through EMP2. Chemical inhibition of β1 integrin and its signaling components, FAK and Src, indicated that APC controls cyst size and migration, but not polarity, through β1 integrin and its downstream targets. Combined, the current studies have identified two distinct and novel mechanisms required for APC to regulate polarity, cyst size, and cell migration independent of Wnt signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Localization of alpha-dystroglycan on the podocyte: from top to toe.

PubMed

Vogtländer, Nils P J; Dijkman, Henry; Bakker, Marinka A H; Campbell, Kevin P; van der Vlag, Johan; Berden, Jo H M

2005-11-01

alpha-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that alpha-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against alpha-DG. Therefore, we studied the exact localization of glomerular alpha-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of alpha-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that alpha-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that alpha-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

Focus on Kir7.1: physiology and channelopathy

PubMed Central

Kumar, Mohit; Pattnaik, Bikash R

2014-01-01

Genetic studies have linked alterations in Kir7.1 channel to diverse pathologies. We summarize functional relevance of Kir7.1 channel in retinal pigment epithelium (RPE), regulation of channel function by various cytoplasmic metabolites, and mutations that cause channelopathies. At the apical membrane of RPE, K+ channels contribute to subretinal K+ homeostasis and support Na+/K+ pump and Na+-K+-2Cl− cotransporter function by providing a pathway for K+ secretion. Electrophysiological studies have established that barium- and cesium-sensitive inwardly rectifying K+ (Kir) channels make up a major component of the RPE apical membrane K+ conductance. Native human RPE expresses transcripts for Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2, and Kir6.1, albeit at levels at least 50-fold lower than Kir7.1. Kir7.1 is structurally similar to other Kir channels, consisting of 2 trans-membrane domains, a pore-forming loop that contains the selectivity filter, and 2 cytoplasmic polar tails. Within the cytoplasmic structure, clusters of amino acid sequences form regulatory domains that interact with cellular metabolites and control the opening and closing of the channel. Recent evidence indicated that intrinsic sequence motifs present in Kir7.1 control surface expression. Mutant Kir7.1 channels are associated with inherited eye pathologies such as Snowflake Vitreoretinal Degeneration (SVD) and Lebers Congenital Amaurosis (LCA16). Based on the current evidence, mutations implicated in channelopathies have the potential to be used for genetic testing to diagnose blindness due to Kir7.1. PMID:25558901

Pak1 Kinase Maintains Apical Membrane Identity in Epithelia.

PubMed

Aguilar-Aragon, Mario; Elbediwy, Ahmed; Foglizzo, Valentina; Fletcher, Georgina C; Li, Vivian S W; Thompson, Barry J

2018-02-13

Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Differential localization of ErbB2 in different tissues of the rat female reproductive tract: implications for the use of specific antibodies for ErbB2 analysis.

PubMed

Idris, N; Carothers Carraway, C A; Carraway, K L

2001-11-01

ErbB2 has been implicated in numerous functions, including normal and aberrant development of a variety of tissues. Although no soluble ligand has been identified for ErbB2, we have recently shown that ASGP-2, the transmembrane subunit of the cell surface glycoprotein Muc4 (also called sialomucin complex, SMC), can act as an intramembrane ligand for ErbB2 and modulate its activity. Muc4/SMC is abundantly expressed at the apical surface of most epithelia of the rat female reproductive tract. Since Muc4/SMC can interact with ErbB2 when they are expressed in the same cell and membrane, we investigated whether these two proteins are co-expressed and co-localized in tissues of the female reproductive tract. Using an anti-ErbB2 antibody from Dako, we found moderate staining at the basolateral surface of the oviduct and also around the cell membrane of the most superficial and medial layers of the stratified epithelia of the vagina. In contrast, Neomarkers neu Ab1 antibody intensely stained the apical surface of the epithelium of the oviduct and the medial and basal layers of the stratified epithelia of the vagina, substantially overlapping the distribution of Muc4/SMC. Furthermore, Muc4/SMC and ErbB2 association in different tissues of the female reproductive tract was demonstrated by co-immunoprecipitation analysis. Interestingly, phosphorylated ErbB2 detected by anti-phospho-ErbB2 is primarily present at the apical surface of the oviduct. Thus, our results show that differentially localized forms of ErbB2 are recognized by different antibodies and raise interesting questions about the nature of the different forms of ErbB2, the mechanism for differential localization, and possible functions of ErbB2 in the female reproductive tract. They also raise a cautionary note about the use of different ErbB2 antibodies for expression and localization studies. Copyright 2001 Wiley-Liss, Inc.

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

PubMed Central

Zhou, Ge; Liang, Feng-Xia; Romih, Rok; Wang, Zefang; Liao, Yi; Ghiso, Jorge; Luque-Garcia, Jose L.; Neubert, Thomas A.; Kreibich, Gert; Alonso, Miguel A.; Schaeren-Wiemers, Nicole; Sun, Tung-Tien

2012-01-01

The apical surface of mammalian bladder urothelium is covered by large (500–1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin–Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface. PMID:22323295

The Cholangiocyte Glycocalyx Stabilizes the 'Biliary HCO3 Umbrella': An Integrated Line of Defense against Toxic Bile Acids.

PubMed

Maillette de Buy Wenniger, Lucas J; Hohenester, Simon; Maroni, Luca; van Vliet, Sandra J; Oude Elferink, Ronald P; Beuers, Ulrich

2015-01-01

Destruction of cholangiocytes is the hallmark of chronic cholangiopathies such as primary biliary cirrhosis. Under physiologic conditions, cholangiocytes display a striking resistance to the high, millimolar concentrations of toxic bile salts present in bile. We recently showed that a 'biliary HCO3(-) umbrella', i.e. apical cholangiocellular HCO3(-) secretion, prevents cholangiotoxicity of bile acids, and speculated on a role for extracellular membrane-bound glycans in the stabilization of this protective layer. This paper summarizes published and thus far unpublished evidence supporting the role of the glycocalyx in stabilizing the 'biliary HCO3(-) umbrella' and thus preventing cholangiotoxicity of bile acids. The apical glycocalyx of a human cholangiocyte cell line and mouse liver sections were visualized by electron microscopy. FACS analysis was used to characterize the surface glycan profile of cultured human cholangiocytes. Using enzymatic digestion with neuraminidase the cholangiocyte glycocalyx was desialylated to test its protective function. Using lectin assays, we demonstrated that the main N-glycans in human and mouse cholangiocytes were sialylated biantennary structures, accompanied by high expression of the H-antigen (α1-2 fucose). Apical neuraminidase treatment induced desialylation without affecting cell viability, but lowered cholangiocellular resistance to bile acid-induced toxicity: both glycochenodeoxycholate and chenodeoxycholate (pKa ≥4), but not taurochenodeoxycholate (pKa <2), displayed cholangiotoxic effects after desialylation. A 24-hour reconstitution period allowed cholangiocytes to recover to a pretreatment bile salt susceptibility pattern. Experimental evidence indicates that an apical cholangiocyte glycocalyx with glycosylated mucins and other glycan-bearing membrane glycoproteins stabilizes the 'biliary HCO3(-) umbrella', thus aiding in the protection of human cholangiocytes against bile acid toxicity. 2015 S. Karger AG, Basel.

Minimal Effects of VEGF and Anti-VEGF Drugs on the Permeability or Selectivity of RPE Tight Junctions

PubMed Central

Peng, Shaomin; Adelman, Ron A.

2010-01-01

Purpose. Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood–retinal barrier were examined. Methods. Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K+ and Na+ (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. Results. Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K+, but did not discriminate between Na+ and Cl−. VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K+, Na+, and Cl−. They had minimal effects on the expression and distribution of the claudins. Conclusions. RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K+ over Na+ and Cl−. Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab. PMID:20042644

β-Cyclodextrin and permeability to water in the bladder of Bufo arenarum.

PubMed

Orce, G; Castillo, G; Chanampa, Y

2011-05-01

We measured the effect of β-cyclodextrin (BCD, a cholesterol scavenger) on water flow across the isolated toad bladder exposed to an osmotic gradient (J(w)) by a gravimetric technique. BCD, when present in the solution bathing the apical side of the bladder, inhibited the increase in J(w) caused by nystatin, a polyene antibiotic that acts by directly binding apical membrane cholesterol. When present in the basolateral bath, BCD inhibited the increase in J(w) caused by basolateral exposure to oxytocin (which binds membrane receptors and stimulates the synthesis of cAMP), but did not alter the response to theophylline (which inhibits hydrolysis of cAMP by cyclic nucleotide phosphodiesterase). The present data are consistent with the notion that agents that increase J(w) by interacting with membrane receptors, which appear to be clustered in cholesterol-rich domains of the basolateral membrane, are altered by cholesterol depletion, whereas agents that do not interact with receptors or other basolateral membrane components are not affected by this treatment. In either case, cholesterol depletion of the apical membrane does not affect the increase in J(w) brought about by an increase in intracellular cAMP concentration.

The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

PubMed

Consonni, Sarah V; Brouwer, Patricia M; van Slobbe, Eleonora S; Bos, Johannes L

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PubMed Central

Consonni, Sarah V.; Brouwer, Patricia M.; van Slobbe, Eleonora S.; Bos, Johannes L.

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid. PMID:24858808

Differential patterns of inhibition of the sugar transporters GLUT2, GLUT5 and GLUT7 by flavonoids.

PubMed

Gauer, Julia S; Tumova, Sarka; Lippiat, Jonathan D; Kerimi, Asimina; Williamson, Gary

2018-06-01

Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[ 14 C(U)]-glucose or D-[ 14 C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (-)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings. Copyright © 2018 Elsevier Inc. All rights reserved.

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells

PubMed Central

Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J.; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2016-01-01

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change. PMID:26879757

Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

PubMed

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K

2006-01-01

Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

PubMed Central

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K.

2005-01-01

Purpose Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. Methods HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. Results The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. Conclusions These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia. PMID:16384952

Placental glucose transporter (GLUT)-1 is down-regulated in preeclampsia.

PubMed

Lüscher, Benjamin P; Marini, Camilla; Joerger-Messerli, Marianne S; Huang, Xiao; Hediger, Matthias A; Albrecht, Christiane; Baumann, Marc U; Surbek, Daniel V

2017-07-01

Transplacental fetal glucose supply is predominantly regulated by glucose transporter-1 (GLUT1). Altered expression and/or function of GLUT1 may affect the intrauterine environment, which could compromise fetal development and may contribute to fetal programming. To date it is unknown whether placental GLUT1 is affected by preeclampsia, which is often associated with intrauterine growth restriction (IUGR). We addressed the hypothesis that preeclampsia leads to decreased expression and function of placental GLUT1. Placentae were obtained following normal pregnancy and from pregnancies affected by preeclampsia. Washed villous tissue fragments were used to prepare syncytial microvillous (MVM) and basal plasma membranes (BM) microvesicles. GLUT1 protein and mRNA expression was assessed by western blot analysis and qPCR using Fast SYBR Green. A radio-labeled glucose up-take assay using placenta-derived syncytial microvesicles was used to analyze GLUT1 function. GLUT1 protein expression was significantly down-regulated in (apical) MVM of the syncytiotrophoblast in preeclampsia (n = 6) compared to controls (n = 6) (0.40 ± 0.04 versus 1.00 ± 0.06, arbitrary units, P < 0.001, Student's t-test), while GLUT1 mRNA expression did not show a significant difference. In addition, the functional assay in syncytial microvesicles showed a significantly decreased glucose transport activity in preeclampsia (61.78 ± 6.48%, P < 0.05) compared to controls. BM GLUT1 protein expression was unchanged and glucose up-take into BM microvesicles showed no differences between the preeclampsia and control groups. Our study shows for the first time that in preeclampsia placental GLUT1 expression and function are down-regulated at the apical plasma membrane of the syncytiotrophoblast. Further studies are needed to assess whether these changes occur also in vivo and contribute to the development of IUGR in preeclampsia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Voltage-gated sodium channels in taste bud cells.

PubMed

Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

2009-03-12

Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

Differentiation-dependent rearrangements of actin filaments and microtubules hinder apical endocytosis in urothelial cells.

PubMed

Tratnjek, Larisa; Romih, Rok; Kreft, Mateja Erdani

2017-08-01

During differentiation, superficial urothelial cells (UCs) of the urinary bladder form the apical surface, which is almost entirely covered by urothelial plaques containing densely packed uroplakin particles. These urothelial plaques are the main structural components of the blood-urine permeability barrier in the urinary bladder. We have shown previously that endocytosis from the apical plasma membrane decreases during urothelial cell differentiation. Here, we investigated the role of actin filament and microtubule rearrangements in apical endocytosis of differentiating UCs cells using hyperplastic and normoplastic porcine urothelial models. Partially differentiated normal porcine UCs contained actin filaments in the subapical cytoplasm, while microtubules had a net-like appearance. In highly differentiated UCs, actin filaments mostly disappeared from the subapical cytoplasm and microtubules remained as a thin layer close to the apical plasma membrane. Inhibition of actin filament formation with cytochalasin-D in partially differentiated UCs caused a decrease in apical endocytosis. Depolymerisation of microtubules with nocodazole did not prevent endocytosis of the endocytotic marker WGA into the subapical cytoplasm; however, it abolished WGA transport to endolysosomal compartments in the central cytoplasm. Cytochalasin-D or nocodazole treatment did not significantly change apical endocytosis in highly differentiated UCs. In conclusion, we showed that the physiological differentiation-dependent or chemically induced redistribution and reorganization of actin filaments and microtubules impair apical endocytosis in UCs. Importantly, reduced apical endocytosis due to cytoskeletal rearrangements in highly differentiated UCs, together with the formation of rigid urothelial plaques, reinforces the barrier function of the urothelium.

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

PubMed Central

McAlroy, H L; Ahmed, S; Day, S M; Baines, D L; Wong, H Y; Yip, C Y; Ko, W H; Wilson, S M; Collett, A

2000-01-01

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. PMID:11139443

Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells

PubMed Central

Mrozowska, Paulina S.; Fukuda, Mitsunori

2016-01-01

ABSTRACT The characteristic feature of polarity establishment in MDCK II cells is transcytosis of apical glycoprotein podocalyxin (PCX) from the outer plasma membrane to the newly formed apical domain. This transcytotic event consists of multiple steps, including internalization from the plasma membrane, transport through early endosomes and Rab11-positive recycling endosomes, and delivery to the apical membrane. These steps are known to be tightly coordinated by Rab small GTPases, which act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. However, our knowledge regarding which sets of Rabs regulate particular steps of PCX trafficking was rather limited. Recently, we have performed a comprehensive analysis of Rab GTPase engagement in the transcytotic pathway of PCX during polarity establishment in 2-dimensional (2D) and 3-dimensional (3D) MDCK II cell cultures. In this Commentary we summarize our findings and set them in the context of previous reports. PMID:27463697

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance.

PubMed

Lange, K; Gartzke, J

2001-08-01

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin

PubMed Central

Masedunskas, Andrius; Chen, Yun; Stussman, Rebecca; Weigert, Roberto; Mather, Ian H.

2017-01-01

The lipid droplet (LD) fraction of milk has attracted special attention because it supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism. Because many aspects of this key process remain uncharacterized, we developed a facile method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. Using these techniques, we describe the first kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0–2 μm/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in apical nucleation centers, either close to or in association with the apical surface. Droplets even continued to expand as they were emerging from the cell. Contrary to expectations, LDs attached to the apical plasma membrane but still associated with the cytoplasm were released after oxytocin-mediated contraction of the myoepithelium. Thus milk LD secretion is an intermittently regulated process. This novel procedure will have broad application for investigating trafficking events within the mammary epithelium in real time. PMID:28179456

Trafficking to the Apical and Basolateral Membranes in Polarized Epithelial Cells

PubMed Central

Stoops, Emily H.

2014-01-01

Renal epithelial cells must maintain distinct protein compositions in their apical and basolateral membranes in order to perform their transport functions. The creation of these polarized protein distributions depends on sorting signals that designate the trafficking route and site of ultimate functional residence for each protein. Segregation of newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur at the trans-Golgi network, recycling endosomes, or a growing assortment of stations along the cellular trafficking pathway. The nature of the specific sorting signal and the mechanism through which it is interpreted can influence the route a protein takes through the cell. Cell type–specific variations in the targeting motifs of a protein, as are evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting pathways to different developmental states or physiologic requirements. This review summarizes our current understanding of apical and basolateral trafficking routes in polarized epithelial cells. PMID:24652803

Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif.

PubMed

Jespersen, Thomas; Rasmussen, Hanne B; Grunnet, Morten; Jensen, Henrik S; Angelo, Kamilla; Dupuis, Delphine S; Vogel, Lotte K; Jorgensen, Nanna K; Klaerke, Dan A; Olesen, Søren-Peter

2004-09-01

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE beta-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38-40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel.

Vigabatrin transport across the human intestinal epithelial (Caco-2) brush-border membrane is via the H+-coupled amino-acid transporter hPAT1

PubMed Central

Abbot, Emily L; Grenade, Danielle S; Kennedy, David J; Gatfield, Kelly M; Thwaites, David T

2005-01-01

The aim of this investigation was to determine if the human proton-coupled amino-acid transporter 1 (hPAT1 or SLC36A1) is responsible for the intestinal uptake of the orally-administered antiepileptic agent 4-amino-5-hexanoic acid (vigabatrin). The Caco-2 cell line was used as a model of the human small intestinal epithelium. Competition experiments demonstrate that [3H]GABA uptake across the apical membrane was inhibited by vigabatrin and the GABA analogues trans-4-aminocrotonic acid (TACA) and guvacine, whereas 1-(aminomethyl)cyclohexaneacetic acid (gabapentin) had no affect. Experiments with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded Caco-2 cells demonstrate that apical exposure to vigabatrin and TACA induce comparable levels of intracellular acidification (due to H+/amino-acid symport) to that generated by GABA, suggesting that they are substrates for a H+-coupled absorptive transporter such as hPAT1. In hPAT1 and mPAT1-expressing Xenopus laevis oocytes [3H]GABA uptake was inhibited by vigabatrin, TACA and guvacine, whereas gabapentin failed to inhibit [3H]GABA uptake. In Na+-free conditions, vigabatrin and TACA evoked similar current responses (due to H+/amino-acid symport) in hPAT1-expressing oocytes under voltage-clamp conditions to that induced by GABA (whereas no current was observed in water-injected oocytes) consistent with the ability of these GABA analogues to inhibit [3H]GABA uptake. This study demonstrates that hPAT1 is the carrier responsible for the uptake of vigabatrin across the brush-border membrane of the small intestine and emphasises the therapeutic potential of hPAT1 as a delivery route for orally administered, clinically significant GABA-related compounds. PMID:16331283

Genuine functions of P-glycoprotein (ABCB1).

PubMed

Mizutani, Takaharu; Masuda, Masatoshi; Nakai, Emi; Furumiya, Kenji; Togawa, Hiroshi; Nakamura, Yutaka; Kawai, Yuko; Nakahira, Keiko; Shinkai, Shigeko; Takahashi, Kazuhiko

2008-02-01

P-glycoprotein (P-gp, ABCB1, MDR1) was recognized as a drug-exporting protein from cancer cells three decade ago. Apart from the multidrug transporter side effects of P-gp, normal physiological functions of P-gp have been reported. P-gp could be responsible for translocating platelet-activating factor (PAF) across the plasma membrane and PAF inhibited drug transport mediated by P-gp in cancer cells. P-gp regulated the translocation of sphingomyelin (SM) and GlcCer, and short chain C(6)-NBD-GlcCer was found in the apical medium of P-gp cells exclusively and not in the basolateral membrane. SM plays an important role in the esterification of cholesterol. High expression of P-gp prevents stem-cell differentiation, leading to the proliferation and amplification of this cell repertoire, and functional P-gp plays a fundamental role in regulating programmed cell death, apoptosis. The transporter function of P-gp is therefore necessary to protect cells from death. P-gp can translocate both C(6)-NBD-PC and C(6)-NBD-PE across the apical membrane. This PC translocation was also confirmed with [(3)H]choline radioactivity. Progesterone is not transported by P-gp, but blocks P-gp-mediated efflux of other drugs and P-gp can mediate the transport of a variety of steroids. Cells transfected with human P-gp esterified more cholesterol. P-gp might also be involved in the transport of cytokines, particularly IL-1beta, IL-2, IL-4 and IFNgamma, out of activated normal lymphocytes into the surrounding medium. P-gp expression is also associated with a volume-activated chloride channel, thus P-gp is bifunctional with both transport and channel regulators. We also present information about P-gp polymorphism and new structural concepts, "gate" and "twist", of the P-gp structure.

Expression of aquaporin water channels in rat taste buds.

PubMed

Watson, Kristina J; Kim, Insook; Baquero, Arian F; Burks, Catherine A; Liu, Lidong; Gilbertson, Timothy A

2007-06-01

In order to gain insight into the molecular mechanisms that allow taste cells to respond to changes in their osmotic environment, we have used primarily immunocytochemical and molecular approaches to look for evidence of the presence of aquaporin-like water channels in taste cells. Labeling of isolated taste buds from the fungiform, foliate, and vallate papillae in rat tongue with antibodies against several of the aquaporins (AQPs) revealed the presence of AQP1, AQP2, and AQP5 in taste cells from these areas. AQP3 antibodies failed to label isolated taste buds from any of the papillae. There was an apparent difference in the regional localization of AQP labeling within the taste bud. Antibodies against AQP1 and AQP2 labeled predominantly the basolateral membrane, whereas the AQP5 label was clearly evident on both the apical and basolateral membranes of cells within the taste bud. Double labeling revealed that AQP1 and AQP2 labeled many, but not all, of the same taste cells. Similar double-labeling experiments with anti-AQP2 and anti-AQP5 clearly showed that AQP5 was expressed on or near the apical membranes whereas AQP2 was absent from this area. The presence of these 3 types of AQPs in taste buds but not in non-taste bud-containing epithelia was confirmed using reverse transcription-polymerase chain reaction. Experiments using patch clamp recording showed that the AQP inhibitor, tetraethylammonium, significantly reduced hypoosmotic-induced currents in rat taste cells. We hypothesize that the AQPs may play roles both in the water movement underlying compensatory mechanisms for changes in extracellular osmolarity and, in the case of AQP5 in particular, in the gustatory response to water.

Investigating CFTR and KCa3.1 Protein/Protein Interactions

PubMed Central

Trinh, Nguyen Thu Ngan; Luo, Yishan; Wiseman, Paul W.; Hanrahan, John W.; Brochiero, Emmanuelle; Sauvé, Rémy

2016-01-01

In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca2+. PMID:27092946

Identification of moesin as NKCC2-interacting protein and analysis of its functional role in the NKCC2 apical trafficking.

PubMed

Carmosino, Monica; Rizzo, Federica; Procino, Giuseppe; Zolla, Lello; Timperio, Anna Maria; Basco, Davide; Barbieri, Claudia; Torretta, Silvia; Svelto, Maria

2012-11-01

The renal Na(+) -K(+) -2Cl(-) co-transporter (NKCC2) is expressed in kidney thick ascending limb cells, where it mediates NaCl re-absorption regulating body salt levels and blood pressure. In this study, we used a well-characterised NKCC2 construct (c-NKCC2) to identify NKCC2-interacting proteins by an antibody shift assay coupled with blue native/SDS-PAGE and mass spectrometry. Among the interacting proteins, we identified moesin, a protein belonging to ezrin, eadixin and moesin family. Co-immunoprecipitation experiments confirmed that c-NKCC2 interacts with the N-terminal domain of moesin in LLC-PK1 cells. Moreover, c-NKCC2 accumulates in intracellular and sub-apical vesicles in cells transfected with a moesin dominant negative green fluorescent protien (GFP)-tagged construct. In addition, moesin knock-down by short interfering RNA decreases by about 50% c-NKCC2 surface expression. Specifically, endocytosis and exocytosis assays showed that moesin knock-down does not affect c-NKCC2 internalisation but strongly reduces exocytosis of the co-transporter. Our data clearly demonstrate that moesin plays a critical role in apical membrane insertion of NKCC2, suggesting a possible involvement of moesin in regulation of Na(+) and Cl(-) absorption in the kidney. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Transport of choline by Madin-Darby canine kidney cells.

PubMed

Zlatkine, P; Moll, G; Blais, A; Loiseau, A; Le Grimellec, C

1993-12-12

Choline is an essential precursor for the synthesis of phosphatidylcholine, the most abundant phospholipid classes in renal cells, as well as for the synthesis of the osmolyte glycerophosphorylcholine. The characteristics of choline uptake in the renal epithelial cell line MDCK were investigated. In the range of physiological concentrations, choline entered MDCK cells, grown as a monolayer on solid support, via a specific sodium-independent transport system (apparent Km = 43 microM, apparent Vmax = 284 pmol/mg protein per 5 min). Cell ATP depletion, addition of KCl to the medium to reduce the cell membrane potential, and hemicholinium-3 (HC-3) inhibited choline uptake. Specific binding of [3H]HC-3 was detected on the apical membrane of cells grown on plastic dishes, whereas it occurred only on the basolateral domain of cells grown on permeant support. When growing cells on filter, choline uptake from the basolateral side was 10-times the apical uptake. This suggests that the choline carrier present at the apical domain of cells grown on solid support is either inactivated or no longer targeted to the apical but to the basolateral membrane of MDCK cells grown on filter.

Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

PubMed

Michalec, Katarzyna; Mysiorek, Caroline; Kuntz, Mélanie; Bérézowski, Vincent; Szczepankiewicz, Andrzej A; Wilczyński, Grzegorz M; Cecchelli, Roméo; Nałęcz, Katarzyna A

2014-07-15

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated. Copyright © 2014 Elsevier Inc. All rights reserved.

Numb controls E-cadherin endocytosis through p120 catenin with aPKC

PubMed Central

Sato, Kazuhide; Watanabe, Takashi; Wang, Shujie; Kakeno, Mai; Matsuzawa, Kenji; Matsui, Toshinori; Yokoi, Keiko; Murase, Kiyoko; Sugiyama, Ikuko; Ozawa, Masayuki; Kaibuchi, Kozo

2011-01-01

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity. PMID:21775625

Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

PubMed Central

Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

2015-01-01

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

Larval body patterning and apical organs are conserved in animal evolution

PubMed Central

2014-01-01

Background Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data. Results To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla. Conclusions The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae. PMID:24476105

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

PubMed Central

Lapierre, Lynne A.; Kumar, Ravindra; Hales, Chadwick M.; Navarre, Jennifer; Bhartur, Sheela G.; Burnette, Jason O.; Provance, D. William; Mercer, John A.; Bähler, Martin; Goldenring, James R.

2001-01-01

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems. PMID:11408590

Plasma membrane-targeted PIN proteins drive shoot development in a moss.

PubMed

Bennett, Tom A; Liu, Maureen M; Aoyama, Tsuyoshi; Bierfreund, Nicole M; Braun, Marion; Coudert, Yoan; Dennis, Ross J; O'Connor, Devin; Wang, Xiao Y; White, Chris D; Decker, Eva L; Reski, Ralf; Harrison, C Jill

2014-12-01

Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture

PubMed Central

Rice, William L.; Li, Wei; Mamuya, Fahmy; McKee, Mary; Păunescu, Teodor G.; Lu, Hua A. Jenny

2015-01-01

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro. PMID:26147297

MST4 kinase phosphorylates ACAP4 protein to orchestrate apical membrane remodeling during gastric acid secretion.

PubMed

Yuan, Xiao; Yao, Phil Y; Jiang, Jiying; Zhang, Yin; Su, Zeqi; Yao, Wendy; Wang, Xueying; Gui, Ping; Mullen, McKay; Henry, Calmour; Ward, Tarsha; Wang, Wenwen; Brako, Larry; Tian, Ruijun; Zhao, Xuannv; Wang, Fengsong; Cao, Xinwang; Wang, Dongmei; Liu, Xing; Ding, Xia; Yao, Xuebiao

2017-09-29

Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,K-ATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr 545 Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts with MST4 and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr 545 by mass spectrometric analyses. Importantly, phosphorylation of Thr 545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylation-deficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr 545 enables ACAP4 to interact with ezrin. Given the location of Thr 545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus

PubMed Central

Kamsteeg, Erik-Jan; Bichet, Daniel G.; Konings, Irene B.M.; Nivet, Hubert; Lonergan, Michelle; Arthus, Marie-Françoise; van Os, Carel H.; Deen, Peter M.T.

2003-01-01

Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease. PMID:14662748

Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270

PubMed Central

1985-01-01

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells. PMID:2409219

Drug transporters, the blood–testis barrier, and spermatogenesis

PubMed Central

Su, Linlin; Mruk, Dolores D; Cheng, C Yan

2015-01-01

The blood–testis barrier (BTB), which is created by adjacent Sertoli cells near the basement membrane, serves as a ‘gatekeeper’ to prohibit harmful substances from reaching developing germ cells, most notably postmeiotic spermatids. The BTB also divides the seminiferous epithelium into the basal and adluminal (apical) compartment so that postmeiotic spermatid development, namely spermiogenesis, can take place in a specialized microenvironment in the apical compartment behind the BTB. The BTB also contributes, at least in part, to the immune privilege status of the testis, so that anti-sperm antibodies are not developed against antigens that are expressed transiently during spermatogenesis. Recent studies have shown that numerous drug transporters are expressed by Sertoli cells. However, many of these same drug transporters are also expressed by spermatogonia, spermatocytes, round spermatids, elongating spermatids, and elongated spermatids, suggesting that the developing germ cells are also able to selectively pump drugs ‘in’ and/or ‘out’ via influx or efflux pumps. We review herein the latest developments regarding the role of drug transporters in spermatogenesis. We also propose a model utilized by the testis to protect germ cell development from ‘harmful’ environmental toxicants and xenobiotics and/or from ‘therapeutic’ substances (e.g. anticancer drugs). We also discuss how drug transporters that are supposed to protect spermatogenesis can work against the testis in some instances. For example, when drugs (e.g. male contraceptives) that can perturb germ cell adhesion and/or maturation are actively pumped out of the testis or are prevented from entering the apical compartment, such as by efflux pumps. PMID:21134990

Pendred syndrome.

PubMed

Wémeau, Jean-Louis; Kopp, Peter

2017-03-01

Pendred syndrome is an autosomal recessive disorder that is classically defined by the combination of sensorineural deafness/hearing impairment, goiter, and an abnormal organification of iodide with or without hypothyroidism. The hallmark of the syndrome is the impaired hearing, which is associated with inner ear malformations such as an enlarged vestibular aqueduct (EVA). The thyroid phenotype is variable and may be modified by the nutritional iodine intake. Pendred syndrome is caused by biallelic mutations in the SLC26A4/PDS gene, which encodes the multifunctional anion exchanger pendrin. Pendrin has affinity for chloride, iodide, and bicarbonate, among other anions. In the inner ear, pendrin functions as a chloride/bicarbonate exchanger that is essential for maintaining the composition and the potential of the endolymph. In the thyroid, pendrin is expressed at the apical membrane of thyroid cells facing the follicular lumen. Functional studies have demonstrated that pendrin can mediate iodide efflux in heterologous cells. This, together with the thyroid phenotype observed in humans (goiter, impaired iodine organification) suggests that pendrin could be involved in iodide efflux into the lumen, one of the steps required for thyroid hormone synthesis. Iodide efflux can, however, also occur in the absence of pendrin suggesting that other exchangers or channels are involved. It has been suggested that Anoctamin 1 (ANO1/TMEM16A), a calcium-activated anion channel, which is also expressed at the apical membrane of thyrocytes, could participate in mediating apical efflux. In the kidney, pendrin is involved in bicarbonate secretion and chloride reabsorption. While there is no renal phenotype under basal conditions, severe metabolic alkalosis has been reported in Pendred syndrome patients exposed to an increased alkali load. This review provides an overview on the clinical spectrum of Pendred syndrome, the functional data on pendrin with a focus on its potential role in the thyroid, as well as the controversy surrounding the relative physiological roles of pendrin and anoctamin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aldosterone regulation of sodium and potassium transport in the cortical collecting duct.

PubMed

O'Neil, R G

1990-07-01

The aldosterone-induced up-regulation of Na absorption and K secretion in the CCD is complex and involves the regulation of numerous transport proteins. Some aspects of the response may be species dependent. For example, stimulation of Na and K transport in the rabbit CCD involves a marked up-regulation in the apical cell membrane Na and K conductances, the basolateral cell membrane K conductance, and the basolateral membrane NaK-ATPase activity. In the rat CCD, aldosterone causes a similar up-regulation in the NaK-ATPase and the apical membrane Na conductance, but supposedly has little influence on the apical and basolateral membrane K conductances as evaluated by indirect methods. Furthermore, the marked hyperpolarization of the basolateral membrane with long-term aldosterone treatment in the rabbit CCD is blunted or absent in the rat CCD. Other differences between the CCD of these two species have been outlined. Nonetheless, the basic responses of the CCDs from the two species show similar trends. The actions of aldosterone in the CCD principal cell are summarized in Figure 5. The initial steps have been described previously. Aldosterone (A) diffuse across the cell membrane and binds to a cytoplasmic receptor (R). The receptor complex moves into the nucleus and binds to an acceptor site on chromatin, initiating transcription and the subsequent synthesis of a myriad of new proteins referred to as aldosterone-induced proteins (AIP). The initial observed action of aldosterone is an upregulation of the apical membrane Na conductance during the early phase, which occurs within 1 to 2 hours. The increase in Na conductance likely reflects activation of preexisting latent Na channels and not synthesis of new channels, although activation does require protein synthesis. The increased Na influx during the early phase presents a larger Na load to the Na pump, which is likely reflected as a modest transient increase in intracellular Na activity. Based on kinetic considerations alone, this should cause an increased transport turnover of the pump with a greater Na extrusion rate and K uptake rate. The stimulated Na influx also causes a modest depolarization of the apical membrane during the early phase, which when combined with the increased K uptake via the pump and an apparent modest elevation in the intracellular K activity, results in a more favorable gradient for K secretion (increased driving force) into the tubule lumen.(ABSTRACT TRUNCATED AT 400 WORDS)

Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes

PubMed Central

Blank, Christian A.; Anderson, David A.; Beard, Michael; Lemon, Stanley M.

2000-01-01

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ± 12.5% of the total virus yield compared with 0.23% ± 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms. PMID:10864660

Sweet taste receptors in rat small intestine stimulate glucose absorption through apical GLUT2.

PubMed

Mace, Oliver J; Affleck, Julie; Patel, Nick; Kellett, George L

2007-07-01

Natural sugars and artificial sweeteners are sensed by receptors in taste buds. T2R bitter and T1R sweet taste receptors are coupled through G-proteins, alpha-gustducin and transducin, to activate phospholipase C beta2 and increase intracellular calcium concentration. Intestinal brush cells or solitary chemosensory cells (SCCs) have a structure similar to lingual taste cells and strongly express alpha-gustducin. It has therefore been suggested over the last decade that brush cells may participate in sugar sensing by a mechanism analogous to that in taste buds. We provide here functional evidence for an intestinal sensing system based on lingual taste receptors. Western blotting and immunocytochemistry revealed that all T1R members are expressed in rat jejunum at strategic locations including Paneth cells, SCCs or the apical membrane of enterocytes; T1Rs are colocalized with each other and with alpha-gustducin, transducin or phospholipase C beta2 to different extents. Intestinal glucose absorption consists of two components: one is classical active Na+-glucose cotransport, the other is the diffusive apical GLUT2 pathway. Artificial sweeteners increase glucose absorption in the order acesulfame potassium approximately sucralose > saccharin, in parallel with their ability to increase intracellular calcium concentration. Stimulation occurs within minutes by an increase in apical GLUT2, which correlates with reciprocal regulation of T1R2, T1R3 and alpha-gustducin versus T1R1, transducin and phospholipase C beta2. Our observation that artificial sweeteners are nutritionally active, because they can signal to a functional taste reception system to increase sugar absorption during a meal, has wide implications for nutrient sensing and nutrition in the treatment of obesity and diabetes.

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

PubMed Central

Li, Cunxi; Hao, Mingming; Cao, Zheng; Ding, Wei; Graves-Deal, Ramona; Hu, Jianyong; Piston, David W.

2007-01-01

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells. PMID:17553928

Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

PubMed Central

Kenworthy, A.K.; Edidin, M.

1998-01-01

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins. PMID:9660864

Conductive choline transport by alveolar epithelial plasma membrane vesicles.

PubMed

Oelberg, D G; Xu, F

1998-11-01

Choline is an important substrate in alveolar epithelia for both surfactant production and cellular maintenance. The underlying mechanisms of uptake and sites of membrane transport remain uncertain. To test the hypothesis that choline transport occurs at the basolateral side of alveolar epithelia by both Na+-independent and -dependent mechanisms, plasma membrane vesicles were prepared from the apical and basolateral membranes of mature porcine type II pneumocytes. Choline+ transport was assayed by uptake of [3H]choline+ by enriched apical or basolateral vesicles. In the presence of imposed, inside-negative charge gradients, basolateral vesicles exhibited early overshoot of [3H]choline+ uptake unaffected by the presence or absence of external Na+ (541 +/- 53 vs 564 +/- 79 pmol/mg protein (NS)). High sensitivity to hemicholinium-3 was observed in the presence or absence of Na+. In the absence of inside-negative charge gradients, uptake was reduced 12-fold in the presence or absence of Na+, and external choline+ induced internal alkalization of acidified basolateral vesicles. Accumulative [3H]choline+ uptakes by apical vesicles in the presence or absence of inside-negative charge gradients and Na+ were insignificant. We conclude that predominant choline+ uptake by type II pneumocytes occurs at the basolateral membrane by Na+-independent, electrogenic choline+ conductance. The presence of electroneutral choline+/H+ exchange is suggested. Copyright 1998 Academic Press.

Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis.

PubMed

Takahama, A; Rôças, I N; Faustino, I S P; Alves, F R F; Azevedo, R S; Gomes, C C; Araújo-Filho, W R; Siqueira, J F

2018-07-01

To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated. Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant. All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found. Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis. © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

PubMed

Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

2015-04-01

The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Ion transport studies with H+-K+-ATPase-rich vesicles: implications for HCl secretion and parietal cell physiology.

PubMed

Wolosin, J M

1985-06-01

A summary of recent studies on relations between the properties of the membrane incorporating the H+-K+-ATPase, the H+ motive force in gastric acid secretion, and the secretory state of the parietal cell is presented. Depending on tissue secretory state, two distinct H+-K+-ATPase-rich membranes predominate in tissue homogenates, the gastric microsomes derived from the intracellular tubulovesicles of the resting cell and the stimulation-associated (SA) vesicle derived from the apical membrane of the acid-secreting cell. Structural and chemical differences between both vesicular types lend support to the notion that the formation of an expanded, elaborated apical membrane in the secreting parietal cell results from fusion of tubulovesicles containing the H+-K+-ATPase to an apical membrane of different chemical composition. Comparison of polypeptide composition of microsomes and SA membranes provides a way to identify and isolate membrane and cytoskeletal components putatively involved in the membrane interconversion process. Comparison of transport properties between gastric microsomes and SA vesicles demonstrates that stimulation triggers the appearance of rapid K+ and Cl- permeabilities in the H+-K+-ATPase membrane, allowing efficient acid accumulation in SA vesicles by the combination of rapid KCl influx followed by ATPase-driven H+ for K+ exchange, i.e., by K+ recycling. These stimulation-triggered conductances are functionally independent. Nevertheless, their concurrent inhibition by certain divalent cations (Mn2+,Zn2+) suggests their location within a single physical domain. The compatibility of the K+-recycling model for HCl accumulation in SA vesicles with gastric HCl secretion and selected electrophysiological observations and certain implications of the findings for cellular mechanisms of transport regulation in the context of a membrane fusion and recycling model are discussed.

Ion transport by primary cultures of canine tracheal epithelium: methodology, morphology, and electrophysiology.

PubMed

Welsh, M J

1985-01-01

Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of C1 secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of C1 secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of C1 secretion by airway epithelia.

Trafficking to the apical and basolateral membranes in polarized epithelial cells.

PubMed

Stoops, Emily H; Caplan, Michael J

2014-07-01

Renal epithelial cells must maintain distinct protein compositions in their apical and basolateral membranes in order to perform their transport functions. The creation of these polarized protein distributions depends on sorting signals that designate the trafficking route and site of ultimate functional residence for each protein. Segregation of newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur at the trans-Golgi network, recycling endosomes, or a growing assortment of stations along the cellular trafficking pathway. The nature of the specific sorting signal and the mechanism through which it is interpreted can influence the route a protein takes through the cell. Cell type-specific variations in the targeting motifs of a protein, as are evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting pathways to different developmental states or physiologic requirements. This review summarizes our current understanding of apical and basolateral trafficking routes in polarized epithelial cells. Copyright © 2014 by the American Society of Nephrology.

Morphology, histochemistry and glycosylation of the placenta and associated tissues in the European hedgehog (Erinaceus europaeus).

PubMed

Jones, Carolyn J P; Carter, A M; Allen, W R; Wilsher, Sandra A

2016-12-01

There are few descriptions of the placenta and associated tissues of the European hedgehog (Erinaceus europaeus) and here we present findings on a near-term pregnant specimen. Tissues were examined grossly and then formalin fixed and wax-embedded for histology and immunocytochemistry (cytokeratin) and resin embedded for lectin histochemistry. Each of four well-developed and near term hoglets displayed a discoid, haemochorial placenta with typical labyrinth and spongy zones. In addition there was a paraplacenta incorporating Reichert's membrane and a largely detached yolk sac. The trophoblast of the placenta contained diverse populations of granule which expressed most classes of glycan. Intercellular membranes were also glycosylated and this tended to be heavier in the labyrinth zone. Fetal capillary endothelium had glycosylated apical surfaces expressing sialic acid and various other glycans. Glycogen was present in large cells situated between the spongy zone and the endometrium. Trophoblast cells in the placental disc and under Reichert's membrane, as well as yolk sac endoderm and mesothelium, were cytokeratin positive. Reichert's membrane was heavily glycosylated. Yolk sac inner and outer endoderm expressed similar glycans except for N-acetylgalactosamine residues in endodermal acini. New features of near-term hedgehog placenta and associated tissues are presented, including their glycosylation, and novel yolk sac acinar structures are described. The trophoblast of the placental disc showed significant differences from that underlying Reichert's membrane while the glycan composition of the membrane itself showed some similarity to that of rat thereby implying a degree of biochemical conservation of this structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

PubMed

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

PubMed Central

Zihni, Ceniz; Vlassaks, Evi; Terry, Stephen; Carlton, Jeremy; Leung, Thomas King Chor; Olson, Michael; Pichaud, Franck; Balda, Maria Susana; Matter, Karl

2017-01-01

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates Myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling. PMID:28825699

Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport.

PubMed

Ehrenfeld, J; Garcia-Romeu, F; Harvey, B J

1985-02-01

Kinetic and electrophysiological studies were carried out in the in vitro Rana esculenta skin, bathed in dilute sodium solution, to characterize the proton pump and coupling between sodium absorption (JNa+n) and proton excretion (JH+n). JNa+n and JH+n were both dependent on transepithelial potential (psi ms); hyperpolarizing the skin decreased JNa+n and increased JH+n; depolarization produced the opposite effects. Amiloride (5 X 10(-5) M) at a clamped psi ms of +50 mV inhibited JNa+n without affecting JH+n. Variations of psi ms or pH had identical effects on JH+n. Ethoxzolamide inhibited JH+n and simultaneously increased psi ms by 15-30 mV. These changes were accompanied by depolarization of the apical membrane potential psi mc from -47 to -25 mV and an increase in apical membrane resistance of 30%; no significant effects on basolateral membrane potential (psi cs) and resistance (Rb) nor on shunt resistance (Rj) were observed. The proton pump appears to be localized at the apical membrane. The proton pump was also inhibited by deoxygenation, oligomycin, dicyclohexylcarbodiimide and vanadate (100, 78, 83 and 100% inhibition respectively). The variations of JH+n and of the measured electrical currents were significantly correlated. These findings are supportive evidence of a primary active proton pump, electrogenic and strictly linked to aerobic metabolism. The current-voltage (I-V) relation of the proton pump was obtained as the difference in the I-V curves of the apical membrane extracted before and after proton-pump inhibition by ethoxzolamide during amiloride block of sodium transport. The proton-pump current (IP) was best described by a saturable exponential function of psi mc. Maximal pump current (ImaxP) was calculated to be 200 nequiv h-1 cm-2 at a psi mc of +50 mV and the pump reversal potential ERP was -130 mV. The effect of ethoxzolamide to depolarize psi mc was dependent on the relation between psi mc and ERP. Maximal induced depolarization occurred at a psi mc of +50 mV whereas ethoxzolamide exerted minimal effect on psi mc when the ERP was approached either by voltage clamping the apical membrane or by the addition of amiloride. We show that electroneutral sodium-proton countertransport is not the mechanism of active proton excretion in frog skin but that it is the proton excretion which provides a favourable electrical driving force for passive apical sodium entry.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport.

PubMed Central

Ehrenfeld, J; Garcia-Romeu, F; Harvey, B J

1985-01-01

Kinetic and electrophysiological studies were carried out in the in vitro Rana esculenta skin, bathed in dilute sodium solution, to characterize the proton pump and coupling between sodium absorption (JNa+n) and proton excretion (JH+n). JNa+n and JH+n were both dependent on transepithelial potential (psi ms); hyperpolarizing the skin decreased JNa+n and increased JH+n; depolarization produced the opposite effects. Amiloride (5 X 10(-5) M) at a clamped psi ms of +50 mV inhibited JNa+n without affecting JH+n. Variations of psi ms or pH had identical effects on JH+n. Ethoxzolamide inhibited JH+n and simultaneously increased psi ms by 15-30 mV. These changes were accompanied by depolarization of the apical membrane potential psi mc from -47 to -25 mV and an increase in apical membrane resistance of 30%; no significant effects on basolateral membrane potential (psi cs) and resistance (Rb) nor on shunt resistance (Rj) were observed. The proton pump appears to be localized at the apical membrane. The proton pump was also inhibited by deoxygenation, oligomycin, dicyclohexylcarbodiimide and vanadate (100, 78, 83 and 100% inhibition respectively). The variations of JH+n and of the measured electrical currents were significantly correlated. These findings are supportive evidence of a primary active proton pump, electrogenic and strictly linked to aerobic metabolism. The current-voltage (I-V) relation of the proton pump was obtained as the difference in the I-V curves of the apical membrane extracted before and after proton-pump inhibition by ethoxzolamide during amiloride block of sodium transport. The proton-pump current (IP) was best described by a saturable exponential function of psi mc. Maximal pump current (ImaxP) was calculated to be 200 nequiv h-1 cm-2 at a psi mc of +50 mV and the pump reversal potential ERP was -130 mV. The effect of ethoxzolamide to depolarize psi mc was dependent on the relation between psi mc and ERP. Maximal induced depolarization occurred at a psi mc of +50 mV whereas ethoxzolamide exerted minimal effect on psi mc when the ERP was approached either by voltage clamping the apical membrane or by the addition of amiloride. We show that electroneutral sodium-proton countertransport is not the mechanism of active proton excretion in frog skin but that it is the proton excretion which provides a favourable electrical driving force for passive apical sodium entry.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 6 Fig. 7 PMID:2582114

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells.

PubMed

Procino, Giuseppe; Carmosino, Monica; Tamma, Grazia; Gouraud, Sabine; Laera, Antonia; Riccardi, Daniela; Svelto, Maria; Valenti, Giovanna

2004-12-01

Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.

Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation

PubMed Central

He, Bing; Doubrovinski, Konstantin; Polyakov, Oleg; Wieschaus, Eric

2014-01-01

Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is employed throughout the development in most animals1. Little is known, however, how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow (VF) formation2, 3. We find that cytoplasmic redistribution during the lengthening phase of VF formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to or driving force on the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells prior to gastrulation (“acellular” embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild type embryos. Our results suggest that during the lengthening phase of VF formation, hydrodynamic behavior of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable. PMID:24590071

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells.

PubMed

Saksena, Seema; Theegala, Saritha; Bansal, Nikhil; Gill, Ravinder K; Tyagi, Sangeeta; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

2009-11-01

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM ( approximately 60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V(max)) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K(m)). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans▿†

PubMed Central

Hervás-Aguilar, América; Peñalva, Miguel A.

2010-01-01

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA. PMID:20693304

Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

PubMed

Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

2017-09-01

Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ

PubMed Central

Koyama, E.; Saunders, C.; Salhab, I.; Decker, R.S.; Chen, I.; Um, H.; Pacifici, M.; Nah, H.D.

2014-01-01

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4 –/– mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K- expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life. PMID:24834922

Functional characteristics of a renal H+/lipophilic cation antiport system in porcine LLC-PK1 cells and rats.

PubMed

Matsui, Ryutaro; Hattori, Ryutaro; Usami, Youhei; Koyama, Masumi; Hirayama, Yuki; Matsuba, Emi; Hashimoto, Yukiya

2018-02-01

We have recently found an H + /quinidine (a lipophilic cation, QND) antiport system in Madin-Darby canine kidney (MDCK) cells. The primary aim of the present study was to evaluate whether the H + /lipophilic cation antiport system is expressed in porcine LLC-PK 1 cells. That is, we investigated uptake and/or efflux of QND and another cation, bisoprolol, in LLC-PK 1 cells. In addition, we studied the renal clearance of bisoprolol in rats. Uptake of QND into LLC-PK 1 cells was decreased by acidification of the extracellular pH or alkalization of the intracellular pH. Cellular uptake of QND from the apical side was much greater than from the basolateral side. In addition, apical efflux of QND from LLC-PK 1 cells was increased by acidification of the extracellular pH. Furthermore, lipophilic cationic drugs significantly reduced uptake of bisoprolol in LLC-PK 1 cells. Renal clearance of bisoprolol in rats was approximately 7-fold higher than that of creatinine, and was markedly decreased by alkalization of the urine pH. The present study suggests that the H + /lipophilic cation antiport system is expressed in the apical membrane of LLC-PK 1 cells. Moreover, the H + /lipophilic cation antiport system may be responsible for renal tubular secretion of bisoprolol in rats. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Petunia Phospholipase C1 Is Involved in Pollen Tube Growth[W

PubMed Central

Dowd, Peter E.; Coursol, Sylvie; Skirpan, Andrea L.; Kao, Teh-hui; Gilroy, Simon

2006-01-01

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)–cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC δ1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell. PMID:16648366

Molecular characterization of the apical organ of the anthozoan Nematostella vectensis

PubMed Central

Sinigaglia, Chiara; Busengdal, Henriette; Lerner, Avi; Oliveri, Paola; Rentzsch, Fabian

2015-01-01

Apical organs are sensory structures present in many marine invertebrate larvae where they are considered to be involved in their settlement, metamorphosis and locomotion. In bilaterians they are characterised by a tuft of long cilia and receptor cells and they are associated with groups of neurons, but their relatively low morphological complexity and dispersed phylogenetic distribution have left their evolutionary relationship unresolved. Moreover, since apical organs are not present in the standard model organisms, their development and function are not well understood. To provide a foundation for a better understanding of this structure we have characterised the molecular composition of the apical organ of the sea anemone Nematostella vectensis. In a microarray-based comparison of the gene expression profiles of planulae with either a wildtype or an experimentally expanded apical organ, we identified 78 evolutionarily conserved genes, which are predominantly or specifically expressed in the apical organ of Nematostella. This gene set comprises signalling molecules, transcription factors, structural and metabolic genes. The majority of these genes, including several conserved, but previously uncharacterized ones, are potentially involved in different aspects of the development or function of the long cilia of the apical organ. To demonstrate the utility of this gene set for comparative analyses, we further analysed the expression of a subset of previously uncharacterized putative orthologs in sea urchin larvae and detected expression for twelve out of eighteen of them in the apical domain. Our study provides a molecular characterization of the apical organ of Nematostella and represents an informative tool for future studies addressing the development, function and evolutionary history of apical organ cells. PMID:25478911

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

PubMed Central

Bennett, Kaila M.; Walker, Sharon L.

2014-01-01

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli (“microvillus-minus,” or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions. PMID:24778113

Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation.

PubMed

Wu, Alex Man Lai; Dedina, Liana; Dalvi, Pooja; Yang, Mingdong; Leon-Cheon, John; Earl, Brian; Harper, Patricia A; Ito, Shinya

2016-04-01

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second (SLC52A2) and third (SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction ofSlc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk. Copyright © 2016 the American Physiological Society.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

PubMed

Zhang, Nan; Khan, Liakot A; Membreno, Edward; Jafari, Gholamali; Yan, Siyang; Zhang, Hongjie; Gobel, Verena

2017-10-03

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

Ammonia exposure affects the mRNA and protein expression levels of certain Rhesus glycoproteins in the gills of climbing perch.

PubMed

Chen, Xiu L; Zhang, Biyan; Chng, You R; Ong, Jasmine L Y; Chew, Shit F; Wong, Wai P; Lam, Siew H; Nakada, Tsutomu; Ip, Yuen K

2017-08-15

The freshwater climbing perch, Anabas testudineus , is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia, this study aimed to obtain the complete cDNA coding sequences of various rhgp isoforms from the gills of A. testudineus , and to determine their mRNA and protein expression levels during 6 days of exposure to 100 mmol l -1 NH 4 Cl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four rhgp ( rhag , rhbg , rhcg1 and rhcg2 ) had been identified from the gills of A. testudineus They had conserved amino acid residues for NH 4 + binding, NH 4 + deprotonation, channel gating and lining of the vestibules. Despite inwardly directed NH 3 and NH 4 + gradients, there were significant increases in the mRNA expression levels of the four branchial rhgp in A. testudineus at certain time points during 6 days of ammonia exposure, with significant increases in the protein abundances of Rhag and Rhcg2 on day 6. Immunofluorescence microscopy revealed a type of ammonia-inducible Na + /K + -ATPase α1c-immunoreactive ionocyte with apical Rhag and basolateral Rhcg2 in the gills of fish exposed to ammonia for 6 days. Hence, active ammonia excretion may involve NH 4 + entering the ionocyte through the basolateral Rhcg2 and being excreted through the apical Rhag, driven by a transapical membrane electrical potential generated by the apical cystic fibrosis transmembrane conductance regulator Cl - channel, as suggested previously. © 2017. Published by The Company of Biologists Ltd.

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

PubMed

Dezerega, A; Osorio, C; Mardones, J; Mundi, V; Dutzan, N; Franco, M; Gamonal, J; Oyarzún, A; Overall, C M; Hernández, M

2010-10-01

To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry.   MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis. © 2010 International Endodontic Journal.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus.

PubMed

Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K; Lemon, Stanley M

2016-12-06

Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1 -/- Ifngr1 -/- and Mavs -/- mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus. HAV is a hepatotropic, fecally/orally transmitted picornavirus that can cause severe hepatitis in humans. Recent work reveals that it has an unusual life cycle. Virus is found in cell culture supernatant fluids in two mature, infectious forms: one wrapped in membranes (quasi-enveloped) and another that is nonenveloped. Membrane-wrapped virions circulate in blood during acute infection and are resistant to neutralizing antibodies, likely facilitating HAV dissemination within the liver. On the other hand, virus shed in feces is nonenveloped and highly stable, facilitating epidemic spread and transmission to naive hosts. Factors controlling the biogenesis of these two distinct forms of the virus in infected humans are not understood. Here we characterize vectorial release of quasi-enveloped virions from polarized epithelial cell cultures and provide evidence that bile acids strip membranes from eHAV following its secretion into the biliary tract. These results enhance our understanding of the life cycle of this unusual picornavirus. Copyright © 2016 Hirai-Yuki et al.

Ion pathways in the taste bud and their significance for transduction.

PubMed

DeSimone, J A; Ye, Q; Heck, G L

1993-01-01

Taste buds share a topology with ion-transporting epithelial and evidence now indicates that neural responses in rats to Na+ salts of differing anion are mediated by both transcellular and paracellular ion transport. Na+ exerts its effects mainly on the transcellular pathway. Neural responses to Na+ salts are enhanced by negative voltage clamp and suppressed by positive clamp in a manner indicating modulation of the apical membrane potential of receptor cells. Anion effects are mainly paracellular. Under zero current clamp increasing anion size reduces the neural response at constant Na+ concentration. Below about 50 mM this difference is entirely eliminated under voltage clamp. This suggests that paracellular transepithelial potentials normally create an anion difference. At higher concentrations the relatively high permeability of the paracellular shunt to Cl- permits sufficient electroneutral diffusion of NaCl below the tight junctions to stimulate cells that do not make direct contact with the oral cavity. In general, the sensitivity of a response to perturbations in the apical membrane potential indicates that some phase of Na+ salt taste transduction is accompanied by changes in an apical membrane channel conductance.

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

PubMed

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

PubMed Central

Priyamvada, Shubha; Kumar, Anoop; Natarajan, Arivarasu A.; Gill, Ravinder K.; Alrefai, Waddah A.; Dudeja, Pradeep K.

2012-01-01

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30–120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [14C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased Vmax of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor. PMID:22982338

Smear Layer Outcome on Healing

DTIC Science & Technology

2015-07-31

apical tissues, symptomatic apical periodontitis , asymptomatic apical periodontitis , chronic apical abscess or condensing osteomyelitis - Presence...percha • Patients presenting with periodontal disease or acute apical abscess ·Patients currently taking antibiotics If a patient expressed an... periodontitis : A randomized clinical trial. Joumal of Endodontics, 33, 1145-48. 23. Moss D, Allemang J, Johnson J. (2001). Philosophies and Practices

Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor

NASA Astrophysics Data System (ADS)

Fong-Ngern, Kedsarin; Thongboonkerd, Visith

2016-10-01

To search for a strategy to prevent kidney stone formation/recurrence, this study addressed the role of α-enolase on apical membrane of renal tubular cells in mediating calcium oxalate monohydrate (COM) crystal adhesion. Its presence on apical membrane and in COM crystal-bound fraction was confirmed by Western blotting and immunofluorescence staining. Pretreating MDCK cells with anti-α-enolase antibody, not isotype-controlled IgG, dramatically reduced cell-crystal adhesion. Immunofluorescence staining also confirmed the direct binding of purified α-enolase to COM crystals at {121} > {100} > {010} crystal faces. Coating COM crystals with urinary proteins diminished the crystal binding capacity to cells and purified α-enolase. Moreover, α-enolase selectively bound to COM, not other crystals. Chemico-protein interactions analysis revealed that α-enolase interacted directly with Ca2+ and Mg2+. Incubating the cells with Mg2+ prior to cell-crystal adhesion assay significantly reduced crystal binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to α-enolase. Taken together, we successfully confirmed the role of α-enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation.

Subcellular localization and distribution of the reduced folate carrier in normal rat tissues.

PubMed

Hinken, M; Halwachs, S; Kneuer, C; Honscha, W

2011-01-27

The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

FACTORS CONTROLLING THE REASSEMBLY OF THE MICROVILLOUS BORDER OF THE SMALL INTESTINE OF THE SALAMANDER

PubMed Central

Tilney, Lewis G.; Cardell, Robert R.

1970-01-01

Hydrostatic pressure, when applied to segments of the small intestine of the salamander, causes a tremendous reduction in number of microvilli and a loss of the terminal web. The intestinal epithelium strips off from its deeper layers at the level of the basement membrane. When the pressure is released and this epithelial sheet is allowed to recover, the microvilli and its terminal web reappear. Stages in the reformation of microvilli are described. In the earliest stages, foci of dense material seem to associate with the cytoplasmic surface of the apical plasma membrane. From this material, filaments appear and their regrowth is correlated with the extension of the microvilli. We suggest that the dense material nucleates the assembly of the filaments which, in turn, appear instrumental in the redevelopment of microvilli. This concept is supported by the existing literature. Further, since neither the microvilli nor the terminal web reappear on any surface but the apical surface, even though the apical and basal surfaces are bathed with the same medium, we suggest that information in the membrane itself or directly associated with the membrane dictates the distribution of the dense material which leads to the formation of the microvilli and ultimately to the polarity of the cell. PMID:19866740

Tamm-Horsfall protein translocates to the basolateral domain of thick ascending limbs, interstitium, and circulation during recovery from acute kidney injury

PubMed Central

McCracken, Ruth; Liu, Yan; Heitmeier, Monique R.; Bourgeois, Soline; Ryerse, Jan; Wu, Xue-Ru

2013-01-01

Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidney's thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross talk between the two functionally distinct tubular segments. However, the extent, timing, specificity, and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is downregulated at the peak of injury, it is significantly upregulated 48 h after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium, and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of Na+-K+-2Cl cotransporter (NKCC2) and basolateral targeting of Na+-K+-ATPase. Compared with the wild-type, THP−/− mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of proinflammatory cytokines and chemokines such as monocyte chemoattractant protein-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI. PMID:23389456

Dexamethasone concentration gradients along scala tympani after application to the round window membrane.

PubMed

Plontke, Stefan K; Biegner, Thorsten; Kammerer, Bernd; Delabar, Ursular; Salt, Alec N

2008-04-01

Local application of dexamethasone-21-dihydrogen-phosphate (Dex-P) to the round window (RW) membrane of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph. In recent years, intratympanically applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after RW application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur after RW application, with lower concentrations expected in apical turns. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph. Dexamethasone-21-dihydrogen-phosphate (10 mg/ml) was administered to the RW membrane of guinea pigs (n = 9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which 10 samples, each of approximately 1 mul, were taken sequentially from the cochlear apex into capillary tubes. Dexamethasone-21-dihydrogen-phosphate concentration of the samples was analyzed by high-performance liquid chromatography. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in ST to be quantified. The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along ST. After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17,000. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially. The existence of substantial basal-apical concentration gradients of Dex-P in ST perilymph were demonstrated experimentally. If the variability in peak concentration and gradient is also present under clinical conditions, this may contribute to the heterogeneity of outcome that is observed after intratympanic application of glucocorticoids for various inner ear diseases.

Specification of ion transport cells in the Xenopus larval skin

PubMed Central

Quigley, Ian K.; Stubbs, Jennifer L.; Kintner, Chris

2011-01-01

Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of a `kidney-specific' form of the H+v-ATPase that localizes to the plasma membrane, orthologs of the Cl–/HCO –3 antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H+v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H+v-ATPase cytosolically or basolaterally. These two subtypes are specified during early PSC differentiation by a binary switch that can be regulated by Notch signaling and by the expression of ubp1, a transcription factor of the grainyhead family. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous. PMID:21266406

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD.

PubMed

Li, Liuzhe; Lynch, I Jeanette; Zheng, Wencui; Cash, Melanie N; Teng, Xueling; Wingo, Charles S; Verlander, Jill W; Xia, Shen-Ling

2007-08-03

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

PubMed Central

1988-01-01

The vacuolar apical compartment (VAC) is an organelle found in Madin- Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo. PMID:3053735

Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine

PubMed Central

Klett, Eric L; Lee, Mi-Hye; Adams, David B; Chavin, Kenneth D; Patel, Shailendra B

2004-01-01

Background The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level. Methods Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses. Results We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins. Conclusion We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function. PMID:15383151

A functional role of the extracellular domain of Crumbs in cell architecture and apicobasal polarity.

PubMed

Letizia, Annalisa; Ricardo, Sara; Moussian, Bernard; Martín, Nicolás; Llimargas, Marta

2013-05-15

Regulated cell shape changes in epithelial cells, which contribute to most organs and tissues, are at the basis of morphogenesis. Crumbs (Crb) is an essential apical determinant controlling epithelial apicobasal polarity. Here we provide evidence for a novel role of Crb apical localisation and stabilisation in controlling cell shape through apical domain organisation and adherens junction positioning. We find that Crb apical stabilisation requires the extracellular domain. In vivo results from Drosophila suggest that the extracellular domain assists Crb apical stabilisation by mediating Crb-Crb interactions at opposing cell membranes. We further confirm Crb-Crb extracellular interactions by showing that the extracellular domain of Crb is sufficient to promote cell aggregation in vitro. Furthermore, we report that Crb apical stabilisation mediated by the extracellular domain is also required for maintenance of Crb apicobasal polarity. Our results provide new insights into the mechanisms of apicobasal polarity and the cellular mechanisms of tissue architecture.

Modulation of small intestinal phosphate transporter by dietary supplements of mineral phosphorus and phytase in broilers.

PubMed

Huber, Korinna; Zeller, Ellen; Rodehutscord, Markus

2015-05-01

Dietary phosphorus (P) is known as a main modulator of phosphate (Pi) transporter expression. The effect of supplemented mineral P with or without phytase on protein expression of two sodium-dependent Pi (NaPi) transporters and a calcium channel was studied in the small intestine of broilers. Thirty-six broilers were randomly assigned to six different diets at 15 days of age. Two levels of total P (tP, adjusted by monocalcium phosphate (MCP) supplementation), 0.39% (BD-) and 0.47% (BD+) were fed until day 25; and at each tP level, three levels of phytase were used with 0, 500, and 12,500 FTU/kg of an E. coli phytase. Mucosa samples from jejunum and ileum were taken and apical membranes were isolated by MgCl2 precipitation. Protein expression of NaPi IIb, NaPi type III (PiT1) and the calcium channel TRPV6 were semiquantitatively measured by Western blotting and jejunal mucosal phytase activity by measurement of Pi release. The jejunal NaPi IIb transporter was expressed with two distinct bands, which were modulated differently by diet. NaPi IIb Band1 increased (P < 0.05) and Band2 decreased (P < 0.05) with phytase supplementation but was not affected by MCP supplementation. This inverse modulation of Band1 and Band2 was significantly related to the amount of net absorbed P with higher expression of Band1 at higher amounts of net absorbed P. In addition, a second Pi transporter, PiT1, was detected in which ileal expression decreased (P < 0.05) in response to higher phytase supplementation. The expression of the calcium channel TRPV6 was increased in BD+ groups. A trend for an interaction between MCP and phytase supplementation on mucosal phytase activity was observed (P = 0.079) with a decrease in activity when BD+ with 12,500 FTU/kg phytase was fed. Chicken intestinal epithelial cells responded to dietary supplemented phytase and MCP by changing the Pi transporter expression in apical membranes. In conclusion, availability of Pi is most likely the key modulator of transporter protein expression. However, a contribution of lower inositol phosphates generated by phytases and other phosphatases may also be relevant. © 2015 Poultry Science Association Inc.

Apical Na(+)-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

USDA-ARS?s Scientific Manuscript database

Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus a...

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

PubMed

van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

2014-09-01

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

An Ichor-dependent apical extracellular matrix regulates seamless tube shape and integrity

PubMed Central

Rosa, Jeffrey B.; Metzstein, Mark M.

2018-01-01

During sprouting angiogenesis in the vertebrate vascular system, and primary branching in the Drosophila tracheal system, specialized tip cells direct branch outgrowth and network formation. When tip cells lumenize, they form subcellular (seamless) tubes. How these seamless tubes are made, shaped and maintained remains poorly understood. Here we characterize a Drosophila mutant called ichor (ich), and show that ich is essential for the integrity and shape of seamless tubes in tracheal terminal cells. We find that Ich regulates seamless tubulogenesis via its role in promoting the formation of a mature apical extracellular matrix (aECM) lining the lumen of the seamless tubes. We determined that ich encodes a zinc finger protein (CG11966) that acts, as a transcriptional activator required for the expression of multiple aECM factors, including a novel membrane-anchored trypsin protease (CG8213). Thus, the integrity and shape of seamless tubes are regulated by the aECM that lines their lumens. PMID:29309404

Model for the dynamic responses of taste receptor cells to salty stimuli. I. Function of lipid bilayer membranes.

PubMed Central

Naito, M; Fuchikami, N; Sasaki, N; Kambara, T

1991-01-01

The dynamic response of the lipid bilayer membrane is studied theoretically using a microscopic model of the membrane. The time courses of membrane potential variations due to monovalent salt stimulation are calculated explicitly under various conditions. A set of equations describing the time evolution of membrane surface potential and diffusion potential is derived and solved numerically. It is shown that a rather simple membrane such as lipid bilayer has functions capable of reproducing the following properties of dynamic response observed in gustatory receptor potential. Initial transient depolarization does not occur under Ringer adaptation but does under water. It appears only for comparatively rapid flows of stimuli, the peak height of transient response is expressed by a power function of the flow rate, and the membrane potential gradually decreases after reaching its peak under long and strong stimulation. The dynamic responses in the present model arise from the differences between the time dependences in the surface potential phi s and the diffusion potential phi d across a membrane. Under salt stimulation phi d cannot immediately follow the variation in phi s because of the delay due to the charging up of membrane capacitance. It is suggested that lipid bilayer in the apical membrane is the most probable agency producing the initial phasic response to the stimulation. PMID:1873461

A nonsurgical endodontic treatment in open-apex and immature teeth affected by dens invaginatus: using a collagen membrane as an apical barrier.

PubMed

Gharechahi, Maryam; Ghoddusi, Jamileh

2012-02-01

The authors' objective in this case report is to demonstrate an effective nonsurgical endodontic treatment in open-apex teeth affected by dens invaginatus (DI) by using a collagen membrane as an apical barrier and using a mineral trioxide aggregate (MTA) apical plug. . The authors present two cases of DI with open apexes in maxillary lateral incisors. In the first case, an adolescent had bilateral Oehlers type II DI and extensive periradicular radiolucency, internal root resorption and a vestibular fistula in the left maxillary lateral incisor. In the second case, an adult had Oehlers type II DI and an incomplete apex in the left maxillary lateral incisor. For both patients, the clinician placed a collagen membrane through the apexes of the left maxillary incisors to provide a resorbable extraradicular barrier against which MTA cement could be packed. The clinician obturated the adolescent's right lateral incisor. In the adolescent, the vestibular sinus tract was closed after one week. At subsequent follow-up examinations, the periradicular regions were completely healed, and postoperative radiographs revealed good bone healing in the lateral incisors. The teeth were asymptomatic and healing was achieved without any need for further endodontic surgical intervention. In the adult patient, the tooth was symptom free after one week, and radiography performed six months after the procedure showed complete healing. and Despite complex anatomy and diagnoses of DI and open apexes, both patients successfully underwent nonsurgical endodontic treatment involving the use of a collagen membrane and an MTA apical plug. Using an extraradicular barrier clinically can help improve the adaptation of MTA in the apexes of open-apex teeth to achieve a complete seal.

Co-localization of endogenous Arf6 and its activator EFA6D in the granular convoluted tubule cells of mouse submandibular glands under normal conditions and when stimulated by isoproterenol, noradrenaline and carbachol.

PubMed

Tachow, Apussara; Thoungseabyoun, Wipawee; Phuapittayalert, Laorrat; Petcharat, Kanoktip; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-10-01

This study proposed to investigate the localization at light and electron microscopic levels of Arf6 and its activator EFA6D in the mouse submandibular gland (SMG) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of male adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and noradrenalin were used as adrenergics, while carbachol was used for the cholinergic stimulant. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Immunoreactivities for both Arf6 and its activator EFA6D were similarly intense in the basolateral domain of GCTs, but no significant immunoreactivities were seen in the apical domain of GCT cells or any domain of acinar cells under normal conditions. In immuno-electron microscopy, the immunoreactive materials were mainly deposited on the basolateral plasma membranes and subjacent cytoplasm. Shortly after injection of isoproterenol and noradrenaline, but not carbachol, the immunoreactivities for both molecules were additionally seen on the apical plasmalemma of most, if not all, GCT cells, but not acinar cells. The present findings suggest that the direct involvement of Arf6/EFA6D in regulatory exocytosis at the apical plasma membrane of acinar and GCT cells is apparently to be smaller, if present, than that of endocytosis at the basolateral membranes of GCT cells under normal conditions. This also suggests that the two molecules function additionally at the apical membrane of GCT cells for modulation of saliva secretion under β-adrenoceptor stimulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Role of SGLT1 and GLUT2 in Intestinal Glucose Transport and Sensing

PubMed Central

Röder, Pia V.; Geillinger, Kerstin E.; Zietek, Tamara S.; Thorens, Bernard; Koepsell, Hermann; Daniel, Hannelore

2014-01-01

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion. PMID:24587162

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth1[OPEN

PubMed Central

Bloch, Daria; Pleskot, Roman; Vukašinović, Nemanja

2016-01-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. PMID:27516531

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

PubMed

Bloch, Daria; Pleskot, Roman; Pejchar, Přemysl; Potocký, Martin; Trpkošová, Pavlína; Cwiklik, Lukasz; Vukašinović, Nemanja; Sternberg, Hasana; Yalovsky, Shaul; Žárský, Viktor

2016-10-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP 2 ) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP 2 However, the interaction with PIP 2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. © 2016 American Society of Plant Biologists. All Rights Reserved.

Apical membrane permeability and kinetic properties of the sodium pump in rabbit urinary bladder.

PubMed

Lewis, S A; Wills, N K

1983-08-01

Previous studies have shown that aldosterone stimulates the rate of Na+ transport across the rabbit urinary bladder epithelium by increasing the apical membrane permeability to Na+. Paradoxically, ion-sensitive and conventional micro-electrode measurements demonstrated that intracellular Na+ activity aiNa+ was essentially unchanged by aldosterone, i.e. aiNa+ was constant regardless of the rate of Na+ transport. The present study was designed to resolve this apparent contradiction. The effects of elevated, endogenous aldosterone levels produced by low-Na+ diet (Lewis & Diamond, 1976) on urinary bladder Na+ transport were investigated in vitro using Ussing-type chambers and intracellular conventional and ion-sensitive microelectrodes. Apical membrane selectivity and kinetics of the Na+ pump were assessed as a function of hormone stimulation. The aldosterone-stimulated increase in Na+ transport was accounted for by increases in both the relative selective permeability of the apical membrane to Na+ and an increase in its absolute Na+ permeability. The kinetics of the Na+ pump were evaluated electrically by loading the cells with Na+ (monitored with Na+-sensitive micro-electrodes) or alternatively by manipulating serosal solution K+ concentration and measuring changes in the basolateral membrane electromotive forces and resistance. From these measurements the current generated by the pump was calculated as a function of intracellular Na+ or extracellular K+. The kinetics of the pump were not altered by aldosterone. A model of highly co-operative binding estimated Km for Na+ as 14.2 mM and 2.3 mM for K+. Hill coefficients for these ions were 2.8 and 1.8, respectively, consistent with a pump stoichiometry of 3 Na+ to 2 K+. The kinetic properties of the Na-K pump indicate that physiological levels of aiNa+ are poised at the foot of a step kinetic curve which energetically favours Na+ extrusion.

Temporal expression and mitochondrial localization of a Foxp2 isoform lacking the forkhead domain in developing Purkinje cells.

PubMed

Tanabe, Yuko; Fujiwara, Yuji; Matsuzaki, Ayumi; Fujita, Eriko; Kasahara, Tadashi; Yuasa, Shigeki; Momoi, Takashi

2012-07-01

FOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of 'apical cytoplasmic swelling' (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2-P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

PubMed

Kominsky, Douglas J; Campbell, Eric L; Ehrentraut, Stefan F; Wilson, Kelly E; Kelly, Caleb J; Glover, Louise E; Collins, Colm B; Bayless, Amanda J; Saeedi, Bejan; Dobrinskikh, Evgenia; Bowers, Brittelle E; MacManus, Christopher F; Müller, Werner; Colgan, Sean P; Bruder, Dunja

2014-02-01

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

PubMed

Klein, Janet D; Wang, Yanhua; Blount, Mitsi A; Molina, Patrick A; LaRocque, Lauren M; Ruiz, Joseph A; Sands, Jeff M

2016-05-15

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations. Copyright © 2016 the American Physiological Society.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts

PubMed Central

Wang, Yanhua; Blount, Mitsi A.; Molina, Patrick A.; LaRocque, Lauren M.; Ruiz, Joseph A.

2016-01-01

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations. PMID:26962099

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

PubMed

Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

NPC1L1 and Cholesterol Transport

PubMed Central

Betters, Jenna L.; Yu, Liqing

2010-01-01

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, fatty liver, in addition to atherosclerosis. PMID:20307540

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3

PubMed Central

Zhou, Gaofeng; Ryan, Peter R.

2014-01-01

Malate and citrate efflux from root apices is a mechanism of Al3+ tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al3+-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al3+ tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al3+-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al3+-activated citrate efflux from root apices and greater tolerance to Al3+ toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al3+ tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al3+ tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al3+ tolerance of an important crop species. PMID:24692647

Blood-urine barrier formation in mouse urinary bladder development.

PubMed

Jezernik, K; Pipan, N

1993-04-01

Formation of the blood-urine permeability barrier in differentiating mouse transitional urothelium was studied. It was established that the development of superficial cell barrier is a two-phase process: beginning with formation of the tight junctions, followed by formation of fusiform vesicles and asymmetric apical plasma membranes. Fusiform vesicles differentiate during days 15 and 17 of gestation and fuse with the apical plasmalemma. Thus a thick membrane is formed before the excretion of hypertonic urine into the embryonic bladder. Through some degenerative superficial cells slough between fetal day 17 and the day of birth, the bladder epithelium in mice does not lack an effective permeability barrier.

Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

PubMed Central

Robertson, Sarah Y. T.; Wen, Xin; Yin, Kaifeng; Chen, Junjun; Smith, Charles E.; Paine, Michael L.

2017-01-01

Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization. PMID:28588505

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

PubMed

Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

PubMed Central

Luo, Shuanghui; Wang, Zhiying; Kansara, Viral; Pal, Dhananjay; Mitra, Ashim. K.

2008-01-01

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [14C]ascorbic acid ([14C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di- substituted benzenes. Transepithelial transport of [14C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a Km of 83.2 μM and Vmax of 94.2 pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca2+/CaM mediated pathways. [14C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group i.e. halogen- and nitro- substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A−B) as well as basal to apical side (B−A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs. PMID:18417304

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.

PubMed

Robben, J H; Knoers, N V A M; Deen, P M T

2004-12-01

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.

Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin.

PubMed

Lecuit, Marc; Nelson, D Michael; Smith, Steve D; Khun, Huot; Huerre, Michel; Vacher-Lavenu, Marie-Cécile; Gordon, Jeffrey I; Cossart, Pascale

2004-04-20

Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. We used immunohistochemistry to examine the location of L. monocytogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L. monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium. Because epidemiological studies indicate that the bacterial surface protein, internalin (InlA), may play a role in human fetoplacental listeriosis, we investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found on the basal and apical plasma membranes of syncytiotrophoblasts and in villous cytotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InlA+ or InlA- strains of L. monocytogenes, and to L. innocua expressing or not InlA. Quantitative assays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InlA-E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces histopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InlA-E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans is also exploited by L. monocytogenes to target and cross the placental barrier. Such a ligand-receptor interaction allowing a pathogen to specifically cross the placental villous trophoblast barrier has not been reported previously.

Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: Role of internalin interaction with trophoblast E-cadherin

PubMed Central

Lecuit, Marc; Nelson, D. Michael; Smith, Steve D.; Khun, Huot; Huerre, Michel; Vacher-Lavenu, Marie-Cécile; Gordon, Jeffrey I.; Cossart, Pascale

2004-01-01

Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. We used immunohistochemistry to examine the location of L. monocytogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L. monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium. Because epidemiological studies indicate that the bacterial surface protein, internalin (InlA), may play a role in human fetoplacental listeriosis, we investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found on the basal and apical plasma membranes of syncytiotrophoblasts and in villous cytotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InlA+ or InlA- strains of L. monocytogenes, and to L. innocua expressing or not InlA. Quantitative assays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InlA–E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces histopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InlA–E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans is also exploited by L. monocytogenes to target and cross the placental barrier. Such a ligand–receptor interaction allowing a pathogen to specifically cross the placental villous trophoblast barrier has not been reported previously. PMID:15073336

Localization of water channels in the skin of two species of desert toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius.

PubMed

Shibata, Yuki; Takeuchi, Hiro-Aki; Hasegawa, Takahiro; Suzuki, Masakazu; Tanaka, Shigeyasu; Hillyard, Stanley D; Nagai, Takatoshi

2011-09-01

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrell, Permila C.; McCawley, Lisa J.; Fingleton, Barbara

2005-02-15

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment asmore » compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7{sup HIGH}-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.« less

Regulation of Cl^- Channels in Normal and Cystic Fibrosis Airway Epithelial Cells by Extracellular ATP

NASA Astrophysics Data System (ADS)

Stutts, M. J.; Chinet, T. C.; Mason, S. J.; Fullton, J. M.; Clarke, L. L.; Boucher, R. C.

1992-03-01

The rate of Cl^- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl^- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl^- conductance decreases the capability to secrete Cl^-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl^- secretion and apical membrane Cl^- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl^- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl^- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells.

PubMed

Le Grimellec, C; Friedlander, G; Giocondi, M C

1988-07-01

Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells. Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers. Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime. Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.

CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.

PubMed

Lant, Benjamin; Yu, Bin; Goudreault, Marilyn; Holmyard, Doug; Knight, James D R; Xu, Peter; Zhao, Linda; Chin, Kelly; Wallace, Evan; Zhen, Mei; Gingras, Anne-Claude; Derry, W Brent

2015-03-06

The mechanisms governing apical membrane assembly during biological tube development are poorly understood. Here, we show that extension of the C. elegans excretory canal requires cerebral cavernous malformation 3 (CCM-3), independent of the CCM1 orthologue KRI-1. Loss of ccm-3 causes canal truncations and aggregations of canaliculular vesicles, which form ectopic lumen (cysts). We show that CCM-3 localizes to the apical membrane, and in cooperation with GCK-1 and STRIPAK, promotes CDC-42 signalling, Golgi stability and endocytic recycling. We propose that endocytic recycling is mediated through the CDC-42-binding kinase MRCK-1, which interacts physically with CCM-3-STRIPAK. We further show canal membrane integrity to be dependent on the exocyst complex and the actin cytoskeleton. This work reveals novel in vivo roles of CCM-3·STRIPAK in regulating tube extension and membrane integrity through small GTPase signalling and vesicle dynamics, which may help explain the severity of CCM3 mutations in patients.

The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression

PubMed Central

Balda, Maria S.; Matter, Karl

2000-01-01

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation. PMID:10790369

SNX31: A Novel Sorting Nexin Associated with the Uroplakin-Degrading Multivesicular Bodies in Terminally Differentiated Urothelial Cells

PubMed Central

Liang, Feng-Xia; Liao, Yi; Chang, Jennifer; Zhou, Ge; Zheng, Weiyue; Simon, Jean-Pierre; Ding, Mingxiao; Wu, Xue-Ru; Romih, Rok; Kreibich, Gert; Sun, Tung-Tien

2014-01-01

Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but ‘softened’) membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation. PMID:24914955

The core planar cell polarity gene, Vangl2, maintains apical-basal organisation of the corneal epithelium.

PubMed

Panzica, D Alessio; Findlay, Amy S; van Ladesteijn, Rianne; Collinson, J Martin

2017-08-17

The role of the core planar cell polarity (PCP) pathway protein, Vangl2, was investigated in the corneal epithelium of the mammalian eye, a paradigm anatomical model of planar cell migration. The gene was conditionally knocked out in vivo and knocked down by siRNA, followed by immunohistochemical, behavioural and morphological analysis of corneal epithelial cells. The primary defects observed in vivo were of apical-basal organisation of the corneal epithelium, with abnormal stratification throughout life, mislocalisation of the cell membrane protein, Scribble, to the basal side of cells, and partial loss of the epithelial basement membrane. Planar defects in migration after wounding and in the presence of an applied electric field were noted. However, knockdown of Vangl2 also retarded cell migration in individual cells that had no contact with their neighbours, which precluded a classic PCP mechanism. It is concluded that some of the planar polarity phenotypes in PCP mutants may arise from disruption of apical-basal polarity. © 2017 Anatomical Society.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

PubMed

Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

2015-01-01

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

A test of current models for the mechanism of milk-lipid droplet secretion

PubMed Central

Jeong, Jaekwang; Lisinski, Ivonne; Kadegowda, Anil K.G.; Shin, Hyunsu; Wooding, F.B. Peter; Daniels, Brian R.; Schaack, Jerome; Mather, Ian H.

2013-01-01

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts, either with itself, or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced-green-fluorescent-protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5–0.7%, (w/w) of the total protein, i.e., over fifty-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets, mark it as a potential mobile signaling molecule in milk. PMID:23738536

Production of the Subdomains of the Plasmodium falciparum Apical Membrane Antigen 1 Ectodomain and Analysis of the Immune Response

DTIC Science & Technology

2004-08-01

expression in shake flasks , cells were grown to an optical density at 600 nm of 0.5 and then induced with a final concentration of 0.5 mM isopropyl--D...proteolytically cleaved into smaller fragments (8, 16, 17, 25). Studies of animal malarias have heightened interest in the development of AMA-1 as a vaccine for... bioreactor (New Brunswick Scientific, Edison, NJ) used Terrific Broth media (1.2% tryp- tone, 2.4% yeast extract, 72 mM K2HPO4, 28 mM KH2PO4 [pH 7.2

Fine structure of the copulatory apparatus of the tapeworm Tetrabothrius erostris (Cestoda: Tetrabothriidea).

PubMed

Korneva, Janetta V; Jones, Malcolm K; Kuklin, Vadim V

2015-05-01

The organization and fine structure of the complex copulatory apparatus of Tetrabothrius erostris (Tetrabothriidea) is investigated by light and transmission electron microscopy. A diversity of microstructures was found on the surface of genital ducts. The apical surfaces of male gonadoducts possess tubular and blade-like microtriches that have specific structure in each section of the duct. The apical part of the tubular microtriches contains numerous constrictions in the proximal section of the sperm duct; blade-like microtriches of cirrus possess longitudinal striation in the apical part, and their basal part is reinforced with electron-dense strands. Two types of microtriches occur on the surface of cirrus, and their presence may be considered as systematic features. Prostate glands containing granules of medium electron density (up to 130 nm diameter) are localized in the cirrus sac. The genital atrium contains numerous non-ciliated receptors. Paramyosin-like fibers (up to 200 nm) were found in the muscle fibers surrounding the male atrium canal. Microtriches on the surface of the distal region of the male atrial canal are covered by a glycocalyx. Electron-dense, membrane-like structures (up to 40 nm) lie under the apical membrane of the genital atrium and vagina. These structures do not form a continuous layer; its edges turn down and sink into the apical invaginations of epithelium. Hypotheses on the possible ways of copulation in T. erostris based on the observed ultrastructure are discussed.

Roles of cyclic AMP and Ca in epithelial ion transport across corneal epithelium: a review.

PubMed

Reinach, P S

1985-04-01

The messenger roles of cyclic AMP and the calcium ion in stimulus-secretion coupling are considered in the frog and bovine corneal epithelium, respectively. In the frog cornea, epinephrine stimulates net C1 transport by increasing cyclic AMP content. This stimulation is associated with a larger apical membrane C1 conductance and basolateral membrane ionic conductance. The response of the apical membrane conductance is thought to result from an increase in cyclic AMP content whereas the basolateral membrane ionic conductance increase is unrelated based on measurements of the effects of the calcium channel antagonist, diltiazem, and the beta agonist, isoproterenol, on the electrical parameters and cyclic AMP content. The basolateral membrane is essentially K permselective since the K channel blocker, Ba, depolarized the intracellular potential difference and increased the basolateral membrane resistance. Diltiazem had even larger effects on these parameters suggesting that this compound is a more effective inhibitor of K channel activity than barium. In broken cell preparations of bovine corneal epithelium, a high affinity form of Ca + Mg activated ATPase is present (Km = .06 microM for Ca) and is essentially of plasma membrane origin. This ATPase activation is at a Ca activity similar to the expected intracellular value and suggests that this activity is the enzymatic basis for net Ca transport.

Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

PubMed Central

Marunaka, Yoshinori; Niisato, Naomi; Taruno, Akiyuki; Ohta, Mariko; Miyazaki, Hiroaki; Hosogi, Shigekuni; Nakajima, Ken-ichi; Kusuzaki, Katsuyuki; Ashihara, Eishi; Nishio, Kyosuke; Iwasaki, Yoshinobu; Nakahari, Takashi; Kubota, Takahiro

2011-01-01

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane. PMID:22028593

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

PubMed

Bartholomé, Kilian; Rius, Maria; Letschert, Katrin; Keller, Daniela; Timmer, Jens; Keppler, Dietrich

2007-09-01

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol.

PubMed

Thoungseabyoun, Wipawee; Tachow, Apussara; Pakkarato, Sawetree; Rawangwong, Atsara; Krongyut, Suthankamon; Sakaew, Waraporn; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-09-01

We wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Selective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol. The present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

A perfusion study of the handling of urea and urea analogues by the gills of the dogfish shark (Squalus acanthias).

PubMed

Wood, Chris M; Liew, Hon Jung; De Boeck, Gudrun; Walsh, Patrick J

2013-01-01

The branchial mechanism of urea retention in elasmobranchs was investigated using an in vitro isolated-perfused head preparation, as well as in vivo samples, in the spiny dogfish shark. Both in vivo and in control saline perfusions containing 350 mmol L(-1) urea, calculated intracellular urea concentrations in gill epithelial cells were close to extracellular concentrations. Urea efflux to the external water fell only non-significantly, and calculated gill intracellular urea concentration did not change when perfusate urea concentration was reduced from 350 to 175 mmol L(-1) with osmotic compensation by 175 mmol L(-1) mannitol. However, when the urea analogues thiourea or acetamide were present in the perfusate at concentrations equimolar (175 mmol L(-1)) to those of urea (175 mmol L(-1)), urea efflux rates were increased 4-fold and 6.5-fold respectively, and calculated gill intracellular urea concentrations were depressed by about 55%. Analogue efflux rates were similar to urea efflux rates. Previous studies have argued that either the basolateral or apical membranes provided the limiting permeability barrier, and/or that a back-transporter on the basolateral membranes of gill cells is responsible for urea retention. The present results provide new evidence that the apical membrane is the limiting factor in maintaining gill urea impermeability, and raise the prospect that a urea back-transporter, which can be competitively inhibited by thiourea and acetamide, operates at the apical membrane.

Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

PubMed

Jarial, M S; Wilkins, J H

2003-10-01

The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

Crumbs3 Is Essential for Proper Epithelial Development and Viability

PubMed Central

Whiteman, Eileen L.; Fan, Shuling; Harder, Jennifer L.; Walton, Katherine D.; Liu, Chia-Jen; Soofi, Abdul; Fogg, Vanessa C.; Hershenson, Marc B.; Dressler, Gregory R.; Deutsch, Gail H.; Gumucio, Deborah L.

2014-01-01

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton. PMID:24164893

The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

PubMed Central

Tran, Thuy Thanh; Mittal, Aditya; Aldinger, Tanya; Polli, Joseph W.; Ayrton, Andrew; Ellens, Harma; Bentz, Joe

2005-01-01

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized. PMID:15501934

Loss of Elp3 Impairs the Acetylation and Distribution of Connexin-43 in the Developing Cerebral Cortex

PubMed Central

Laguesse, Sophie; Close, Pierre; Van Hees, Laura; Chariot, Alain; Malgrange, Brigitte; Nguyen, Laurent

2017-01-01

The Elongator complex is required for proper development of the cerebral cortex. Interfering with its activity in vivo delays the migration of postmitotic projection neurons, at least through a defective α-tubulin acetylation. However, this complex is already expressed by cortical progenitors where it may regulate the early steps of migration by targeting additional proteins. Here we report that connexin-43 (Cx43), which is strongly expressed by cortical progenitors and whose depletion impairs projection neuron migration, requires Elongator expression for its proper acetylation. Indeed, we show that Cx43 acetylation is reduced in the cortex of Elp3cKO embryos, as well as in a neuroblastoma cell line depleted of Elp1 expression, suggesting that Cx43 acetylation requires Elongator in different cellular contexts. Moreover, we show that histones deacetylase 6 (HDAC6) is a deacetylase of Cx43. Finally, we report that acetylation of Cx43 regulates its membrane distribution in apical progenitors of the cerebral cortex. PMID:28507509

The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

USDA-ARS?s Scientific Manuscript database

Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3.

PubMed

Zhou, Gaofeng; Pereira, Jorge F; Delhaize, Emmanuel; Zhou, Meixue; Magalhaes, Jurandir V; Ryan, Peter R

2014-06-01

Malate and citrate efflux from root apices is a mechanism of Al(3+) tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al(3+)-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al(3+) tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al(3+)-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al(3+)-activated citrate efflux from root apices and greater tolerance to Al(3+) toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al(3+) tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al(3+) tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al(3+) tolerance of an important crop species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

PubMed

Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

2017-11-01

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

Regulation of apical blebbing in the porcine epididymis.

PubMed

Hughes, Jennifer R; Berger, Trish

2018-03-01

Apical blebbing, a non-classical secretion mechanism, occurs in the mature porcine epididymis as part of its normal function. Proteins secreted by this mechanism contribute to the modification of the sperm plasma membrane during epididymal transit and are thought to contribute to acquisition of fertilizing ability. However, little is known about the regulation of this secretion mechanism in an in vivo model. Previous work demonstrated apical blebbing in the epididymis developed pubertally, suggesting androgens, sperm or other luminal factors regulated this process. Hence, the objective was to evaluate the hypothesized regulation of apical blebbing in the epididymis of pubertal boars by androgens and luminal factors. Androgen receptor blockade (flutamide) and surgical interventions (efferent duct ligation, orchidectomy or transection of the caput epididymis) were used to alter signaling, and the subsequent effects on apical blebbing were evaluated histologically. Apical blebbing was not altered by androgen receptor blockade with flutamide, but was significantly reduced 24 h after efferent duct ligation and after orchidectomy, treatments that eliminated luminal flow from the testis (P < 0.05). Like efferent duct ligation, epididymal transection altered luminal flow without removing the androgen source and significantly reduced the appearance of apical blebbing (P < 0.05). In conclusion, apical blebbing in the porcine epididymis appears to be regulated by luminal factors. © 2017 Anatomical Society.

Functional characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia)

PubMed Central

2010-01-01

Background Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model. Results We investigated structural as well as physiological properties of the pronephric duct. The key elements of our methodology were: using histology, light and transmission electron microscopy as well as confocal laser scanning microscopy on fixed tissue and applying the microperfusion technique on isolated pronephric ducts in combination with single cell microelectrode impalements. Our data show that the fully differentiated pronephric duct is composed of a single layered epithelium consisting of one cell type comparable to the principal cell of the renal collecting duct system. The cells are characterized by a prominent basolateral labyrinth and a relatively smooth apical surface with one central cilium. Cellular impalements demonstrate the presence of apical Na+ and K+ conductances, as well as a large K+ conductance in the basolateral cell membrane. Immunolabeling experiments indicate heavy expression of Na+/K+-ATPase in the basolateral labyrinth. Conclusions We propose that the pronephric duct is important for the subsequent modification of urine produced by the pronephros. Our results indicate that it reabsorbs sodium and secretes potassium via channels present in the apical cell membrane with the driving force for ion movement provided by the Na+/K+ pump. This is to our knowledge the first characterization of the pronephric duct, the precursor of the collecting duct system, which provides a model of cell structure and basic mechanisms for ion transport. Such information may be important in understanding the evolution of vertebrate kidney systems and human diseases associated with congenital malformations. PMID:20507566

Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung-Min; Department of Nutritional Science and Toxicology, University of California, Berkeley, CA; Attieh, Zouhair K.

2012-05-11

Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicatesmore » hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a cellular compartment in close proximity but not overlapping with the basolateral surface. Surface biotinylation studies indicate that hephaestin in the peri-basolateral location is accessible to the extra-cellular environment. These results support the hypothesis that hephaestin is involved in iron mobilization of iron from the intestine to circulation.« less

Interaction of the new monofunctional anticancer agent Phenanthriplatin with transporters for organic cations

NASA Astrophysics Data System (ADS)

Hucke, Anna; Park, Ga Young; Bauer, Oliver B.; Beyer, Georg; Köppen, Christina; Zeeh, Dorothea; Wehe, Christoph A.; Sperling, Michael; Schröter, Rita; Kantauskaitè, Marta; Hagos, Yohannes; Karst, Uwe; Lippard, Stephen J.; Ciarimboli, Giuliano

2018-05-01

Cancer treatment with platinum compounds is an important achievement of modern chemotherapy. However, despite the beneficial effects, the clinical impact of these agents is hampered by the development of drug resistance as well as dose-limiting side effects. The efficacy but also side effects of platinum complexes can be mediated by uptake through plasma membrane transporters. In the kidneys, plasma membrane transporters are involved in their secretion into the urine. Renal secretion is accomplished by uptake from the blood into the proximal tubules cells, followed by excretion into the urine. The uptake process is mediated mainly by organic cation transporters (OCT), which are expressed in the basolateral domain of the plasma membrane facing the blood. The excretion of platinum into the urine is mediated by exchange with protons via multidrug and toxin extrusion proteins (MATE) expressed in the apical domain of plasma membrane. Recently, the monofunctional, cationic platinum agent phenanthriplatin, which is able to escape common cellular resistance mechanisms, has been synthesized and investigated. In the present study, the interaction of phenanthriplatin with transporters for organic cations has been evaluated. Phenanthriplatin is a high affinity substrate for OCT2, but has a lower apparent affinity for MATEs. The presence of these transporters increased cytotoxicity of phenanthriplatin. Therefore, phenanthriplatin may be especially effective in the treatment of cancers that express OCTs, such as colon cancer cells. However, the interaction of phenanthriplatin with OCTs suggests that its use as chemotherapeutic agent may be complicated by OCT-mediated toxicity. Unlike cisplatin, phenanthriplatin interacts with high specificity with hMATE1 and hMATE2K in addition to hOCT2. This interaction may facilitate its efflux from the cells and thereby decrease overall efficacy and/or toxicity.

Amino acid sequence and the cellular location of the Na(+)-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell.

PubMed Central

Tarpey, P S; Wood, I S; Shirazi-Beechey, S P; Beechey, R B

1995-01-01

The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7492327

Survival of the Apical Papilla and Its Resident Stem Cells in a Case of Advanced Pulpal Necrosis and Apical Periodontitis.

PubMed

Chrepa, Vanessa; Pitcher, Brandon; Henry, Michael A; Diogenes, Anibal

2017-04-01

Apical papilla represents a source of an enriched mesenchymal stem cell (MSC) population (stem cells of the apical papilla [SCAPs]) that modulates root development and may participate in regenerative endodontic procedures in immature teeth with pulp necrosis. The characteristics and phenotype of this tissue in the presence of inflammation are largely unknown. The purpose of this study was to characterize a human apical papilla sample that was isolated from an immature tooth with pulp necrosis and apical periodontitis. Inflamed periapical tissue that included part of the apical papilla (apical papilla clinical sample [CS]) was collected from an immature mandibular premolar previously diagnosed with pulp necrosis and apical periodontitis during an apexification procedure. Harvested cells from this tissue (SCAP CS) were compared with inflamed periapical progenitor cells (IPAPCs) and normal SCAP (SCAP-RP89) in flow cytometry and quantitative osteogenesis experiments. Part of the issue was further processed for immunohistochemistry and compared with apical papilla and coronal pulp sections from normal immature teeth as well as inflamed periapical tissues from mature teeth. Similar to SCAP-RP89, 96.6% of the SCAP CS coexpressed the MSC markers CD73, CD90, and CD105, whereas only 66.3% of IPAPCs coexpressed all markers. The SCAP CS showed a significantly greater mineralization potential than both SCAP-RP89 and IPAPCs. Finally, immunohistochemical analysis revealed moderate infiltration of cells expressing the inflammatory markers CD45/68 in the apical papilla CS and prominent CD24, CD105, and von Willebrand factor expression. Under inflammatory conditions, human apical papilla was found moderately inflamed with retained SCAP vitality and stemness and increased osteogenic and angiogenesis potential. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

PubMed

Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

2018-06-01

In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.

Transcellular oxalate and Cl− absorption in mouse intestine is mediated by the DRA anion exchanger Slc26a3, and DRA deletion decreases urinary oxalate

PubMed Central

Freel, Robert W.; Whittamore, Jonathan M.

2013-01-01

Active transcellular oxalate transport in the mammalian intestine contributes to the homeostasis of this important lithogenic anion. Several members of the Slc26a gene family of anion exchangers have a measurable oxalate affinity and are expressed along the gut, apically and basolaterally. Mouse Slc26a6 (PAT1) targets to the apical membrane of enterocytes in the small intestine, and its deletion results in net oxalate absorption and hyperoxaluria. Apical exchangers of the Slc26a family that mediate oxalate absorption have not been established, yet the Slc26a3 [downregulated in adenoma (DRA)] protein is a candidate mediator of oxalate uptake. We evaluated the role of DRA in intestinal oxalate and Cl− transport by comparing unidirectional and net ion fluxes across short-circuited segments of small (ileum) and large (cecum and distal colon) intestine from wild-type (WT) and DRA knockout (KO) mice. In WT mice, all segments demonstrated net oxalate and Cl− absorption to varying degrees. In KO mice, however, all segments exhibited net anion secretion, which was consistently, and solely, due to a significant reduction in the absorptive unidirectional fluxes. In KO mice, daily urinary oxalate excretion was reduced 66% compared with that in WT mice, while urinary creatinine excretion was unchanged. We conclude that DRA mediates a predominance of the apical uptake of oxalate and Cl− absorbed in the small and large intestine of mice under short-circuit conditions. The large reductions in urinary oxalate excretion underscore the importance of transcellular intestinal oxalate absorption, in general, and, more specifically, the importance of the DRA exchanger in oxalate homeostasis. PMID:23886857

Effects of Ag+ on ion transport by the corneal epithelium of the rabbit.

PubMed

Klyce, S D; Marshall, W S

1982-01-01

Exposure of the in vitro rabbit corneal epithelium to Ag+ by the addition of AgNO3 (10(-7)-10(-5) M) to the apical surface or by the use of imperfectly chlorided Ag/AgCl half-cells in Ussing-style membrane chambers, greatly increases short-circuit current and transepithelial potential. The early phase (the first 30 min) of the short-circuit current stimulation by Ag+ is linearly dependent on tear-side sodium concentration, is largely a result of a tenfold increase in net Na+ uptake and is incompletely inhibited by ouabain, suggesting that Ag+ increases cation (primarily Na+) conductance of the apical membrane. This mechanism for the Ag+ effect is supported by microelectrode experiments, wherein Ag+ depolarizes specifically the apical barrier potential and increases apical barrier conductance. A later phase in the effect (0.5-3 hr) is characterized by a gradual increase in 36Cl- and 14C-mannitol unidirectional fluxes, by a decline in epithelial resting potential and short-circuit current, by complete ouabain inhibition and by fit to saturation kinetics with respect to Na+ concentration in the bathing media. This phase of the effect apparently reflects a nonselective opening of the paracellular pathway in the epithelium and is rate-limited by Na+ pump activity at the basolateral membrane. Both phases are associated with swelling of the corneal stroma and may be rapidly reversed using thiol agents (reduced glutathione and dithiothreitol). The results suggest that Ag+ may be useful in the study of cation transport by epithelia and the work provides basic physiological information that is pertinent to the prophylactic use of AgNO3 in clinical ophthalmology.

The Spectrin cytoskeleton regulates the Hippo signalling pathway.

PubMed

Fletcher, Georgina C; Elbediwy, Ahmed; Khanal, Ichha; Ribeiro, Paulo S; Tapon, Nic; Thompson, Barry J

2015-04-01

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Identification of a spatially specific enhancer element in the chicken Msx-2 gene that regulates its expression in the apical ectodermal ridge of the developing limb buds of transgenic mice.

PubMed

Sumoy, L; Wang, C K; Lichtler, A C; Pierro, L J; Kosher, R A; Upholt, W B

1995-07-01

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the apical ectodermal ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the apical ectodermal ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an apical ectodermal ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.

Ultrastructural study of the semicircular canal cells of the frog Rana esculenta.

PubMed

Oudar, O; Ferrary, E; Feldmann, G

1988-03-01

The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in an attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes.

Prediction of intestinal absorption and metabolism of pharmacologically active flavones and flavanones.

PubMed

Serra, H; Mendes, T; Bronze, M R; Simplício, Ana Luísa

2008-04-01

Three glycosilated flavonoids (diosmin, hesperidin and naringin) and respective aglycones were characterized in terms of their apparent ionisation constants and bidirectional permeability using the cellular model Caco-2 as well as the artificial membrane model PAMPA. Ionisation curves were established by capillary electrophoresis. It was confirmed that significant amounts of the aglycones are ionised at physiological pH whereas the glycosides are in the neutral form. Permeation was not detected for the glycosides in either the apical-to-basolateral or basolateral-to-apical directions confirming the need for metabolism before absorption through the intestinal membrane. The aglycones permeated in both directions with apparent permeabilities (P(app)) in the range of 1-8x10(-5) cm/s. The results from both in vitro methods correlated providing some evidence of passive transport; however, the hypothesis of active transport cannot be excluded particularly in the case of diosmetin. Metabolism of the aglycones was detected with the cell model, more extensively when loading in the apical side. Some of the metabolites were identified as glucuronide conjugates by enzymatic hydrolysis.

Posterior midgut epithelial cells differ in their organization of the membrane skeleton from other drosophila epithelia.

PubMed

Baumann, O

2001-11-01

In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton. Copyright 2001 Academic Press.

Regulation of Sodium Transport in the Inner Ear

PubMed Central

Kim, Sung Huhn; Marcus, Daniel C.

2011-01-01

Na+ concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K+-selective. Flooding or fluctuations of the hair cell cytosol with Na+ would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na+homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na+into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na+ absorption is controlled by gate -keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na+-selective epithelial sodium channels (ENaC) control absorption by Reissner’s membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Nonselective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na+ and parasensory K+ absorption. The K+-mediated transduction current in hair cells is also accompanied by a Na+ flux since the transduction channels are nonselective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical nonselective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na+ with regulatory pathways that include glucocorticoids and purinergic agonists. PMID:21620939

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

PubMed

Collaco, Anne M; Jakab, Robert L; Hoekstra, Nadia E; Mitchell, Kisha A; Brooks, Amos; Ameen, Nadia A

2013-08-01

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

A perfusion study of the handling of urea and urea analogues by the gills of the dogfish shark (Squalus acanthias)

PubMed Central

Liew, Hon Jung; De Boeck, Gudrun; Walsh, Patrick J.

2013-01-01

The branchial mechanism of urea retention in elasmobranchs was investigated using an in vitro isolated-perfused head preparation, as well as in vivo samples, in the spiny dogfish shark. Both in vivo and in control saline perfusions containing 350 mmol L−1 urea, calculated intracellular urea concentrations in gill epithelial cells were close to extracellular concentrations. Urea efflux to the external water fell only non-significantly, and calculated gill intracellular urea concentration did not change when perfusate urea concentration was reduced from 350 to 175 mmol L−1 with osmotic compensation by 175 mmol L−1 mannitol. However, when the urea analogues thiourea or acetamide were present in the perfusate at concentrations equimolar (175 mmol L−1) to those of urea (175 mmol L−1), urea efflux rates were increased 4-fold and 6.5-fold respectively, and calculated gill intracellular urea concentrations were depressed by about 55%. Analogue efflux rates were similar to urea efflux rates. Previous studies have argued that either the basolateral or apical membranes provided the limiting permeability barrier, and/or that a back-transporter on the basolateral membranes of gill cells is responsible for urea retention. The present results provide new evidence that the apical membrane is the limiting factor in maintaining gill urea impermeability, and raise the prospect that a urea back-transporter, which can be competitively inhibited by thiourea and acetamide, operates at the apical membrane. PMID:23638369

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Rab11 in Recycling Endosomes Regulates the Sorting and Basolateral Transport of E-CadherinV⃞

PubMed Central

Lock, John G.; Stow, Jennifer L.

2005-01-01

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion. PMID:15689490

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

PubMed Central

Paula-Silva, Francisco Wanderley Garcia; da Silva, Léa Assed Bezerra; Kapila, Yvonne Lorraine

2009-01-01

Objectives To investigate the expression of matrix metalloproteinases (MMPs) in apical periodontitis and during the periapical healing phase following root canal treatment. Methods Apical periodontitis was induced in dog teeth and root canal treatment was performed in a single visit or using an additional calcium hydroxide root canal dressing. One hundred and eighty days following treatment the presence of inflammation was examined and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed using one-way ANOVA followed by Tukey test or Kruskal-Wallis followed by Dunn. Results Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment using calcium hydroxide presented a lower inflammatory cell infiltrate. This group had a moderately organized connective tissue, a lower prevalence of bacteria, and a lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusion Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized ECM, unlike those treated in a single visit. This suggests that calcium hydroxide may be beneficial in tissue repair processes. PMID:20113780

Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro.

PubMed

Bikson, Marom; Inoue, Masashi; Akiyama, Hiroki; Deans, Jackie K; Fox, John E; Miyakawa, Hiroyoshi; Jefferys, John G R

2004-05-15

The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage-sensitive dye recordings. Small electric fields (1 s) changes in neuronal excitability. Electric fields perpendicular to the apical-dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time-dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non-linear properties of the cell membrane, and effects on afferents.

Cochlear potential difference between endolymph fluid and the hair cell's interior: a retold interpretation based on the Goldman equation.

PubMed

Kurbel, Sven; Borzan, Vladimir; Golem, Hilda; Dinjar, Kristijan

2017-02-01

Reported cochlear potential values of near 150 mV are often attributed to endolymph itself, although membrane potentials result from ion fluxes across the adjacent semipermeable membranes due to concentration gradients. Since any two fluids separated by a semipermeable membrane develop potential due to differences in solute concentrations, a proposed interpretation here is that positive potential emanates from the Reissner membrane due to small influx of sodium from perilymph to endolymph. Basolateral hair cell membranes leak potassium into the interstitial fluid and this negative potential inside hair cells further augments the electric gradient of cochlear potential. Taken together as a sum, these two potentials are near the reported values of cochlear potential. This is based on reported data for cochlear fluids used for the calculation of Nernst and Goldman potentials. The reported positive potential of Reissner membrane can be explained almost entirely by the traffic of Na+ that enters endolymph through this membrane. At the apical membrane of hair cells, acoustic stimulation modulates stereocillia permeability to potassium. Potassium concentration gradients on the apical membrane are low (the calculated Nernst value is <+3 mV), suggesting that the potassium current is not caused by the local potassium concentration gradient, but an electric field between the positive sodium generated potential on the Reissner membrane and negative inside hair cells. Potassium is forced by this overall electric field to enter hair cells when stereocilia are permeable due to mechanical bending. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

Vasopressin Receptor Signaling and Cycling of Water Channels in Renal Epithelia (HBC)

DTIC Science & Technology

1993-08-27

in the cellular ultrastructure that results in mucosal membrane reorganization to numerous microvilli from the normal phase of microridges ( DiBona ...a restructuring of the apical membrane microridges into numerous microvilli with an increase in membrane surface area ( DiBona , 1981; DiBona et aL...markers in freeze-fracture studies of toad urinary bladder. J. Histochem. Cytochem., 35, 1405-1414. DiBona , D.R. 1981. Vasopressin action on the

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

PubMed

Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

Different natriuretic responses in obese and lean rats in response to nitric oxide reduction.

PubMed

Ambrozewicz, Marta A; Khraibi, Ali A; Simsek-Duran, Fatma; DeBose, Sophia C; Baydoun, Hind A; Dobrian, Anca D

2011-08-01

Nitric oxide (NO) is an important regulator of renal sodium transport and participates in the control of natriuresis and diuresis. In obesity, the nitric oxide bioavailability was reportedly reduced, which may contribute to the maintenance of hypertension. The aim of this study was to determine the effect of NO depletion on renal sodium handling in a model of diet-induced obesity hypertension. Obese hypertensive (obesity-prone (OP)) and lean normotensive (obesity-resistant (OR)) Sprague-Dawley rats were treated with 1.2 mg/kg/day N(G)-nitro-L-arginine-methyl ester (L-NAME) for 4 weeks to inhibit NO synthesis. Acute pressure natriuresis and diuresis were measured in response to an increase in perfusion pressure. NHE3 and Na(+), K(+)-ATPase protein expression were measured by Western blot and NHE3 activity was determined as the rate of pH change in brush border membrane vesicles. NHE3 membrane localization was determined by confocal microscopy. L-NAME did not significantly attenuate the natriuretic and diuretic responses to increases in renal perfusion pressure (RPP) in OP rats while inducing a significant reduction in OR rats. Following chronic NO inhibition, NHE3 protein expression and activity and Na(+), K(+)-ATPase protein expression were significantly increased in the OR but not in the OP group. Immunofluorescence studies indicated that the increase in NHE3 activity could be, at least in part, due to NHE3 membrane trafficking. Obese hypertensive rats have a weaker natriuretic response in response to NO inhibition compared to lean rats and the mechanism involves different regulation of the apical sodium exchanger NHE3 expression, activity, and trafficking.

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

PubMed

Seiler, C; Nicolson, T

1999-11-15

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

Pollen-tube tip growth requires a balance of lateral propagation and global inhibition of Rho-family GTPase activity

PubMed Central

Hwang, Jae-Ung; Wu, Guang; Yan, An; Lee, Yong-Jik; Grierson, Claire S.; Yang, Zhenbiao

2010-01-01

Rapid tip growth allows for efficient development of highly elongated cells (e.g. neuronal axons, fungal hyphae and pollen tubes) and requires an elaborate spatiotemporal regulation of the growing region. Here, we use the pollen tube as a model to investigate the mechanism regulating the growing region. ROPs (Rho-related GTPases from plants) are essential for pollen tip growth and display oscillatory activity changes in the apical plasma membrane (PM). By manipulating the ROP activity level, we showed that the PM distribution of ROP activity as an apical cap determines the tip growth region and that efficient tip growth requires an optimum level of the apical ROP1 activity. Excessive ROP activation induced the enlargement of the tip growth region, causing growth depolarization and reduced tube elongation. Time-lapse analysis suggests that the apical ROP1 cap is generated by lateral propagation of a localized ROP activity. Subcellular localization and gain- and loss-of-function analyses suggest that RhoGDI- and RhoGAP-mediated global inhibition limits the lateral propagation of apical ROP1 activity. We propose that the balance between the lateral propagation and the global inhibition maintains an optimal apical ROP1 cap and generates the apical ROP1 activity oscillation required for efficient pollen-tube elongation. PMID:20053639

Effect of entacapone on colon motility and ion transport in a rat model of Parkinson's disease.

PubMed

Li, Li-Sheng; Liu, Chen-Zhe; Xu, Jing-Dong; Zheng, Li-Fei; Feng, Xiao-Yan; Zhang, Yue; Zhu, Jin-Xia

2015-03-28

To study the effects of entacapone, a catechol-O-methyltransferase inhibitor, on colon motility and electrolyte transport in Parkinson's disease (PD) rats. Distribution and expression of catechol-O-methyltransferase (COMT) were measured by immunohistochemistry and Western blotting methods. The colonic smooth muscle motility was examined in vitro by means of a muscle motility recording device. The mucosal electrolyte transport of PD rats was examined by using a short-circuit current (ISC ) technique and scanning ion-selective electrode technique (SIET). Intracellular detection of cAMP and cGMP was accomplished by radioimmunoassay testing. COMT was expressed in the colons of both normal and PD rats, mainly on the apical membranes of villi and crypts in the colon. Compared to normal controls, PD rats expressed less COMT. The COMT inhibitor entacapone inhibited contraction of the PD rat longitudinal muscle in a dose-dependent manner. The β2 adrenoceptor antagonist ICI-118,551 blocked this inhibitory effect by approximately 67% (P < 0.01). Entacapone increased mucosal ISC in the colon of rats with PD. This induction was significantly inhibited by apical application of Cl(-) channel blocker diphenylamine-2, 2'-dicarboxylic acid, basolateral application of Na(+)-K(+)-2Cl(-)co-transporter antagonist bumetanide, elimination of Cl(-) from the extracellular fluid, as well as pretreatment using adenylate cyclase inhibitor MDL12330A. As an inhibitor of prostaglandin synthetase, indomethacin can inhibit entacapone-induced ISC by 45% (P < 0.01). When SIET was applied to measure Cl(-) flux changes, this provided similar results. Entacapone significantly increased intracellular cAMP content in the colonic mucosa, which was greatly inhibited by indomethacin. COMT expression exists in rat colons. The β2 adrenoceptor is involved in the entacapone-induced inhibition of colon motility. Entacapone induces cAMP-dependent Cl(-) secretion in the PD rat.

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

PubMed Central

Kotha, Poornima L. N.; Sharma, Priyanka; Kolawole, Abimbola O.; Yan, Ran; Alghamri, Mahmoud S.; Brockman, Trisha L.; Gomez-Cambronero, Julian; Excoffon, Katherine J. D. A.

2015-01-01

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection. PMID:25768646

A differentially amplified motion in the ear for near-threshold sound detection

PubMed Central

Chen, Fangyi; Zha, Dingjun; Fridberger, Anders; Zheng, Jiefu; Choudhury, Niloy; Jacques, Steven L.; Wang, Ruikang K.; Shi, Xiaorui; Nuttall, Alfred L.

2011-01-01

The ear is a remarkably sensitive pressure fluctuation detector. In guinea pigs, behavioral measurements indicate a minimum detectable sound pressure of ~20 μPa at 16 kHz. Such faint sounds produce 0.1 nm basilar membrane displacements, a distance smaller than conformational transitions in ion channels. It seems that noise within the auditory system would swamp such tiny motions, making weak sounds imperceptible. Here, a new mechanism contributing to a resolution of this problem is proposed and validated through direct measurement. We hypothesize that vibration at the apical end of hair cells is enhanced compared to the commonly measured basilar membrane side. Using in vivo optical coherence tomography, we demonstrated that apical-side vibrations peak at a higher frequency, had different timing, and were enhanced compared to the basilar membrane. These effects depend nonlinearly on the stimulus level. The timing difference and enhancement are important for explaining how the noise problem is circumvented. PMID:21602821

A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.

PubMed

Park, Sungjin; Szumlanski, Amy L; Gu, Fangwei; Guo, Feng; Nielsen, Erik

2011-07-17

In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP-CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.

Mammary gland involution is associated with rapid down regulation of major mammary Ca**2+-ATPases

USDA-ARS?s Scientific Manuscript database

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca**2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca**2+-ATPases and mammary calcium transport is unknown. We found that 24 hours after stopping milk prod...

Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

NASA Astrophysics Data System (ADS)

Tateishi, Kazuhiro; Nishida, Tomoki; Inoue, Kanako; Tsukita, Sachiko

2017-03-01

The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Impaired Monoamine and Organic Cation Uptake in Choroid Plexus in Mice with Targeted Disruption of the Plasma Membrane Monoamine Transporter (Slc29a4) Gene*

PubMed Central

Duan, Haichuan; Wang, Joanne

2013-01-01

The choroid plexus (CP) forms the blood-cerebrospinal fluid (CSF) barrier and protects the brain from circulating metabolites, drugs, and toxins. The plasma membrane monoamine transporter (PMAT, SLC29A4) is a new polyspecific organic cation transporter that transports a wide variety of organic cations including biogenic amines, cationic drugs, and neurotoxins. PMAT is known to be expressed in the CP, but its specific role in CP transport of organic cations has not been clearly defined. Here we showed that PMAT transcript is highly expressed in human and mouse CPs, whereas transcripts of other functionally related transporters are minimally expressed in the CPs. Immunofluorescence staining further revealed that PMAT protein is localized to the apical (CSF-facing) membrane of the CP epithelium, consistent with a role of transporting organic cations from the CSF into CP epithelial cells. To further evaluate the role of PMAT in the CP, mice with targeted deletion of the Slc29a4 gene were generated and validated. Although Pmat−/− mice showed no overt abnormalities, the uptake of monoamines and the neurotoxin 1-methyl-4-phenylpyridinium was significantly reduced in CP tissues isolated from the knock-out mice. Together, our data demonstrated that PMAT is a major transporter for CP uptake of bioactive amines and xenobiotic cations. By removing its substrates from the CSF, PMAT may play an important role in protecting the brain from cationic neurotoxins and other potentially toxic organic cations. PMID:23255610

Inhibition of intestinal dipeptide transport by the neuropeptide VIP is an anti-absorptive effect via the VPAC1 receptor in a human enterocyte-like cell line (Caco-2)

PubMed Central

Anderson, Catriona M H; Mendoza, Maria E; Kennedy, David J; Raldua, Demetrio; Thwaites, David T

2003-01-01

Optimal dipeptide and peptidomimetic drug transport across the intestinal mucosal surface is dependent upon the co-operative functional activity of the di/tripeptide transporter hPepT1 and the Na+/H+ exchanger NHE3. The ability of the anti-absorptive enteric neuropeptide VIP (vasoactive intestinal peptide) to modulate dipeptide uptake was determined using human intestinal (Caco-2) epithelial cell monolayers. Uptake of glycylsarcosine (Gly-Sar) across the apical membrane of Caco-2 cell monolayers is inhibited by basolateral exposure to either VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), or the VPAC1 receptor agonist [11,22,28Ala]-VIP. Inhibition of Gly-Sar uptake is observed only in the presence of extracellular Na+. Reverse-transcription polymerase chain reaction (RT–PCR) demonstrates that VPAC1 mRNA is expressed in Caco-2 cells whereas VPAC2 mRNA is not detected. The VIP-induced inhibition of Gly-Sar uptake is abolished in the presence of the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl). 22Na+ uptake across the apical membrane is inhibited by the selective NHE3 inhibitor S1611. Experiments with BCECF [2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that VIP reduces the NHE3-dependent recovery of intracellular pH (pHi) after dipeptide-induced acidification. Western blot of Caco-2 cell protein demonstrates expression of the NHE regulatory factor NHERF1 (expression of which is thought to be required for PKA-mediated inhibition of NHE3). VIP has no effect on Gly-Sar uptake in the presence of S1611 suggesting that VIP and S1611 both modulate dipeptide uptake via the same mechanism. These observations demonstrate that VIP (and PACAP) modulate activity of the H+/dipeptide transporter hPepT1 in a Na+-dependent manner consistent with the modulation being indirect through inhibition of NHE3. PMID:12598410

Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.

PubMed

Osypiw, J C; Gleeson, D; Lobley, R W; Pemberton, P W; McMahon, R F

1994-09-01

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.

Dexamethasone concentration gradients along scala tympani after application to the round window membrane

PubMed Central

Salt, Alec N

2008-01-01

Hypothesis Local application of dexamethasone-21-dihydrogene-phosphate (Dex-P) to the round window membrane (RWM) of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph. Background In recent years, intratympanically-applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after round window (RW) application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur with lower concentrations expected in apical turns after RW application. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph. Methods Dex-P (10mg/ml) was administered to the RWM of guinea pigs (n=9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which ten samples, each of approximately 1μl, were taken sequentially from the cochlear apex into capillary tubes. Dex-P concentration of the samples was determined by HPLC. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in scala tympani to be quantified. Results The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along scala tympani (ST). After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17•103. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially. Conclusion The existence of substantial basal-apical concentration gradients of Dex-P in ST perilymph was demonstrated experimentally. If the variability in peak concentration and gradient is also present under clinical conditions this may contribute to the heterogeneity of outcome that is observed after intratympanic application of glucocorticoids for various inner ear diseases. PMID:18277312

Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips.

PubMed

Pensabene, Virginia; Costa, Lino; Terekhov, Alexander Y; Gnecco, Juan S; Wikswo, John P; Hofmeister, William H

2016-08-31

The basal lamina or basement membrane (BM) is a key physiological system that participates in physicochemical signaling between tissue types. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology. In vitro models of the BM (e.g., Boyden and transwell chambers) are common in cell biology and lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied in these models. The goal of the present study was to integrate a semipermeable ultrathin polymer membrane with precisely positioned pores of 2 μm diameter in a microfluidic device with apical and basolateral chambers. We selected poly(l-lactic acid) (PLLA), a transparent biocompatible polymer, to prepare the semipermeable ultrathin membranes. The pores were generated by pattern transfer using a three-step method coupling femtosecond laser machining, polymer replication, and spin coating. Each step of the fabrication process was characterized by scanning electron microscopy to investigate reliability of the process and fidelity of pattern transfer. In order to evaluate the compatibility of the fabrication method with organs-on-a-chip technology, porous PLLA membranes were embedded in polydimethylsiloxane (PDMS) microfluidic devices and used to grow human umbilical vein endothelial cells (HUVECS) on top of the membrane with perfusion through the basolateral chamber. Viability of cells, optical transparency of membranes and strong adhesion of PLLA to PDMS were observed, thus confirming the suitability of the prepared membranes for use in organs-on-a-chip devices.

Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression.

PubMed

Pandey, Dhananjay K; Chaudhary, Bhupendra

2016-05-13

Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development. Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor. Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP-genes, indicating for its role upstream in the apical-to-floral meristem signalling cascade.

C-reactive protein expression is up-regulated in apical lesions of endodontic origin in association with interleukin-6.

PubMed

Garrido, Mauricio; Dezerega, Andrea; Bordagaray, María José; Reyes, Montserrat; Vernal, Rolando; Melgar-Rodríguez, Samantha; Ciuchi, Pía; Paredes, Rodolfo; García-Sesnich, Jocelyn; Ahumada-Montalva, Pablo; Hernández, Marcela

2015-04-01

C-reactive protein (CRP) is the prototype component of acute-phase proteins induced ultimately by interleukin (IL)-6 in the liver, but it is unknown whether periradicular tissues locally express CRP. The present study aimed to identify whether CRP messenger RNA synthesis occurs in situ within apical lesions of endodontic origin (ALEOs) and healthy periodontal ligament and its association with IL-6 and to determine their protein levels and tissue localization. Patients with asymptomatic apical periodontitis and healthy volunteers presenting at the School of Dentistry, University of Chile, Santiago, Chile, were enrolled. ALEOs and healthy teeth were obtained and processed for either immunohistochemistry and double immunofluorescence to assess IL-6 and CRP tissue localization, whereas healthy periodontal ligaments were processed as controls for real-time reverse-transcription polymerase chain reaction for their RNA expression levels and multiplex assay to determine their protein levels. Statistic analysis was performed using the unpaired t test or Mann-Whitney test according to data distribution and Pearson correlation. IL-6 and CRP were synthesized in ALEOs, whereas their RNA expression and protein levels were significantly higher when compared with healthy periodontal ligament. IL-6 and CRP immunolocalized to the inflammatory cells, vascular endothelial cells, and mesenchymal cells. Both, IL-6 and CRP colocalized in ALEOs, and a positive correlation was found between their expression levels (P < .05). IL-6 and CRP messenger RNA are constitutively expressed in periodontal ligament and up-regulated in ALEOs along with higher protein levels. Given their pleiotropic effects, IL-6 and CRP protein levels in apical tissues might partially explain the development and progression of ALEOs as well as potentially asymptomatic apical periodontitis-associated systemic low-grade inflammation. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression.

PubMed

Shen, Hong; Liu, Tongtong; Morse, Bridget L; Zhao, Yue; Zhang, Yueping; Qiu, Xi; Chen, Cliff; Lewin, Anne C; Wang, Xi-Tao; Liu, Guowen; Christopher, Lisa J; Marathe, Punit; Lai, Yurong

2015-07-01

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

PubMed

Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

2014-09-05

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Intercalated Cell Nedd4-2 in BP Regulation, Ion Transport, and Transporter Expression.

PubMed

Nanami, Masayoshi; Pham, Truyen D; Kim, Young Hee; Yang, Baoli; Sutliff, Roy L; Staub, Olivier; Klein, Janet D; Lopez-Cayuqueo, Karen I; Chambrey, Regine; Park, Annie Y; Wang, Xiaonan; Pech, Vladimir; Verlander, Jill W; Wall, Susan M

2018-06-01

Background Nedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP. Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/ Nedd4-2 null mice by breeding global Nedd4-2 null ( Nedd4-2 -/- ) mice with global pendrin null ( Slc26a4 -/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl - and total CO 2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry. Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl - /HCO 3 - exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 ( Slc4a9 ), H + -ATPase, barttin, or the Na + -dependent Cl - /HCO 3 - exchanger ( Slc4a8 ). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice. Conclusions IC Nedd4-2 regulates Cl - /HCO 3 - exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells. Copyright © 2018 by the American Society of Nephrology.

CALCIUM BINDING TO INTESTINAL MEMBRANES

PubMed Central

Oschman, James L.; Wall, Betty J.

1972-01-01

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl2 (as low as 5 x 10-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg++, Sr++, Ba++, Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization. PMID:4569411

Pro-apoptotic Bid induces membrane perturbation by inserting selected lysolipids into the bilayer.

PubMed

Goonesinghe, Alexander; Mundy, Elizabeth S; Smith, Melanie; Khosravi-Far, Roya; Martinou, Jean-Claude; Esposti, Mauro D

2005-04-01

Bid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial membranes. Bid appears to link the mitochondrial pathway with the death receptor-mediated pathway of cell death. It is generally assumed that the f.l. (full-length) protein becomes activated after proteolytic cleavage, especially by apical caspases like caspase 8. The cleaved protein then relocates to mitochondria and promotes membrane permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins that facilitate the release of apoptogenic proteins like cytochrome c. Although the major action may reside in the C-terminus part, tBid (cleaved Bid), un-cleaved Bid also has pro-apoptotic potential when ectopically expressed in cells or in vitro. This pro-apoptotic action of f.l. Bid has remained unexplained, especially at the biochemical level. In the present study, we show that f.l. (full-length) Bid can insert specific lysolipids into the membrane surface, thereby priming mitochondria for the release of apoptogenic factors. This is most effective for lysophosphatidylcholine species that we report to accumulate in mitochondria during apoptosis induction. A Bid mutant that is not pro-apoptotic in vivo is defective in lysophosphatidylcholine-mediated membrane perturbation in vitro. Our results thus provide a biochemical explanation for the pro-apoptotic action of f.l. Bid.

The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; Wu, Qi; Rojas-Vega, Lorena; Gamba, Gerardo; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-10-11

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na + and K + homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

Lubiprostone activates Cl- secretion via cAMP signaling and increases membrane CFTR in the human colon carcinoma cell line, T84.

PubMed

Ao, Mei; Venkatasubramanian, Jayashree; Boonkaewwan, Chaiwat; Ganesan, Nivetha; Syed, Asma; Benya, Richard V; Rao, Mrinalini C

2011-02-01

Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats.

PubMed

Yamamoto, Taeko; Kaku, Masato; Sumi, Hiromi; Yashima, Yuka; Izumino, Jin; Tanimoto, Kotaro

2018-01-01

Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 μM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1β, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1β, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1β, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement.

Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats

PubMed Central

Sumi, Hiromi; Yashima, Yuka; Izumino, Jin

2018-01-01

Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 μM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1β, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1β, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1β, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement. PMID:29694352

Myosin Vb mediates Cu+ export in polarized hepatocytes

PubMed Central

Gupta, Arnab; Schell, Michael J.; Bhattacharjee, Ashima; Lutsenko, Svetlana; Hubbard, Ann L.

2016-01-01

ABSTRACT The cellular machinery responsible for Cu+-stimulated delivery of the Wilson-disease-associated protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the Cu+-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B-myosin Vb interaction reduces the apical surface expression of ATP7B. Overexpression of the myosin Vb tail, which competes for binding of subapical cargos to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular Cu+ export. The myosin-Vb-dependent targeting step occurred in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on γ-tubulin. ATP7B became selectively arrested in this compartment at high [Cu+] in the presence of myosin Vb tail, suggesting that these compartments are precursors of donor–acceptor transfer stations for apically targeted cargos of myosin Vb. Our data suggest that reduced hepatic Cu+ clearance in idiopathic non-Wilsonian types of disease might be associated with the loss of function of myosin Vb. PMID:26823605

Loss of PiT-1 Results in Abnormal Endocytosis in the Yolk Sac Visceral Endoderm

PubMed Central

Wallingford, Mary C.; Giachelli, Cecilia M.

2014-01-01

PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE. PMID:25138534

Effects of Repeated Administration of Pilocarpine and Isoproterenol on Aquaporin-5 Expression in Rat Salivary Glands

PubMed Central

Susa, Taketo; Sawai, Nobuhiko; Aoki, Takeo; Iizuka-Kogo, Akiko; Kogo, Hiroshi; Negishi, Akihide; Yokoo, Satoshi; Takata, Kuniaki; Matsuzaki, Toshiyuki

2013-01-01

Aquaporins are water channel proteins which enable rapid water movement across the plasma membrane. Aquaporin-5 (AQP5) is the major aquaporin and is expressed on the apical membrane of salivary gland acinar cells. We examined the effects of repeated administration of pilocarpine, a clinically useful stimulant for salivary fluid secretion, and isoproterenol (IPR), a stimulant for salivary protein secretion, on the abundance of AQP5 protein in rat salivary glands by immunofluorescence microscopy and semi-quantitative immunoblotting. Unexpectedly AQP5 was decreased in pilocarpine-administered salivary glands, in which fluid secretion must be highly stimulated, implying that AQP5 might not be required for fluid secretion at least in pilocarpine-administered state. The abundance of AQP5, on the other hand, was found to be significantly increased in IPR-administered submandibular and parotid glands. To address the possible mechanism of the elevation of AQP5 abundance in IPR-administered animals, changes of AQP5 level in fasting animals, in which the exocytotic events are reduced, were examined. AQP5 was found to be decreased in fasting animals as expected. These results suggested that the elevation of cAMP and/or frequent exocytotic events could increase AQP5 protein. AQP5 expression seems to be easily changed by salivary stimulants, although these changes do not always reflect the ability in salivary fluid secretion. PMID:24610966

Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

PubMed

Navarro-Moreno, L G; Quintanar-Escorza, M A; González, S; Mondragón, R; Cerbón-Solorzáno, J; Valdés, J; Calderón-Salinas, J V

2009-10-01

Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers.

PubMed

Ho, Norman F H; Nielsen, James; Peterson, Michelle; Burton, Philip S

2016-02-01

An approach to characterizing P-glycoprotein (Pgp) interaction potential for sparingly water-soluble compounds was developed using bidirectional transport kinetics in MDR1-MDCK cell monolayers. Paclitaxel, solubilized in a dilute polysorbate 80 (PS80) micellar solution, was used as a practical example. Although the passage of paclitaxel across the cell monolayer was initially governed by the thermodynamic activity of the micelle-solubilized drug solution, Pgp inhibition was sustained by the thermodynamic activity (i.e., critical micelle concentration) of the PS80 micellar solution bathing the apical (ap) membrane. The mechanistic understanding of the experimental strategies and treatment of data was supported by a biophysical model expressed in the form of transport events occurring at the ap and basolateral (bl) membranes in series whereas the vectorial directions of the transcellular kinetics were accommodated. The derived equations permitted the stepwise quantitative delineation of the Pgp efflux activity (inhibited and uninhibited by PS80) and the passive permeability coefficient of the ap membrane, the passive permeability at the bl membrane and, finally, the distinct coupling of these with efflux pump activity to identify the rate-determining steps and mechanisms. The Jmax/KM(∗) for paclitaxel was in the order of 10(-4) cm/s and the ap- and bl-membrane passive permeability coefficients were asymmetric, with bl-membrane permeability significantly greater than ap. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Functional characterization and cellular dynamics of the CDC-42 - RAC - CDC-24 module in Neurospora crassa.

PubMed

Araujo-Palomares, Cynthia L; Richthammer, Corinna; Seiler, Stephan; Castro-Longoria, Ernestina

2011-01-01

Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 - RAC - CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.

[Microelectrode study of the cellular reactions of the taste bud in the frog Rana temporaria].

PubMed

Lotarev, A N; Samoĭlov, V O

1986-01-01

Microelectrophysiological studies reveal two types of cells in the taste bud of frog which differ by the level of their membrane potential. During vertical implantation of microelectrode through the apical part of the taste bud, the potential difference in the upper layer amounts to 15 mV. Further implantation of the electrode results in a stepwise decrease of the potential difference up to 27 mV. Cells of the deeper layer are located 12-24 micron lower from the apical surface. Stimulation of cells by solutions of chemical substances is accompanied by cell depolarization, its amplitude being proportional to stimulus concentration. The steepness of depolarization depends on the modality of the stimulus, being maximum for salts. The data obtained suggest that cells of the second layer, with a higher resting membrane potential level, are taste ones.

Detergent-mediated protein aggregation

PubMed Central

Neale, Chris; Ghanei, Hamed; Holyoake, John; Bishop, Russell E.; Privé, Gilbert G.; Pomès, Régis

2016-01-01

Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein’s hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation. PMID:23466535

Polarized Ends of Human Macula Densa Cells: Ultrastructural Investigation and Morphofunctional Correlations.

PubMed

Cangiotti, Angela Maria; Lorenzi, Teresa; Zingaretti, Maria Cristina; Fabri, Mara; Morroni, Manrico

2018-05-01

The morphology of the kidney macula densa (MD) has extensively been investigated in animals, whereas human studies are scanty. We studied the fine structure of human MD cells focusing on their apical and basal ends and correlating structure and function. The MD region was examined by transmission electron microscopy in six renal biopsies from patients with kidney disease. Ultrastructural analysis of MD cells was performed on serial sections. MD cells show two polarized ends. The apical portion is characterized by a single, immotile cilium associated with microvilli; apically, cells are joined by adhering junctions. In the basal portion, the cytoplasm contains small, dense granules and numerous, irregular cytoplasmic projections extending to the adjacent extraglomerular mesangium. The projections often contain small, dense granules. A reticulated basement membrane around MD cells separates them from the extraglomerular mesangium. Although the fact that tissue specimens came from patients with kidney disease mandates extreme caution, ultrastructural examination confirmed that MD cells have sensory features due to the presence of the primary cilium, that they are connected by apical adhering junctions forming a barrier that separates the tubular flow from the interstitium, and that they present numerous basal interdigitations surrounded by a reticulated basement membrane. Conceivably, the latter two features are related to the functional activity of the MD. The small, dense granules in the basal cytoplasm and in cytoplasmic projections are likely related to the paracrine function of MD cells. Anat Rec, 301:922-931, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats.

PubMed

Cuello-Carrión, F Darío; Troncoso, Mariana; Guiñazu, Elina; Valdez, Susana R; Fanelli, Mariel A; Ciocca, Daniel R; Kreimann, Erica L

2010-12-01

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins

Treesearch

Algimantas P. Valaitis; John D. Podgwaite

2013-01-01

Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium....

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.

PubMed

Worrell, Roger T; Best, Alison; Crawford, Oscar R; Xu, Jie; Soleimani, Manoocher; Matthews, Jeffrey B

2005-10-01

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.

Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

PubMed Central

Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

2013-01-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.

PubMed

Samal, Sweety; Khattar, Sunil K; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L; Samal, Siba K

2013-09-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.

Low-density lipoproteins modulate endothelial cells to secrete endothelin-1 in a polarized pattern: a study using a culture model system simulating arterial intima.

PubMed

Unoki, H; Fan, J; Watanabe, T

1999-01-01

We investigated the structural and functional properties of human umbilical vein endothelial cells (HUVECs) cultured on a two-chamber culture model system using an amnion membrane. Compared to HUVECs cultured on a plastic dish, HUVECs cultured on the model system exhibited several features similar to those of in vivo vessels, including formation of the intercellular junctional devices and expression of tight junction-associated protein ZO-1 and adherence junction-associated protein alpha-catenin. Furthermore, we found that HUVECs had a property of polar secretion of endothelin-1 (ET-1). About 90% of the total amount of synthesized ET-1 was found in the lower well, designated as the basal side. When HUVECs were incubated with either native low-density lipoproteins (nLDLs) or oxidized LDLs (oxLDLs) at a concentration of 100 microgram/ml, ET-1 secretion was significantly increased, dependent on the cell side (apical vs basal) on which the nLDLs or oxLDLs were loaded. When the LDLs were loaded on the apical side, the secretion of ET-1 from HUVECs on the apical side was increased by 48% (nLDL) and 61% (oxLDL), whereas it was accompanied by a concomitant decrease of ET-1 on the basal side (45% by nLDLs and 38% by oxLDLs). When loaded on the basal side, however, ET-1 was increased by 23% (nLDLs) and 53% (oxLDLs) on the basal side, with a 26% simultaneous decrease of ET-1 on the opposite side for both nLDLs and oxLDLs. On the contrary, high-density lipoproteins (HDLs) inhibited ET-1 secretion from HUVECs on the opposite side of the well on which HDLs were loaded; there was a 57% decrease on the basal side when HDLs were loaded on the apical side, and a 46% decrease on the apical side when loaded on the basal side. These results indicate that modulation of ET-1 secretion from ECs by lipoproteins is virtually dependent on the place (apical vs basal) where these proteins are present. The finding that nLDLs and oxLDLs enhance ET-1 secretion by ECs in a polarized pattern suggests that ET-1 may be involved in pathophysiological processes such as atherogenesis.

Expression of the ctenophore Brain Factor 1 forkhead gene ortholog (ctenoBF-1) mRNA is restricted to the presumptive mouth and feeding apparatus: implications for axial organization in the Metazoa

NASA Technical Reports Server (NTRS)

Yamada, Atsuko; Martindale, Mark Q.

2002-01-01

Ctenophores are thoroughly modern animals whose ancestors are derived from a separate evolutionary branch than that of other eumetazoans. Their major longitudinal body axis is the oral-aboral axis. An apical sense organ, called the apical organ, is located at the aboral pole and contains a highly innervated statocyst and photodetecting cells. The apical organ integrates sensory information and controls the locomotory apparatus of ctenophores, the eight longitudinal rows of ctene/comb plates. In an effort to understand the developmental and evolutionary organization of axial properties of ctenophores we have isolated a forkhead gene from the Brain Factor 1 (BF-1) family. This gene, ctenoBF-1, is the first full-length nuclear gene reported from ctenophores. This makes ctenophores the most basal metazoan (to date) known to express definitive forkhead class transcription factors. Orthologs of BF-1 in vertebrates, Drosophila, and Caenorhabditis elegans are expressed in anterior neural structures. Surprisingly, in situ hybridizations with ctenoBF-1 antisense riboprobes show that this gene is not expressed in the apical organ of ctenophores. CtenoBF-1 is expressed prior to first cleavage. Transcripts become localized to the aboral pole by the 8-cell stage and are inherited by ectodermal micromeres generated from this region at the 16- and 32-cell stages. Expression in subsets of these cells persists and is seen around the edge of the blastopore (presumptive mouth) and in distinct ectodermal regions along the tentacular poles. Following gastrulation, stomodeal expression begins to fade and intense staining becomes restricted to two distinct domains in each tentacular feeding apparatus. We suggest that the apical organ is not homologous to the brain of bilaterians but that the oral pole of ctenophores corresponds to the anterior pole of bilaterian animals.

Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

PubMed

Oufattole, M; Arango, M; Boutry, M

2000-04-01

To analyze in detail the multigene family encoding the plasma-membrane H(+)-ATPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and classified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While the pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricted to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in secretion, and to the cortical parenchyma of the young nodes where the developing leaves and axillary flowering stalks join the stem. In the latter tissues, gene expression was enhanced by mechanical stress, suggesting that H(+)-ATPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed in the apical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the developmental stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identified in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 was found to be expressed in both organs at a lower level. In conclusion, although pma 6 and 9 had a more restricted expression pattern than the previously characterized pma genes, they were nevertheless expressed in cell types in which H(+)-ATPase had not been previously detected.

Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the meninges and the choroid plexus: implications for non-neuronal sources for GABA in the developing mouse brain.

PubMed

Tochitani, Shiro; Kondo, Shigeaki

2013-01-01

Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11-E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.

Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the Meninges and the Choroid Plexus: Implications for Non-Neuronal Sources for GABA in the Developing Mouse Brain

PubMed Central

Tochitani, Shiro; Kondo, Shigeaki

2013-01-01

Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11–E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development. PMID:23437266

Simple image-based no-wash method for quantitative detection of surface expressed CFTR

PubMed Central

Larsen, Mads Breum; Hu, Jennifer; Frizzell, Raymond A.; Watkins, Simon C.

2016-01-01

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR’s anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins. PMID:26361332

The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

PubMed

Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

2016-01-01

Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit are Critical for its Fusion Activity.

PubMed

Hicks, Stephanie N; Chaiwatpongsakorn, Supranee; Costello, Heather M; McLellan, Jason S; Ray, William; Peeples, Mark E

2018-05-09

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both viral-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F 1 and F 2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F 1 subunit to refold dramatically while F 2 changes minimally. Hypothesizing that the most likely site for interaction with a target-cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F 2 by scanning mutagenesis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting they are involved in refolding. Alanine mutation at Ile64, also on the ascent to the apex, and Lys75, did not prevent fusion following forced triggering, suggesting they are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3, causing nearly 33 million acute lower respiratory infections (ALRI) worldwide each year in children younger than 5 (Nair H, et al. 2010. Lancet 375:1545-55). RSV is also the second leading cause of respiratory related death in the elderly (Falsey AR, Walsh EE. 2005. Drugs Aging 22:577-87; Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. 2005. N Engl J Med 352:1749-1759). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (Costello HM, Ray W, Chaiwatpongsakorn S, Peeples ME. 2012. 12:110-128). The essential residues identified in the apical domain of F 2 are adjacent to the apical portion of F 1 which, upon triggering, refolds into the long heptad repeat A (HRA) with the fusion peptide at its N-terminus. These essential residues in F 2 are likely involved in triggering and/or refolding of the F protein and as such may be ideal targets for antiviral drug development. Copyright © 2018 American Society for Microbiology.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

PubMed

Shadforth, Audra M A; Suzuki, Shuko; Theodoropoulos, Christina; Richardson, Neil A; Chirila, Traian V; Harkin, Damien G

2017-06-01

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell ® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na + /K + -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells.

PubMed

Ducharme, Nicole A; Hales, Chadwick M; Lapierre, Lynne A; Ham, Amy-Joan L; Oztan, Asli; Apodaca, Gerard; Goldenring, James R

2006-08-01

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.

TASK-2 K₂p K⁺ channel: thoughts about gating and its fitness to physiological function.

PubMed

López-Cayuqueo, Karen I; Peña-Münzenmayer, Gaspar; Niemeyer, María Isabel; Sepúlveda, Francisco V; Cid, L Pablo

2015-05-01

TASK-2 (K2P5) was one of the earliest members of the K2P two-pore, four transmembrane domain K(+) channels to be identified. TASK-2 gating is controlled by changes in both extra- and intracellular pH through separate sensors: arginine 224 and lysine 245, located at the extra- and intracellular ends of transmembrane domain 4. TASK-2 is inhibited by a direct effect of CO2 and is regulated by and interacts with G protein subunits. TASK-2 takes part in regulatory adjustments and is a mediator in the chemoreception process in neurons of the retrotrapezoid nucleus where its pHi sensitivity could be important in regulating excitability and therefore signalling of the O2/CO2 status. Extracellular pH increases brought about by HCO3 (-) efflux from proximal tubule epithelial cells have been proposed to couple to TASK-2 activation to maintain electrochemical gradients favourable to HCO3 (-) reabsorption. We demonstrate that, as suspected previously, TASK-2 is expressed at the basolateral membrane of the same proximal tubule cells that express apical membrane Na(+)-H(+)-exchanger NHE-3 and basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-A, the main components of the HCO3 (-) transport machinery. We also discuss critically the mechanism by which TASK-2 is modulated and impacts the process of HCO3 (-) reclaim by the proximal tubule epithelium, concluding that more than a mere shift in extracellular pH is probably involved.

Neuroglian stabilizes epithelial structure during Drosophila oogenesis.

PubMed

Wei, Jun; Hortsch, Michael; Goode, Scott

2004-08-01

The vertebrate L1 family of cell adhesion molecules (CAMs) and their fly homolog, Neuroglian, are members of the immunoglobulin (Ig) superfamily of CAMs. In general, Ig CAMs have been found to play critical roles in mediating axon guidance. One Ig CAM, NCAM, has also been implicated in maintaining epithelial integrity and suppressing metastatic dissemination of tumor cells. Other Ig CAMs, such as Nrg, are also expressed in epithelia. We thus tested the hypothesis that, like NCAM, Nrg might also be required for maintaining epithelial integrity and for inhibiting tumor invasion. We used the Drosophila follicular epithelium to determine the function of Nrg in vivo in maintaining epithelial structure, and in regulating the motility of migrating border cells and invasive tumorous follicle cells. Nrg(167) is expressed on the lateral membrane of follicle cells. Loss of Nrg(167) causes border cells to delay delamination and causes other follicle cells to delaminate inappropriately. The delaminated cells have aberrant epithelial polarity manifested as severe mislocalization of apical and basal membrane proteins, and uniform localization of lateral membrane proteins. Furthermore, loss of Nrg(167) dramatically enhances the invasive phenotype associated with loss of Discs Large, a neoplastic tumor suppressor. These results indicate that Nrg(167) stabilizes epithelial polarity by regulating junctional adhesion and function in normal and tumorous epithelia. Our data also suggest that Ig superfamily members have significant functional redundancy in maintaining epithelial polarity, with individual members playing subtle, unique roles during epithelial morphogenesis. Copyright 2004 Wiley-Liss, Inc.

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

PubMed Central

1983-01-01

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment. PMID:6411740

A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S

1994-01-01

We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasismore » suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system. While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.« less

Loss of syntaxin 3 causes variant microvillus inclusion disease.

PubMed

Wiegerinck, Caroline L; Janecke, Andreas R; Schneeberger, Kerstin; Vogel, Georg F; van Haaften-Visser, Désirée Y; Escher, Johanna C; Adam, Rüdiger; Thöni, Cornelia E; Pfaller, Kristian; Jordan, Alexander J; Weis, Cleo-Aron; Nijman, Isaac J; Monroe, Glen R; van Hasselt, Peter M; Cutz, Ernest; Klumperman, Judith; Clevers, Hans; Nieuwenhuis, Edward E S; Houwen, Roderick H J; van Haaften, Gijs; Hess, Michael W; Huber, Lukas A; Stapelbroek, Janneke M; Müller, Thomas; Middendorp, Sabine

2014-07-01

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halwachs, Sandra

In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim,more » chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B > A) directed transport of [{sup 14}C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates. - Highlights: • Generation of MDCKII-rbABCG2 monolayers with epithelial barrier function • Detection of rbABCG2 in the apical plasma membrane of polarized MDCKII cells • Several pesticides interact with the ABCG2 transporter from rabbit placenta. • rbABCG2 mediates transport of the insecticide chlorpyrifos. • MDCKII-rbABCG2 cells are a suitable model to study transport in rabbit placenta.« less

Ultrastructural aspects of the oesophageal and reproductive systems of the equine parasite Strongylus vulgaris.

PubMed

Mobarak, M S; Ryan, M F

2002-06-01

The ultrastructure of the dorsal oesophageal gland ampulla and its relationship with the oesophagus, oesophageal ultrastructure, and control mechanisms in oesophageal activity were studied. Terminal ducts of the sub-ventral glands open through the oesophageal crown at the base of the buccal cavity. The terminal duct of the dorsal oesophageal gland running through the dorsal gutter opens to the exterior at the rim 'groove' of the buccal capsule. The posterior oesophageal region is clavate and the cuticle of the lumen folds to form outlet valves, 'valvulae'. An inconspicuous oesophago-intestinal valve (three lobes) connects oesophagus and intestine and is visualized in the open and shut position. In the female reproductive tract, with the exception of the uterus, the cells lie on a thick, irregular (convoluted) basal lamina. The apical plasma membrane of the uterus, and seminal receptacle, extend into the lumen by microvilli-like projections with which spermatozoa make intimate contact. The lumen of the uterus is filled with oocytes, fertilized and unfertilized. Testicular cells have two parts linked by a rachis. Spermatocytes are elongated with a large nucleus, distinct nuclear membrane, and many granules. The apical membrane of the rachis forms long microvilli-like projections with balloon-like tips. The amoeboid spermatozoa contain membrane specializations, a nucleus devoid of a membrane, and are enclosed by a pseudopodial-like extension.

Basolateral membrane K+ channels in renal epithelial cells

PubMed Central

Devor, Daniel C.

2012-01-01

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K+ channels play critical roles in normal physiology. Over 90 different genes for K+ channels have been identified in the human genome. Epithelial K+ channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K+ channels is to recycle K+ across the basolateral membrane for proper function of the Na+-K+-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K+ channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a “K+ channel gene family” approach in presenting the representative basolateral K+ channels of the nephron. The basolateral K+ channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families. PMID:22338089

Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-08-01

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.

Breast cancer resistance protein (Bcrp) and the testis—an unexpected turn of events

PubMed Central

Qian, Xiaojing; Cheng, Yan-Ho; Mruk, Dolores D; Cheng, C Yan

2013-01-01

Breast cancer resistance protein (Bcrp) is an ATP-dependent efflux drug transporter. It has a diverse spectrum of hydrophilic and hydrophobic substrates ranging from anticancer, antiviral and antihypertensive drugs, to organic anions, antibiotics, phytoestrogens (e.g., genistein, daidzein, coumestrol), xenoestrogens and steroids (e.g., dehydroepiandrosterone sulfate). Bcrp is an integral membrane protein in cancer and normal cells within multiple organs (e.g., brain, placenta, intestine and testis) that maintains cellular homeostasis by extruding drugs and harmful substances from the inside of cells. In the brain, Bcrp is a major component of the blood–brain barrier located on endothelial cells near tight junctions (TJs). However, Bcrp is absent at the Sertoli cell blood–testis barrier (BTB); instead, it is localized almost exclusively to the endothelial TJ in microvessels in the interstitium and the peritubular myoid cells in the tunica propria. Recent studies have shown that Bcrp is also expressed stage specifically and spatiotemporally by Sertoli and germ cells in the seminiferous epithelium of rat testes, limited only to a testis-specific cell adhesion ultrastructure known as the apical ectoplasmic specialisation (ES) in stage VI–early VIII tubules. These findings suggest that Bcrp is equipped by late spermatids and Sertoli cells to protect late-stage spermatids completing spermiogenesis. Furthermore, Bcrp was found to be associated with F (filamentous)-actin and several actin regulatory proteins at the apical ES and might be involved in the organisation of actin filaments at the apical ES in stage VII–VIII tubules. These findings will be carefully evaluated in this brief review. PMID:23665760

Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic.

PubMed

Rosenbaum, Jason N; Duggan, Anne; García-Añoveros, Jaime

2011-02-01

Insm1 is a zinc-finger transcription factor transiently expressed throughout the developing nervous system in late progenitors and nascent neurons. Insm1 is also highly expressed in medulloblastomas and other neuroendocrine tumors. We generated mice lacking the Insm1 gene and used them to elucidate its role in neurogenic proliferation of the embryonic olfactory epithelium. We found that deletion of Insm1 results in more apical cells and fewer nascent and mature neurons. In the embryonic olfactory epithelium of Insm1 mutants we detect fewer basal progenitors, which produce neurons, and more apical progenitors, which at this stage produce additional progenitors. Furthermore, in the mutants we detect fewer progenitors expressing NEUROD1, a marker of terminally dividing, neuronogenic (neuron-producing) progenitors (immediate neuronal precursors), and more progenitors expressing ASCL1, a marker of the transit amplifying progenitors that migrate from the apical to the basal edges of the epithelium while dividing to generate the terminal, neuronogenic progenitors. Finally, with timed administration of nucleoside analogs we demonstrate that the Insm1 mutants contain fewer terminally dividing progenitors at embryonic day 12.5. Altogether, these results suggest a role for Insm1 in promoting the transition of progenitors from apical and proliferative to basal, terminal and neuronogenic. This role appears partially conserved with that of its nematode ortholog, egl-46. The similar effects of Insm1 deletion on progenitors of embryonic olfactory epithelium and cortex point to striking parallels in the development of these neuroepithelia, and particularly between the basal progenitors of olfactory epithelium and the subventricular zone progenitors of cortex.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation1[C][W

PubMed Central

Campo, Sonia; Baldrich, Patricia; Messeguer, Joaquima; Lalanne, Eric; Coca, María; San Segundo, Blanca

2014-01-01

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage. PMID:24784760

Expression and subcellular localization of NHE3 in the human gallbladder epithelium

PubMed Central

Chen, Yongsheng; Kong, Jing; Wu, Shuodong

2014-01-01

Background: Enhanced gallbladder concentrating function is an important factor for the pathogenesis of cholesterol gallstone disease (CGD), but the mechanism is unknown. Potential candidates for regulation of gallbladder ion absorption are suggested to be Na+/H+ exchanger isoform 3 (NHE3). In this study, we investigated the expression and subcellular localization of NHE3 in both acalculous and calculous human gallbladders. Methods: Adult human gallbladder tissue was obtained from 23 patients (7 men, 16 women) who had undergone cholecystectomy. The patients were divided into two groups: Group A (acalculous group) and Group B (calculous group). Gene expression of NHE3 was quantitatively estimated by real-time PCR. Protein expression was studied by Western blotting assays. Furthermore, expression of immunoreactive NHE3 was investigated by immunohistochemistry. Results: There was no significant difference in the NHE3 mRNA expression between calculous and acalculous human gallbladders. NHE3 protein expression in gallbladders from patients with cholelithiasis is increased compared to those without gallstones. Immunohistochemistry studies prove that NHE3 is located both on the apical plasma membrane and in the intracellular pool in human GBECs. Conclusions: NHE3 may play a role in the pathogenesis of human CGD. Additional studies are required to further delineate the underlying mechanisms. PMID:25674247

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

PubMed Central

Annaba, Fadi; Sarwar, Zaheer; Kumar, Pradeep; Saksena, Seema; Turner, Jerrold R.; Dudeja, Pradeep K.; Gill, Ravinder K.; Alrefai, Waddah A.

2016-01-01

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. PMID:18063707

Embryonic integument and "molts" in Manduca sexta (Insecta, Lepidoptera).

PubMed

Ziese, Stefanie; Dorn, August

2003-02-01

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed. Copyright 2002 Wiley-Liss, Inc.

Analysis of Microtubule Mediated Functions of Prostate Specific Membrane Antigen

DTIC Science & Technology

2006-04-01

localization of vesicles containing these markers increased to approximately 43% (38/88) when cells are incubated with tunicamycin, indicating a role...PSMA at both plasma membrane domains following nocodazole treatment. Polarity of the basolateral marker Na,K- ATPase was unaffected by nocodazole...restricted to the apical surface facing the lumen. This staining was clearly distinct from that of the endothelial cell marker CD 34 and CD31

Tight junctions and human diseases.

PubMed

Sawada, Norimasa; Murata, Masaki; Kikuchi, Keisuke; Osanai, Makoto; Tobioka, Hirotoshi; Kojima, Takashi; Chiba, Hideki

2003-09-01

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

Redistribution of distal tubule Na+-Cl- cotransporter (NCC) in response to a high-salt diet.

PubMed

Sandberg, Monica B; Maunsbach, Arvid B; McDonough, Alicia A

2006-08-01

The distal convoluted tubule (DCT) apical Na(+)-Cl(-) cotransporter (NCC) is responsible for the reabsorption of 5-10% of filtered NaCl and is the target for thiazide diuretics. NCC abundance is increased during dietary NaCl restriction and by aldosterone and decreased during a high-salt (HS) diet and mineralocorticoid blockade. This study tested the hypothesis that subcellular distribution of NCC is also regulated in response to changes in dietary salt. Six-week-old Sprague-Dawley rats were fed a normal-salt diet (NS; 0.4% NaCl) for 3 wk, then switched to a HS diet (4% NaCl) for 3 wk or a low-salt diet (LS; 0.07% NaCl) for 1 wk. Under anesthesia, kidneys were excised, renal cortex was dissected, and NCC was analyzed with specific antibodies after either 1) density gradient centrifugation followed by immunoblotting or 2) fixation followed by immunoelectron microscopy. The HS diet decreased NCC abundance to 0.50 +/- 0.10 of levels in LS diet (1.00 +/- 0.23). The HS diet also caused a redistribution of NCC from low to higher density membranes. Immunoelectron microscopy revealed that NCC resides predominantly in the apical membrane in rats fed the LS diet and increases in subapical vesicles in rats fed the HS diet. In conclusion, a HS diet provokes a rapid and persistent redistribution of NCC from apical to subapical membranes, a mechanism that would facilitate a homeostatic decrease in NaCl reabsorption in the DCT to compensate for increased dietary salt.

Variants in Solute Carrier SLC26A9 Modify Prenatal Exocrine Pancreatic Damage in Cystic Fibrosis

PubMed Central

Miller, Melissa R.; Soave, David; Li, Weili; Gong, Jiafen; Pace, Rhonda G.; Boëlle, Pierre-Yves; Cutting, Garry R.; Drumm, Mitchell L.; Knowles, Michael R.; Sun, Lei; Rommens, Johanna M.; Accurso, Frank; Durie, Peter R.; Corvol, Harriet; Levy, Hara; Sontag, Marci K.; Strug, Lisa J.

2015-01-01

Objectives To test the hypothesis that multiple constituents of the apical plasma membrane residing alongside the causal CF Transmembrane Conductance Regulator (CFTR) protein, including known cystic fibrosis (CF) modifiers SLC26A9, SLC6A14, and SLC9A3, would be associated with prenatal exocrine pancreatic damage as measured by newborn screened (NBS) IRT levels. Study design NBS IRT measures and genome-wide genotype data were available on 111 subjects from Colorado, 37 subjects from Wisconsin, and 80 subjects from France. Multiple linear regression was used to determine whether any of eight SNPs in SLC26A9, SLC6A14 and SLC9A3 were associated with IRT and whether other constituents of the apical plasma membrane contributed to IRT. Results In the Colorado sample, three SLC26A9 SNPs were associated with NBS IRT (min P = 1.16 × 10−3; rs7512462), but no SLC6A14 or SLC9A3 SNPs were associated (P > 0.05). The rs7512462 association replicated in the Wisconsin sample (P = 0.03) but not in the French sample (P = 0.76). Furthermore, rs7512462 was the top ranked apical membrane constituent in the combined Colorado and Wisconsin sample. Conclusions NBS IRT is a biomarker of prenatal exocrine pancreatic disease in patients with CF, and a SNP in SLC26A9 accounts for significant IRT variability. This suggests SLC26A9 as a potential therapeutic target to ameliorate exocrine pancreatic disease. PMID:25771386

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogel, Lotte K.; Larsen, Jakob E.; Hansen, Martin

2005-05-13

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity ofmore » secretion is determined by conformational sensitive sorting signals.« less

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

PubMed

Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucose Uptake in the Human Pathogen Schistosoma mansoni Is Regulated Through Akt/Protein Kinase B Signaling.

PubMed

McKenzie, Maxine; Kirk, Ruth S; Walker, Anthony J

2018-06-05

In Schistosoma mansoni, the facilitated glucose transporter SGTP4, which is expressed uniquely in the apical surface tegumental membranes of the parasite, imports glucose from host blood to support its growth, development, and reproduction. However, the molecular mechanisms that underpin glucose uptake in this blood fluke are not understood. In this study we employed techniques including Western blotting, immunolocalization, confocal laser scanning microscopy, pharmacological assays, and RNA interference to functionally characterize and map activated Akt in S mansoni. We find that Akt, which could be activated by host insulin and l-arginine, was active in the tegument layer of both schistosomules and adult worms. Blockade of Akt attenuated the expression and evolution of SGTP4 at the surface of the host-invading larval parasite life-stage, and suppressed SGTP4 expression at the tegument in adults; concomitant glucose uptake by the parasite was also attenuated in both scenarios. These findings shed light on crucial mechanistic signaling processes that underpin the energetics of glucose uptake in schistosomes, which may open up novel avenues for antischistosome drug development.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

PubMed

De Berdt, P; Vanacker, J; Ucakar, B; Elens, L; Diogenes, A; Leprince, J G; Deumens, R; des Rieux, A

2015-11-01

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel. © International & American Associations for Dental Research 2015.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells

PubMed Central

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe

2017-01-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. PMID:29046391

Cochlear partition tuning within the 2nd apical turn of the intact gerbil cochlea

NASA Astrophysics Data System (ADS)

Dong, Wei; Xia, Anping; Puria, Sunil; Applegate, Brian E.; Oghalai, John S.

2018-05-01

Our understanding of cochlear amplification has been mainly informed by observations of the high-frequency basal region. Results from the few existing in vivo studies from the low-frequency apical region have suggested that cochlear amplification may be more broadly tuned and not as nonlinear as in the base. The current study explored micromechanics via sound evoked vibrations of the cochlear partition, including the organ of Corti (OoC), the basilar membrane (BM) and the tectorial membrane (TM), within the 2nd apical turn, corresponding to the 2.5 kHz best frequency region, of the gerbil cochlea using volumetric optical coherence tomography vibrometry (VOCTV), imaged non-invasively through the otic capsule bone in vivo. Sound induced radial displacements below 4 kHz showed similarities and differences to the well-established BM transverse responses at high-frequency basal region. Responses showed broader tuning, but similar gains at the best frequency and similar phase accumulation. The distinct difference found was the presence of compressive nonlinear growth with SPL below the best frequency down to 0.1 kHz observed at the outer hair cell (OHC) and reticular laminar (RL) locations but not in the BM or inner hair cell regions. These relative motions provide further insight on how OHC somatic forces are distributed within the cochlear partition.

Neuroelectric Tuning of Cortical Oscillations by Apical Dendrites in Loop Circuits

PubMed Central

LaBerge, David; Kasevich, Ray S.

2017-01-01

Bundles of relatively long apical dendrites dominate the neurons that make up the thickness of the cerebral cortex. It is proposed that a major function of the apical dendrite is to produce sustained oscillations at a specific frequency that can serve as a common timing unit for the processing of information in circuits connected to that apical dendrite. Many layer 5 and 6 pyramidal neurons are connected to thalamic neurons in loop circuits. A model of the apical dendrites of these pyramidal neurons has been used to simulate the electric activity of the apical dendrite. The results of that simulation demonstrated that subthreshold electric pulses in these apical dendrites can be tuned to specific frequencies and also can be fine-tuned to narrow bandwidths of less than one Hertz (1 Hz). Synchronous pulse outputs from the circuit loops containing apical dendrites can tune subthreshold membrane oscillations of neurons they contact. When the pulse outputs are finely tuned, they function as a local “clock,” which enables the contacted neurons to synchronously communicate with each other. Thus, a shared tuning frequency can select neurons for membership in a circuit. Unlike layer 6 apical dendrites, layer 5 apical dendrites can produce burst firing in many of their neurons, which increases the amplitude of signals in the neurons they contact. This difference in amplitude of signals serves as basis of selecting a sub-circuit for specialized processing (e.g., sustained attention) within the typically larger layer 6-based circuit. After examining the sustaining of oscillations in loop circuits and the processing of spikes in network circuits, we propose that cortical functioning can be globally viewed as two systems: a loop system and a network system. The loop system oscillations influence the network system’s timing and amplitude of pulse signals, both of which can select circuits that are momentarily dominant in cortical activity. PMID:28659768

Neuroelectric Tuning of Cortical Oscillations by Apical Dendrites in Loop Circuits.

PubMed

LaBerge, David; Kasevich, Ray S

2017-01-01

Bundles of relatively long apical dendrites dominate the neurons that make up the thickness of the cerebral cortex. It is proposed that a major function of the apical dendrite is to produce sustained oscillations at a specific frequency that can serve as a common timing unit for the processing of information in circuits connected to that apical dendrite. Many layer 5 and 6 pyramidal neurons are connected to thalamic neurons in loop circuits. A model of the apical dendrites of these pyramidal neurons has been used to simulate the electric activity of the apical dendrite. The results of that simulation demonstrated that subthreshold electric pulses in these apical dendrites can be tuned to specific frequencies and also can be fine-tuned to narrow bandwidths of less than one Hertz (1 Hz). Synchronous pulse outputs from the circuit loops containing apical dendrites can tune subthreshold membrane oscillations of neurons they contact. When the pulse outputs are finely tuned, they function as a local "clock," which enables the contacted neurons to synchronously communicate with each other. Thus, a shared tuning frequency can select neurons for membership in a circuit. Unlike layer 6 apical dendrites, layer 5 apical dendrites can produce burst firing in many of their neurons, which increases the amplitude of signals in the neurons they contact. This difference in amplitude of signals serves as basis of selecting a sub-circuit for specialized processing (e.g., sustained attention) within the typically larger layer 6-based circuit. After examining the sustaining of oscillations in loop circuits and the processing of spikes in network circuits, we propose that cortical functioning can be globally viewed as two systems: a loop system and a network system. The loop system oscillations influence the network system's timing and amplitude of pulse signals, both of which can select circuits that are momentarily dominant in cortical activity.

Breast cancer resistance protein regulates apical ectoplasmic specialization dynamics stage specifically in the rat testis

PubMed Central

Qian, Xiaojing; Mruk, Dolores D.; Wong, Elissa W. P.

2013-01-01

Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18–19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis. PMID:23403943

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

PubMed Central

Parker, Michelle L.; Penarete-Vargas, Diana M.; Hamilton, Phineas T.; Guérin, Amandine; Dubey, Jitender P.; Perlman, Steve J.; Spano, Furio; Lebrun, Maryse; Boulanger, Martin J.

2016-01-01

Plasmodium falciparum and Toxoplasma gondii are widely studied parasites in phylum Apicomplexa and the etiological agents of severe human malaria and toxoplasmosis, respectively. These intracellular pathogens have evolved a sophisticated invasion strategy that relies on delivery of proteins into the host cell, where parasite-derived rhoptry neck protein 2 (RON2) family members localize to the host outer membrane and serve as ligands for apical membrane antigen (AMA) family surface proteins displayed on the parasite. Recently, we showed that T. gondii harbors a novel AMA designated as TgAMA4 that shows extreme sequence divergence from all characterized AMA family members. Here we show that sporozoite-expressed TgAMA4 clusters in a distinct phylogenetic clade with Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) proteins and forms a high-affinity, functional complex with its coevolved partner, TgRON2L1. High-resolution crystal structures of TgAMA4 in the apo and TgRON2L1-bound forms complemented with alanine scanning mutagenesis data reveal an unexpected architecture and assembly mechanism relative to previously characterized AMA–RON2 complexes. Principally, TgAMA4 lacks both a deep surface groove and a key surface loop that have been established to govern RON2 ligand binding selectivity in other AMAs. Our study reveals a previously underappreciated level of molecular diversity at the parasite–host-cell interface and offers intriguing insight into the adaptation strategies underlying sporozoite invasion. Moreover, our data offer the potential for improved design of neutralizing therapeutics targeting a broad range of AMA–RON2 pairs and apicomplexan invasive stages. PMID:26712012

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

PubMed Central

MacDonald, Kelvin D.; McKenzie, Karen R.; Henderson, Mark J.; Hawkins, Charles E.; Vij, Neeraj; Zeitlin, Pamela L.

2008-01-01

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl− transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 μM lubiprostone was −5.8 ± 2.1 mV (CF, n = 12), −8.1 ± 2.6 mV (C57Bl/6 wild-type, n = 12), and −5.3 ± 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 μM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia. PMID:18805957

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice.

PubMed

MacDonald, Kelvin D; McKenzie, Karen R; Henderson, Mark J; Hawkins, Charles E; Vij, Neeraj; Zeitlin, Pamela L

2008-11-01

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss

PubMed Central

de Groot, John C.M.J.; van Iperen, Liesbeth; Huisman, Margriet A.; Frijns, Johan H.M.

2015-01-01

ABSTRACT Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9–W18) and show the cochlear expression dynamics of key potassium‐regulating proteins. At W12, MITF+/SOX10+/KIT+ neural‐crest‐derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+‐ATPase‐positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1219–1240, 2015 PMID:25663387

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura.

PubMed

Sironi, Chiara; Bodega, Francesca; Porta, Cristina; Monaco, Ario; Zocchi, Luciano; Agostoni, Emilio

2008-05-31

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.

Genome-Wide Analysis of Long Non-Coding RNAs in Potato and Their Potential Role in Tuber Sprouting Process

PubMed Central

Hou, Xiaodong; Du, Yongmei; Liu, Xinmin; Zhang, Hongbo; Liu, Yanhua; Yan, Ning; Zhang, Zhongfeng

2017-01-01

Sprouting is a key factor affecting the quality of potato tubers. The present study aimed to compare the differential expression of long non-coding RNAs (lncRNAs) in the apical meristem during the dormancy release and sprouting stages by using lncRNA sequencing. Microscopic observations and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the changes in the morphology and expression of lncRNAs in potato tubers during sprouting. Meristematic cells of potato tuber apical buds divided continuously and exhibited vegetative cone bulging and vascularisation. In all, 3175 lncRNAs were identified from the apical buds of potato tubers, among which 383 lncRNAs were up-regulated and 340 were down-regulated during sprouting. The GO enrichment analysis revealed that sprouting mainly influenced the expression of lncRNAs related to the cellular components of potato apical buds (e.g., cytoplasm and organelles) and cellular metabolic processes. The KEGG enrichment analysis also showed significant enrichment of specific metabolic pathways. In addition, 386 differentially expressed lncRNAs during sprouting were identified as putative targets of 235 potato miRNAs. Quantitative real-time polymerase chain reaction results agreed with the sequencing data. Our study provides the first systematic study of numerous lncRNAs involved in the potato tuber sprouting process and lays the foundation for further studies to elucidate their precise functions. PMID:29286332

Effect of entacapone on colon motility and ion transport in a rat model of Parkinson’s disease

PubMed Central

Li, Li-Sheng; Liu, Chen-Zhe; Xu, Jing-Dong; Zheng, Li-Fei; Feng, Xiao-Yan; Zhang, Yue; Zhu, Jin-Xia

2015-01-01

AIM: To study the effects of entacapone, a catechol-O-methyltransferase inhibitor, on colon motility and electrolyte transport in Parkinson’s disease (PD) rats. METHODS: Distribution and expression of catechol-O-methyltransferase (COMT) were measured by immunohistochemistry and Western blotting methods. The colonic smooth muscle motility was examined in vitro by means of a muscle motility recording device. The mucosal electrolyte transport of PD rats was examined by using a short-circuit current (ISC) technique and scanning ion-selective electrode technique (SIET). Intracellular detection of cAMP and cGMP was accomplished by radioimmunoassay testing. RESULTS: COMT was expressed in the colons of both normal and PD rats, mainly on the apical membranes of villi and crypts in the colon. Compared to normal controls, PD rats expressed less COMT. The COMT inhibitor entacapone inhibited contraction of the PD rat longitudinal muscle in a dose-dependent manner. The β2 adrenoceptor antagonist ICI-118,551 blocked this inhibitory effect by approximately 67% (P < 0.01). Entacapone increased mucosal ISC in the colon of rats with PD. This induction was significantly inhibited by apical application of Cl- channel blocker diphenylamine-2, 2’-dicarboxylic acid, basolateral application of Na+-K+-2Cl-co-transporter antagonist bumetanide, elimination of Cl- from the extracellular fluid, as well as pretreatment using adenylate cyclase inhibitor MDL12330A. As an inhibitor of prostaglandin synthetase, indomethacin can inhibit entacapone-induced ISC by 45% (P < 0.01). When SIET was applied to measure Cl- flux changes, this provided similar results. Entacapone significantly increased intracellular cAMP content in the colonic mucosa, which was greatly inhibited by indomethacin. CONCLUSION: COMT expression exists in rat colons. The β2 adrenoceptor is involved in the entacapone-induced inhibition of colon motility. Entacapone induces cAMP-dependent Cl- secretion in the PD rat. PMID:25834315

The Endocytic Receptor Megalin and its Associated Proteins in Proximal Tubule Epithelial Cells

PubMed Central

De, Shankhajit; Kuwahara, Shoji; Saito, Akihiko

2014-01-01

Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs) is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases. PMID:25019425

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

PubMed Central

Skop, Ahna R.; Bergmann, Dominique; Mohler, William A.; White, John G.

2013-01-01

Background The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. Results We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. Conclusions We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell. PMID:11378383

Transmigrated neutrophils in the intestinal lumen engage ICAM 1 to regulate the epithelial barrier and neutrophil recruitment

PubMed Central

Sumagin, Ronen; Robin, Alex Z.; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Neutrophil (PMN) transepithelial migration (TEM) and accumulation in luminal spaces is a hallmark of mucosal inflammation. TEM has been extensively modeled, however the functional consequences and molecular basis of PMN interactions with luminal epithelial ligands are not clear. Here we report that cytokine-induced expression of a PMN ligand, intercellular adhesion molecule-1 (ICAM-1), exclusively on the luminal (apical) membrane of the intestinal epithelium results in accumulation and enhanced motility of transmigrated PMN on the apical epithelial surface. Using complementary in-vitro and in-vivo approaches we demonstrate that ligation of epithelial ICAM-1 by PMN or with specific antibodies results in myosin light chain kinase (MLCK)-dependent increases in epithelial permeability that are associated with enhanced PMN TEM. Effects of ICAM-1 ligation on epithelial permeability and PMN migration in-vivo were blocked after intraluminal addition of peptides derived from the cytoplasmic domain of ICAM-1. These findings provide new evidence for functional interactions between PMN and epithelial cells after migration into the intestinal lumen. While such interactions may aid in clearance of invading microorganisms by promoting PMN recruitment, engagement of ICAM-1 under pathologic conditions would increase accumulation of epithelial-associated PMN, thus contributing to mucosal injury as observed in conditions including ulcerative colitis. PMID:24345805

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane.

PubMed

Alli, Abdel A; Bao, Hui-Fang; Liu, Bing-Chen; Yu, Ling; Aldrugh, Summer; Montgomery, Darrice S; Ma, He-Ping; Eaton, Douglas C

2015-09-01

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane. Copyright © 2015 the American Physiological Society.

Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosis: Rhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.

PubMed

Bourgeois, Soline; Bounoure, Lisa; Christensen, Erik I; Ramakrishnan, Suresh K; Houillier, Pascal; Devuyst, Olivier; Wagner, Carsten A

2013-02-22

Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH(3) transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH(3) transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg(+/+) and Rhcg(+/-) mice were able to handle an acute acid load, whereas Rhcg(-/-) mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg(+/-) mice did not fully recover, showing lower blood HCO(3)(-) concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH(3) permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg(-/-) and Rhcg(+/-) mice compared with controls. Basolateral membrane permeability to NH(3) was reduced in CDs from Rhcg(-/-) mice consistent with basolateral Rhcg localization. Rhcg(-/-) responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH(3) transport and uncover an incomplete dRTA phenotype in Rhcg(+/-) mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA.

Nephrogenic diabetes insipidus in mice caused by deleting COOH-terminal tail of aquaporin-2

PubMed Central

Shi, Peijun P.; Cao, Xiao R.; Qu, Jing; Volk, Ken A.; Kirby, Patricia; Williamson, Roger A.; Stokes, John B.; Yang, Baoli

2009-01-01

In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp2Δ230), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221–229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolality in wild-type mice but had no effect on Aqp2Δ230/Δ230 mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the NH2-terminal region of the protein Aqp2 Δ230/Δ230 mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease. PMID:17229678

Participation of IAA in transduction of gravistimulus in apical cells of moss protonema

NASA Astrophysics Data System (ADS)

Oksyniuk, U. A.; Khorkavtsiv, O. Y.; Lesniak, Y. I.

Growth movements of vascular plant axis organs -- photo-, gravi- and other tropisms -- are tightly connected with IAA transport (Hertel, 1983; Medvedev, 1996; Kiss, 2000). Moss protonema synthesizes IAA (indole-3-acetic acid) and transports it basipetally favouring growth and differentiation of caulonema (Bopp, 1979; Rose, Bopp, 1983; Rose et al., 1983). We aimed at studying the role of IAA in moss protonema gravitropism using exogenous IAA, 1-NAA (1-naphthaleneacetic acid), 2,4D (2,4-dichlorophenoxyacetic acid) and inhibitors of polar IAA transport -- phytotropins NPA (N-1-naphthylphthalamic acid) and TIBA (2,3,5-triiodobenzoic acid). Six-day gravitropic protonema of Ceratodon purpureus and Pohlia nutans were taken for experiments. Auxin and phytotropins solutions were laid on protonema mats the latters being kept in solutions for 30 min. Then the surplus of solutions were poured off and Petri dishes were placed vertically for 6 h. 20 μ M of IAA and of other synthetic auxins did not significantly influence the angle of protonema gravity bending, 40 μ M of the agents, howewer, reduced the per cent of apical cells bendings and their angles. The most expressed influence on the angles of bending had the inhibitors of polar IAA transport -- NPA. 0,1 -- 3,0 μ M of this phytotropin did not change the form of apical cell, did not disturb the general distribution of amyloplasts and did not significantly lower the per cent and the value of gravity bending angle, though 10 μ M of the phytotropin - inhibited gravity bending. The mixture of 1-NAA and NPA having been added into the medium the influence of NPA was lowered and gravitropic growth renewed in course of time. 10 μ M of other phytopropin TIBA also inhibited gravitropism of Ceratodon purpureus and Pohlia nutans protonema. The analysis of basipetal transport of IAA in moss rhizoids and protonema may indicate the availability of special IAA transport in these structures (Bopp, Cerier, 1988). On the basis of the carried out experiments it can be suggested that high concentrations of IAA and 1-NAA result in surplus of IAA cells led, probably, to a destruction of the apical-basal gradient in cells. Our results testify that NPA inhibits the gravitropism stronger than the growth of protonema. The peculiarity of moss protonema is that the growth orientation change is a result of a transference of growth zone in the apical cell dome caused by amyloplasts sedimentation inducing lateral asymmetry of Ca2+ and apical-basal IAA flow what in its turn manifests itself in distribution of IAA and/or Ca2+ channels in apical cell dome plasma membrane ( Schwuchow et al., 2001). The transport of IAA in apical cells, probably, functionally polarizes it and just that polarizing function is dominant in cells with tip growth.

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids.

PubMed

Bartnik, E; Sievers, A

1988-11-01

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 μm and is positioned in the center of the apical Golgi-vesicle accumulation ("Spitzenkörper"). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.

Fine structure of the retinal pigment epithelium of the great horned owl (Bubo virginianus).

PubMed

Braekevelt, C R; Thorlakson, I J

1993-01-01

The fine structure of the retinal epithelium (RPE), choriocapillaries and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the great horned owl (Bubo virginianus). The RPE consists of a single layer of cuboidal cells joined laterally in the mid to basal region by a series of tight junctions forming part of the blood-ocular barrier. Basally (sclerally) the epithelial cells show numerous deep infoldings while apically (vitreally) a wealth of microvillar processes interdigitate with the photoreceptor cells. Internally the RPE cells display a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and polysomes with only small scattered profiles of rough endoplasmic reticulum (RER). Numerous pleomorphic mitochondria are basally located. In the light-adapted state the melanosomes are located almost exclusively within the apical processes indicating retinomotor movements. Myeloid bodies are numerous and often show ribosomes on their outer surface. Bruch's membrane is typical of avian species in that it is pentalaminate and the lamina densa is displaced towards the choriocapillaris. The choriocapillaris itself is but minimally fenestrated facing Bruch's membrane. Most fenestrations present show a single layered diaphragm while others display a double-layered diaphragm.

Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

PubMed Central

Kasioulis, Ioannis

2017-01-01

Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment. PMID:29058679

Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

NASA Astrophysics Data System (ADS)

Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

1996-07-01

In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

PubMed

Siegel, Dustin S; Sever, David M; Aldridge, Robert D

2010-12-01

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. © 2010 Wiley-Liss, Inc.

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

PubMed

Zha, L-Y; Xu, Z-R; Wang, M-Q; Gu, L-Y

2008-04-01

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 (CrCl(3)), 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 CrCl(3) to examine their transport and uptake respectively. In the concentration range of 0.2-20 micromol/l, transport of CrNano, CrPic and CrCl(3) 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 (P(app)) of CrNano was between 5.89 and 7.92 x 10(-6) cm/s and that of CrPic and CrCl(3) was between 3.52 and 5.31 x 10(-6) cm/s and between 0.97 and 1.37 x 10(-6) cm/s respectively. Uptake of CrNano, CrPic and CrCl(3) 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 degrees C to 4 degrees C. The transport efficiency of CrNano, CrPic and CrCl(3) after incubation for 120 min at 37 degrees C was 15.83% +/- 0.76%, 9.08% +/- 0.25% and 2.11% +/- 0.53% respectively. The uptake efficiency of CrNano, CrPic and CrCl(3) 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 CrCl(3) across the Caco-2 cell monolayers was mainly via passive transport pathways. In addition, CrNano exhibited considerably higher absorption efficiency than both CrPic and CrCl(3) in Caco-2 cell monolayers.

Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats.

PubMed

Wang, Di; Yuan, Zhenfang; Inoue, Noriko; Cho, Gota; Shono, Masayuki; Ishikawa, Yasuko

2011-05-01

The mechanisms underlying diabetic xerostomia have not been clarified in relation with aquaporin-5 (AQP5) subcellular localization in salivary glands. Western blotting, real-time PCR, and immunocytochemistry were used to analyse AQP5 protein levels and mRNA expression. AQP5 protein levels were measured in the apical plasma membrane (APM) and detergent-insoluble fraction prepared from streptozotocin-diabetic rat parotid glands. Despite an increase in AQP5 mRNA, AQP5 protein levels were decreased in diabetic parotid glands compared with controls. Immunohistochemical studies indicated that AQP5, under unstimulated conditions, colocalised with flotillin-2 and GM1 with a diffuse pattern in the apical cytoplasm of acinar and duct cells in both control and diabetic rats. Ten minutes after intravenous injection of muscarinic agonist cevimeline, AQP5 was dramatically increased together with flotillin-2 and GM1 in the APM of parotid acinar and duct cells of control but not diabetic rats. Sixty minutes after injection, AQP5 was located in a diffuse pattern in the apical cytoplasm in both rats. Treatment of the parotid tissues with cevimeline for 10min increased the Triton X-100 solubility of AQP5 in control but not diabetic rats. Administration of insulin to diabetic rats tended to restore the cevimeline-induced translocation of AQP5. Lack of AQP5 translocation in the salivary gland in response to a muscarinic agonist and downregulation of AQP5 protein might lead to diabetic xerostomia. Cevimeline is useful to cure diabetic xerostomia under insulin administration. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition.

PubMed

Fumoto, Katsumi; Takigawa-Imamura, Hisako; Sumiyama, Kenta; Kaneiwa, Tomoyuki; Kikuchi, Akira

2017-01-01

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages. © 2017. Published by The Company of Biologists Ltd.

MMP-7 and TIMP-1, new targets in predicting poor wound healing in apical periodontitis.

PubMed

Letra, Ariadne; Ghaneh, Ghazaleh; Zhao, Min; Ray, Herbert; Francisconi, Carolina Favaro; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

2013-09-01

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions. Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy. Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus. MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The auditory nerve overlapped waveform (ANOW): A new objective measure of low-frequency hearing

NASA Astrophysics Data System (ADS)

Lichtenhan, Jeffery T.; Salt, Alec N.; Guinan, John J.

2015-12-01

One of the most pressing problems today in the mechanics of hearing is to understand the mechanical motions in the apical half of the cochlea. Almost all available measurements from the cochlear apex of basilar membrane or other organ-of-Corti transverse motion have been made from ears where the health, or sensitivity, in the apical half of the cochlea was not known. A key step in understanding the mechanics of the cochlear base was to trust mechanical measurements only when objective measures from auditory-nerve compound action potentials (CAPs) showed good preparation sensitivity. However, such traditional objective measures are not adequate monitors of cochlear health in the very low-frequency regions of the apex that are accessible for mechanical measurements. To address this problem, we developed the Auditory Nerve Overlapped Waveform (ANOW) that originates from auditory nerve output in the apex. When responses from the round window to alternating low-frequency tones are averaged, the cochlear microphonic is canceled and phase-locked neural firing interleaves in time (i.e., overlaps). The result is a waveform that oscillates at twice the probe frequency. We have demonstrated that this Auditory Nerve Overlapped Waveform - called ANOW - originates from auditory nerve fibers in the cochlear apex [8], relates well to single-auditory-nerve-fiber thresholds, and can provide an objective estimate of low-frequency sensitivity [7]. Our new experiments demonstrate that ANOW is a highly sensitive indicator of apical cochlear function. During four different manipulations to the scala media along the cochlear spiral, ANOW amplitude changed when either no, or only small, changes occurred in CAP thresholds. Overall, our results demonstrate that ANOW can be used to monitor cochlear sensitivity of low-frequency regions during experiments that make apical basilar membrane motion measurements.

LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

PubMed

Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

2014-01-01

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

A missense mutation in solute carrier family 12, member 1 (SLC12A1) causes hydrallantois in Japanese Black cattle.

PubMed

Sasaki, Shinji; Hasegawa, Kiyotoshi; Higashi, Tomoko; Suzuki, Yutaka; Sugano, Sumio; Yasuda, Yasuaki; Sugimoto, Yoshikazu

2016-09-09

Hydrallantois is the excessive accumulation of fluid within the allantoic cavity in pregnant animals and is associated with fetal mortality. Although the incidence of hydrallantois is very low in artificial insemination breeding programs in cattle, recently 38 cows with the phenotypic appearance of hydrallantois were reported in a local subpopulation of Japanese Black cattle. Of these, 33 were traced back to the same sire; however, both their parents were reported healthy, suggesting that hydrallantois is a recessive inherited disorder. To identify autozygous chromosome segments shared by individuals with hydrallantois and the causative mutation in Japanese Black cattle, we performed autozygosity mapping using single-nucleotide polymorphism (SNP) array and exome sequencing. Shared haplotypes of the affected fetuses spanned 3.52 Mb on bovine chromosome 10. Exome sequencing identified a SNP (g.62382825G > A, p.Pro372Leu) in exon 10 of solute carrier family 12, member 1 (SLC12A1), the genotype of which was compatible with recessive inheritance. SLC12A1 serves as a reabsorption molecule of Na(+)-K(+)-2Cl(-) in the apical membrane of the thick ascending limb of the loop of Henle in the kidney. We observed that the concentration of Na(+)-Cl(-) increased in allantoic fluid of homozygous SLC12A1 (g.62382825G > A) in a hydrallantois individual. In addition, SLC12A1-positive signals were localized at the apical membrane in the kidneys of unaffected fetuses, whereas they were absent from the apical membrane in the kidneys of affected fetuses. These results suggested that p.Pro372Leu affects the membrane localization of SLC12A1, and in turn, may impair its transporter activity. Surveillance of the risk-allele frequency revealed that the carriers were restricted to the local subpopulation of Japanese Black cattle. Moreover, we identified a founder individual that carried the mutation (g.62382825G > A). In this study, we mapped the shared haplotypes of affected fetuses using autozygosity mapping and identified a de novo mutation in the SLC12A1 gene that was associated with hydrallantois in Japanese Black cattle. In kidneys of hydrallantois-affected fetuses, the mutation in SLC12A1 impaired the apical membrane localization of SLC12A1 and reabsorption of Na(+)-K(+)-2Cl(-) in the thick ascending limb of the loop of Henle, leading to a defect in the concentration of urine via the countercurrent mechanism. Consequently, the affected fetuses exhibited polyuria that accumulated in the allantoic cavity. Surveillance of the risk-allele frequency indicated that carriers were not widespread throughout the Japanese Black cattle population. Moreover, we identified the founder individual, and thus could effectively manage the disorder in the population.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii.

PubMed

Foe, Ian T; Child, Matthew A; Majmudar, Jaimeen D; Krishnamurthy, Shruthi; van der Linden, Wouter A; Ward, Gary E; Martin, Brent R; Bogyo, Matthew

2015-10-14

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

YAP and TAZ in epithelial stem cells: A sensor for cell polarity, mechanical forces and tissue damage.

PubMed

Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I; Thompson, Barry J

2016-07-01

The YAP/TAZ family of transcriptional co-activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST-LATS or Src family kinase activity to modulate YAP/TAZ activity. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

The fine structure of the rectal pads of Zorotypus caudelli Karny (Zoraptera, Insecta).

PubMed

Dallai, R; Mercati, D; Mashimo, Y; Machida, R; Beutel, R G

2016-07-01

The rectal pads of a species of the controversial polyneopteran order Zoraptera were examined using histological sections and TEM micrographs. Six pads are present along the thin rectal epithelium. Each pad consists of a few large principal cells surrounded by flattened junctional cells, which extend also beneath the principal cells. The cells are lined by a thin apical cuticle. No basal cells and no cavity have been observed beneath the pad. Principal cells have a regular layer of apical microvilli and are joined by intercellular septate junctions, which are interrupted by short dilatations of the intercellular space. At these levels the two adjacent plasma membranes are joined by short zonulae adhaerentes. In the cytoplasm, a rich system of strict associations between lateral plasma membranes and mitochondria forms scalariform junctions. Rectal pads share ultrastructural features with similar excretory organs of several neopteran groups, in particular with Blattodea (roaches and termites) and Thysanoptera, and are involved in fluid reabsorption and ion regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Strain difference in amiloride-sensitivity of salt-induced responses in mouse non-dissociated taste cells.

PubMed

Miyamoto, T; Fujiyama, R; Okada, Y; Sato, T

1999-12-17

The chorda tympani nerve responses to NaCl in a mouse strain, C57BL/6 are known to be much more sensitive than those in BALB/c. We compared the NaCl-induced responses obtained from taste cells of the fungiform papillae in these two strains of mice. Amiloride inhibited, in the same degree, the responses induced by a bath-application of normal extracellular solution (NES) containing 140 mM NaCl in either taste cells of C57BL/6 and BALB/c mice. In contrast, amiloride inhibited 62% of responses induced by an apically applied 0.5 M NaCl in the C57BL/6 strain, but only 33% of responses in the BALB/c strain. These results suggest that the difference in amiloride-sensitivity between taste cells in both strains mainly derives from the difference in density of functional amiloride sensitive Na+ channels at the apical receptive membrane but not at the basolateral membrane.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells.

PubMed

Kimura, Osamu; Ohta, Chiho; Koga, Nobuyuki; Haraguchi, Koichi; Kato, Yoshihisa; Endo, Tetsuya

2014-07-01

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of renal urea transport by vasopressin.

PubMed

Sands, Jeff M; Blount, Mitsi A; Klein, Janet D

2011-01-01

Terrestrial life would be miserable without the ability to concentrate urine. Production of concentrated urine requires complex interactions among the nephron segments and vasculature in the kidney medulla. In addition to water channels (aquaporins) and sodium transporters, urea transporters are critically important to the theories proposed to explain the physiologic processes occurring when urine is concentrated. Vasopressin (anti-diuretic hormone) is the key hormone regulating the production of concentrated urine. Vasopressin rapidly increases water and urea transport in the terminal inner medullary collecting duct (IMCD). Vasopressin rapidly increases urea permeability in the IMCD through increases in phosphorylation and apical plasma-membrane accumulation of the urea transporter A1 (UT-A1). Vasopressin acts through two cAMP-dependent signaling pathways in the IMCD: protein kinase A and exchange protein activated by cAMP Epac. Protein kinase A phosphorylates UT-A1 at serines 486 and 499. In summary, vasopressin regulates urea transport acutely by increasing UT-A1 phosphorylation and the apical plasma-membrane accumulation of UT-A1 through two cAMP-dependent pathways.

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia▿

PubMed Central

Vermeer, Paola D.; McHugh, Julia; Rokhlina, Tatiana; Vermeer, Daniel W.; Zabner, Joseph; Welsh, Michael J.

2007-01-01

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors. PMID:17581984

The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter*

PubMed Central

Moreno, Erika; Plata, Consuelo; Rodríguez-Gama, Alejandro; Argaiz, Eduardo R.; Vázquez, Norma; Leyva-Ríos, Karla; Islas, León; Cutler, Christopher; Pacheco-Alvarez, Diana; Mercado, Adriana; Cariño-Cortés, Raquel; Castañeda-Bueno, María; Gamba, Gerardo

2016-01-01

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter. PMID:27587391

Antibody validation and scoring guidelines for ABCG2 immunohistochemical staining in formalin-fixed paraffin-embedded colon cancer tissue

PubMed Central

Cederbye, Camilla Natasha; Palshof, Jesper Andreas; Hansen, Tine Plato; Duun-Henriksen, Anne Katrine; Linnemann, Dorte; Stenvang, Jan; Nielsen, Dorte Lisbet; Brünner, Nils; Viuff, Birgitte Martine

2016-01-01

Overexpression of the ATP-dependent drug efflux pump ABCG2 is a major molecular mechanism of multidrug resistance in cancer and might be a predictive biomarker for drug response. Contradictory results have been reported for immunohistochemical studies of ABCG2 protein expression in colorectal cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we established scoring guidelines for ABCG2 expression based on the clinically used guidelines for HER2 immunohistochemistry assessment in gastric cancer. The guidelines provide a semi-quantitative measure of the basolateral membrane staining of ABCG2 and disregard the apical membrane staining and the cytoplasmic signal. Intra-tumor heterogeneity in ABCG2 immunoreactivity was observed; however, statistical analyses of tissue microarrays (TMAs) and the corresponding whole sections from primary tumors of 57 metastatic CRC patients revealed a strong positive correlation between maximum TMA scores and whole sections, especially when more than one core was used. In conclusion, here, we provide validated results to guide future studies on the associations between ABCG2 immunoreactivity in tumor cells and the benefits of chemotherapeutic treatment in patients with CRC. PMID:27257141

Uncoupling apical constriction from tissue invagination

PubMed Central

Chung, SeYeon; Kim, Sangjoon; Andrew, Deborah J

2017-01-01

Apical constriction is a widely utilized cell shape change linked to folding, bending and invagination of polarized epithelia. It remains unclear how apical constriction is regulated spatiotemporally during tissue invagination and how this cellular process contributes to tube formation in different developmental contexts. Using Drosophila salivary gland (SG) invagination as a model, we show that regulation of folded gastrulation expression by the Fork head transcription factor is required for apicomedial accumulation of Rho kinase and non-muscle myosin II, which coordinate apical constriction. We demonstrate that neither loss of spatially coordinated apical constriction nor its complete blockage prevent internalization and tube formation, although such manipulations affect the geometry of invagination. When apical constriction is disrupted, compressing force generated by a tissue-level myosin cable contributes to SG invagination. We demonstrate that fully elongated polarized SGs can form outside the embryo, suggesting that tube formation and elongation are intrinsic properties of the SG. DOI: http://dx.doi.org/10.7554/eLife.22235.001 PMID:28263180

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

PubMed

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

2017-12-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

Vitellogenin transcytosis in follicular cells of the honeybee Apis mellifera and the wasp Polistes simillimus.

PubMed

Dohanik, Virgínia Teles; Gonçalves, Wagner Gonzaga; Oliveira, Leandro Licursi; Zanuncio, José Cola; Serrão, José Eduardo

2018-05-13

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.

The immediate large-scale dendritic plasticity of cortical pyramidal neurons subjected to acute epidural compression.

PubMed

Chen, J-R; Wang, T-J; Wang, Y-J; Tseng, G-F

2010-05-05

Head trauma and acute disorders often instantly compress the cerebral cortex and lead to functional abnormalities. Here we used rat epidural bead implantation model and investigated the immediate changes following acute compression. The dendritic arbors of affected cortical pyramidal neurons were filled with intracellular dye and reconstructed 3-dimensionally for analysis. Compression was found to shorten the apical, but not basal, dendrites of underlying layer III and V cortical pyramidal neurons and reduced dendritic spines on the entire dendritic arbor immediately. Dendrogram analysis showed that in addition to distal, proximal apical dendrites also quickly reconfigured. We then focused on apical dendritic trunks and explored how proximal dendrites were rapidly altered. Compression instantly twisted the microtubules and deformed the membrane contour of dendritic trunks likely a result of the elastic nature of dendrites as immediate decompression restored it and stabilization of microtubules failed to block it. Subsequent adaptive remodeling restored plasmalemma and microtubules to normal appearance in 3 days likely via active mechanisms as taxol blocked the restoration of microtubules and in addition partly affected plasmalemmal reorganization which presumably engaged recycling of excess membrane. In short, the structural dynamics and the associated mechanisms that we revealed demonstrate how compression quickly altered the morphology of cortical output neurons and hence cortical functions consequently. (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs.

PubMed

Carlson, M R; Bryant, S V; Gardiner, D M

1998-12-15

Msx genes are transcription factors that are expressed during embryogenesis of developing appendages in regions of epithelial-mesenchymal interactions. Various lines of evidence indicate that these genes function to maintain embryonic tissues in an undifferentiated, proliferative state. We have identified the axolotl homolog of Msx-2, and investigated its expression during limb development, limb regeneration, and wound healing. As in limb buds of higher vertebrates, axolotl Msx-2 is expressed in the apical epidermis and mesenchyme; however, its expression domain is more extensive, reflecting the broader region of the apical epidermal cap in amphibians. Msx-2 expression is downregulated at late stages of limb development, but is reexpressed within one hour after limb amputation. Msx-2 is also reexpressed during wound healing, and may be essential in the early stages of initiation of the limb regeneration cascade.

3D time-lapse analysis of Rab11/FIP5 complex: spatiotemporal dynamics during apical lumen formation.

PubMed

Mangan, Anthony; Prekeris, Rytis

2015-01-01

Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells.

Effects of cholera toxin on human colon carcinoma cell lines.

PubMed

Barkla, D H; Whitehead, R H; Hayward, I P

1992-10-01

This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical properties of urea transporters.

PubMed

Chen, Guangping

2014-01-01

Urea and urea transporters (UT) are critical to the production of concentrated urine and hence in maintaining body fluid balance. The UT-A1 urea transporter is the major and most important UT isoform in the kidney. Native UT-A1, expressed in the terminal inner medullary collecting duct (IMCD) epithelial cells, is a glycosylated protein with two glycoforms of 117 and 97 kDa. Vasopressin is the major hormone in vivo that rapidly increases urea permeability in the IMCD through increases in phosphorylation and apical plasma-membrane accumulation of UT-A1. The cell signaling pathway for vasopressin-mediated UT-A1 phosphorylation and activity involves two cAMP-dependent signaling pathways: protein kinase A (PKA) and exchange protein activated by cAMP (Epac). In this chapter, we will discuss UT-A1 regulation by phosphorylation, ubiquitination, and glycosylation.

Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

PubMed Central

Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang

2013-01-01

A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman’s syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)–mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. NccT58M/T58M mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with NccT58M/T58M mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II. PMID:23833262

Phosphorylation regulates NCC stability and transporter activity in vivo.

PubMed

Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang; Lin, Shih-Hua

2013-10-01

A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

PubMed Central

Chapin, Hannah C.; Rajendran, Vanathy

2010-01-01

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery. PMID:20980620

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

PubMed

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

PubMed

Monks, Jenifer; Dzieciatkowska, Monika; Bales, Elise S; Orlicky, David J; Wright, Richard M; McManaman, James L

2016-10-15

Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia.

PubMed

Wolf, A A; Jobling, M G; Wimer-Mackin, S; Ferguson-Maltzman, M; Madara, J L; Holmes, R K; Lencer, W I

1998-05-18

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

NASA Astrophysics Data System (ADS)

Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

2017-12-01

In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation.

PubMed

Latorre, Eva; Layunta, Elena; Grasa, Laura; Castro, Marta; Pardo, Julián; Gomollón, Fernando; Alcalde, Ana I; Mesonero, José E

2016-01-01

TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT) has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT) activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system.

Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation

PubMed Central

Layunta, Elena; Grasa, Laura; Castro, Marta; Pardo, Julián; Gomollón, Fernando; Mesonero, José E.

2016-01-01

TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT) has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT) activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system. PMID:28033388

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

PubMed Central

Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

2010-01-01

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

Iron homeostasis and its disruption in mouse lung in iron deficiency and overload.

PubMed

Giorgi, Gisela; D'Anna, María Cecilia; Roque, Marta Elena

2015-10-01

What is the central question of this study? The aim was to explore the role and hitherto unclear mechanisms of action of iron proteins in protecting the lung against the harmful effects of iron accumulation and the ability of pulmonary cells to mobilize iron in iron deficiency. What is the main finding and its importance? We show that pulmonary hepcidin appears not to modify cellular iron mobilization in the lung. We propose pathways for supplying iron to the lung in iron deficiency and for protecting the lung against iron excess in iron overload, mediated by the co-ordinated action of iron proteins, such as divalent metal transporter 1, ZRT-IRE-like-protein 14, transferrin receptor, ferritin, haemochromatosis-associated protein and ferroportin. Iron dyshomeostasis is associated with several forms of chronic lung disease, but its mechanisms of action remain to be elucidated. The aim of the present study was to determine the role of the lung in whole-animal models with iron deficiency and iron overload, studying the divalent metal transporter 1 (DMT1), ZRT-IRE-like protein 14 (ZIP14), transferrin receptor (TfR), haemochromatosis-associated protein (HFE), hepcidin, ferritin and ferroportin (FPN) expression. In each model, adult CF1 mice were divided into the following groups (six mice per group): (i) iron-overload model, iron saccharate i.p. and control group (iron adequate), 0.9% NaCl i.p.; and (ii) iron-deficiency model, induced by repeated bleeding, and control group (sham operated). Proteins were assessed by immunohistochemistry and Western blot. In control mice, DMT1 was localized in the cytoplasm of airway cells, and in iron deficiency and overload it was in the apical membrane. Divalent metal transporter 1 and TfR increased in iron deficiency, without changes in iron overload. ZRT-IRE-like protein 14 decreased in airway cells in iron deficiency and increased in iron overload. In iron deficiency, HFE and FPN were immunolocalized close to the apical membrane. Ferroportin increased in iron overload. Prohepcidin was present in control groups, with no changes in iron deficiency and iron overload. In iron overload, ferritin showed intracytoplasmic localization close to the apical membrane of airway cells and intense immunostaining in macrophage-like cells. The results show that pulmonary hepcidin does not appear to modify cellular iron mobilization in the lung. We propose the following two novel pathways in the lung: (i) for supplying iron in iron deficiency, mediated principally by DMT1 and TfR and regulated by the action of FPN and HFE; and (ii) for iron detoxification in order to protect the lung against iron overload, facilitated by the action of DMT1, ZIP14, FPN and ferritin. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

A Model of Differential Growth-Guided Apical Hook Formation in Plants

PubMed Central

Žádníková, Petra; Wabnik, Krzysztof; Abuzeineh, Anas; Prusinkiewicz, Przemysław

2016-01-01

Differential cell growth enables flexible organ bending in the presence of environmental signals such as light or gravity. A prominent example of the developmental processes based on differential cell growth is the formation of the apical hook that protects the fragile shoot apical meristem when it breaks through the soil during germination. Here, we combined in silico and in vivo approaches to identify a minimal mechanism producing auxin gradient-guided differential growth during the establishment of the apical hook in the model plant Arabidopsis thaliana. Computer simulation models based on experimental data demonstrate that asymmetric expression of the PIN-FORMED auxin efflux carrier at the concave (inner) versus convex (outer) side of the hook suffices to establish an auxin maximum in the epidermis at the concave side of the apical hook. Furthermore, we propose a mechanism that translates this maximum into differential growth, and thus curvature, of the apical hook. Through a combination of experimental and in silico computational approaches, we have identified the individual contributions of differential cell elongation and proliferation to defining the apical hook and reveal the role of auxin-ethylene crosstalk in balancing these two processes. PMID:27754878

Solubilization methods and reference 2-DE map of cow milk fat globules.

PubMed

Bianchi, Laura; Puglia, Michele; Landi, Claudia; Matteoni, Silvia; Perini, Daniele; Armini, Alessandro; Verani, Margherita; Trombetta, Claudia; Soldani, Patrizia; Roncada, Paola; Greppi, Gianfranco; Pallini, Vitaliano; Bini, Luca

2009-07-21

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

PubMed

Marunaka, Y; Hagiwara, N; Tohda, H

1992-09-01

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

A K(+)/H (+) P-ATPase transport in the accessory cell membrane of the blowfly taste chemosensilla sustains the transepithelial potential.

PubMed

Sollai, Giorgia; Solari, Paolo; Masala, Carla; Liscia, Anna; Crnjar, Roberto

2008-11-01

An electrogenic K(+) transport in the tormogen cell of insect chemosensilla is involved in the generation and maintenance of the transepithelial potential (TEP). To gain more information about the K(+) transport system underlying the TEP generation and the location of its components in the plasma membrane of the tormogen cell, we studied the effects of inhibitors of K(+)/H(+) P-ATPase (bafilomycin A1, omeprazole and Na-orthovanadate), of K(+)/Cl(-) co-transport (bumetanide), of Cl(-) channels (NPPB) and of a K(+) channel blocker (BaCl(2)). The relationship between TEP amplitude and spike firing activity was also studied. Experiments were performed on the labellar chemosensilla of the blowfly Protophormia terraenovae using a modified tip-recording technique. Results show that: (a) K(+)/H(+) P-ATPase inhibitors significantly decrease the TEP, when properly applied to the labellum for 20 min, so as to reach the basolateral side of the plasma membrane, while no effect was detected when applied to the apical side, (b) bumetanide, NPPB and BaCl(2) decrease the TEP value only when administered to the apical side, (c) spike activity is positively correlated with the TEP. A model is proposed of the active and passive K(+) transports sustaining the TEP associated with the blowfly chemosensilla.

Biophysical Model of Ion Transport across Human Respiratory Epithelia Allows Quantification of Ion Permeabilities

PubMed Central

Garcia, Guilherme J.M.; Boucher, Richard C.; Elston, Timothy C.

2013-01-01

Lung health and normal mucus clearance depend on adequate hydration of airway surfaces. Because transepithelial osmotic gradients drive water flows, sufficient hydration of the airway surface liquid depends on a balance between ion secretion and absorption by respiratory epithelia. In vitro experiments using cultures of primary human nasal epithelia and human bronchial epithelia have established many of the biophysical processes involved in airway surface liquid homeostasis. Most experimental studies, however, have focused on the apical membrane, despite the fact that ion transport across respiratory epithelia involves both cellular and paracellular pathways. In fact, the ion permeabilities of the basolateral membrane and paracellular pathway remain largely unknown. Here we use a biophysical model for water and ion transport to quantify ion permeabilities of all pathways (apical, basolateral, paracellular) in human nasal epithelia cultures using experimental (Ussing Chamber and microelectrode) data reported in the literature. We derive analytical formulas for the steady-state short-circuit current and membrane potential, which are for polarized epithelia the equivalent of the Goldman-Hodgkin-Katz equation for single isolated cells. These relations allow parameter estimation to be performed efficiently. By providing a method to quantify all the ion permeabilities of respiratory epithelia, the model may aid us in understanding the physiology that regulates normal airway surface hydration. PMID:23442922

An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis

PubMed Central

Guo, Hongyan; Zhang, Wei; Tian, Hainan; Zheng, Kaijie; Dai, Xuemei; Liu, Shanda; Hu, Qingnan; Wang, Xianling; Liu, Bao; Wang, Shucai

2015-01-01

Plant hormone auxin regulates most, if not all aspects of plant growth and development, including lateral root formation, organ pattering, apical dominance, and tropisms. Peptide hormones are peptides with hormone activities. Some of the functions of peptide hormones in regulating plant growth and development are similar to that of auxin, however, the relationship between auxin and peptide hormones remains largely unknown. Here we report the identification of OsCLE48, a rice (Oryza sativa) CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) gene, as an auxin response gene, and the functional characterization of OsCLE48 in Arabidopsis and rice. OsCLE48 encodes a CLE peptide hormone that is similar to Arabidopsis CLEs. RT-PCR analysis showed that OsCLE48 was induced by exogenously application of IAA (indole-3-acetic acid), a naturally occurred auxin. Expression of integrated OsCLE48p:GUS reporter gene in transgenic Arabidopsis plants was also induced by exogenously IAA treatment. These results indicate that OsCLE48 is an auxin responsive gene. Histochemical staining showed that GUS activity was detected in all the tissue and organs of the OsCLE48p:GUS transgenic Arabidopsis plants. Expression of OsCLE48 under the control of the 35S promoter in Arabidopsis inhibited shoot apical meristem development. Expression of OsCLE48 under the control of the CLV3 native regulatory elements almost completely complemented clv3-2 mutant phenotypes, suggesting that OsCLE48 is functionally similar to CLV3. On the other hand, expression of OsCLE48 under the control of the 35S promoter in Arabidopsis has little, if any effects on root apical meristem development, and transgenic rice plants overexpressing OsCLE48 are morphologically indistinguishable from wild type plants, suggesting that the functions of some CLE peptides may not be fully conserved in Arabidopsis and rice. Taken together, our results showed that OsCLE48 is an auxin responsive peptide hormone gene, and it regulates shoot apical meristem development when expressed in Arabidopsis. PMID:25983737

Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

PubMed Central

Krishnamurthy, Shruthi; Deng, Bin; del Rio, Roxana; Buchholz, Kerry R.; Treeck, Moritz; Urban, Siniša; Boothroyd, John; Lam, Ying-Wai

2016-01-01

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T. gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. PMID:27624124

Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5.

PubMed

Arakawa, Hiroshi; Shirasaka, Yoshiyuki; Haga, Makoto; Nakanishi, Takeo; Tamai, Ikumi

2012-09-01

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier-mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco-2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8-benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P-glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5-mediated uptake of ciprofloxacin was saturable with a K(m) value of 140 µm, and naringin inhibited the uptake with an IC(50) value of 18 µm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal-to-serosal side, with an IC(50) value of 7.5 µm by the Ussing-type chamber method. The estimated IC(50) values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P-gp and Bcrp. Copyright © 2012 John Wiley & Sons, Ltd.

Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains

PubMed Central

Grego-Bessa, Joaquim; Hildebrand, Jeffrey; Anderson, Kathryn V.

2015-01-01

The genetic control of mammalian epithelial polarity and dynamics can be studied in vivo at cellular resolution during morphogenesis of the mouse neural tube. The mouse neural plate is a simple epithelium that is transformed into a columnar pseudostratified tube over the course of ∼24 h. Apical F-actin is known to be important for neural tube closure, but the precise roles of actin dynamics in the neural epithelium are not known. To determine how the organization of the neural epithelium and neural tube closure are affected when actin dynamics are blocked, we examined the cellular basis of the neural tube closure defect in mouse mutants that lack the actin-severing protein cofilin 1 (CFL1). Although apical localization of the adherens junctions, the Par complex, the Crumbs complex and SHROOM3 is normal in the mutants, CFL1 has at least two distinct functions in the apical and basal domains of the neural plate. Apically, in the absence of CFL1 myosin light chain does not become phosphorylated, indicating that CFL1 is required for the activation of apical actomyosin required for neural tube closure. On the basal side of the neural plate, loss of CFL1 has the opposite effect on myosin: excess F-actin and myosin accumulate and the ectopic myosin light chain is phosphorylated. The basal accumulation of F-actin is associated with the assembly of ectopic basal tight junctions and focal disruptions of the basement membrane, which eventually lead to a breakdown of epithelial organization. PMID:25742799

Uroplakins do not restrict CO2 transport through urothelium.

PubMed

Zocher, Florian; Zeidel, Mark L; Missner, Andreas; Sun, Tung-Tien; Zhou, Ge; Liao, Yi; von Bodungen, Maximilian; Hill, Warren G; Meyers, Susan; Pohl, Peter; Mathai, John C

2012-03-30

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (∼1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (∼7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (∼ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

PubMed Central

Stindt, Jan; Smits, Sander H. J.; Schmitt, Lutz

2013-01-01

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters. PMID:23593265

Genome-wide characterization and expression profiling of NAC transcription factor genes under abiotic stresses in radish (Raphanus sativus L.)

PubMed Central

Muleke, Everlyne M’mbone; Jabir, Bashir Mohammed; Xie, Yang; Zhu, Xianwen; Cheng, Wanwan

2017-01-01

NAC (NAM, no apical meristem; ATAF, Arabidopsis transcription activation factor and CUC, cup-shaped cotyledon) proteins are among the largest transcription factor (TF) families playing fundamental biological processes, including cell expansion and differentiation, and hormone signaling in response to biotic and abiotic stresses. In this study, 172 RsNACs comprising 17 membrane-bound members were identified from the whole radish genome. In total, 98 RsNAC genes were non-uniformly distributed across the nine radish chromosomes. In silico analysis revealed that expression patterns of several NAC genes were tissue-specific such as a preferential expression in roots and leaves. In addition, 21 representative NAC genes were selected to investigate their responses to heavy metals (HMs), salt, heat, drought and abscisic acid (ABA) stresses using real-time polymerase chain reaction (RT-qPCR). As a result, differential expressions among these genes were identified where RsNAC023 and RsNAC080 genes responded positively to all stresses except ABA, while RsNAC145 responded more actively to salt, heat and drought stresses compared with other genes. The results provides more valuable information and robust candidate genes for future functional analysis for improving abiotic stress tolerances in radish. PMID:29259849

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auger, Floriane; Gendron, Marie-Claude; Chamot, Christophe

Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) weremore » exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM{sub 2.5}). DEP and PM{sub 2.5} (10-80 {mu}g/cm{sup 2}) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1{beta} secretion and only weak non-reproducible secretion of TNF-{alpha}. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM{sub 2.5}. ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-{alpha} treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles ({<=} 40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM{sub 2.5}. Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response.« less

Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

PubMed Central

Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

2002-01-01

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faddy, Helen M.; Smart, Chanel E.; Xu, Ren

2008-04-09

The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold duringmore » lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.« less

Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

PubMed Central

Estilo, Genevieve; Liu, Wen; Pastor-Soler, Nuria; Mitchell, Phillip; Carattino, Marcelo D.; Kleyman, Thomas R.; Satlin, Lisa M.

2008-01-01

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading. PMID:18579708

Visualization of highly dynamic F-actin plus ends in growing phaseolus vulgaris root hair cells and their responses to Rhizobium etli nod factors.

PubMed

Zepeda, Isaac; Sánchez-López, Rosana; Kunkel, Joseph G; Bañuelos, Luis A; Hernández-Barrera, Alejandra; Sánchez, Federico; Quinto, Carmen; Cárdenas, Luis

2014-03-01

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.