Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK

2001 ◽  
Vol 280 (4) ◽  
pp. F675-F682 ◽  
Author(s):  
Johannes Loffing ◽  
Marija Zecevic ◽  
Eric Féraille ◽  
Brigitte Kaissling ◽  
Carol Asher ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Surface Expression ◽  
Apical Plasma Membrane ◽  
Sodium Reabsorption ◽  
Xenopus Laevis Oocytes ◽  
Subunit Expression ◽  
Early Portion ◽  
Potassium Secretion ◽  
Early Effects

Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30–60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces α-ENaC subunit expression along the entire ASDN within 2 h, whereas β- and γ-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and α-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of α,β,γ-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.

CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo

1999 ◽  
Vol 112 (6) ◽  
pp. 887-894
Author(s):  
N.A. Ameen ◽  
B. Martensson ◽  
L. Bourguinon ◽  
C. Marino ◽  
J. Isenberg ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Surface Expression ◽  
Apical Plasma Membrane ◽  
Intracellular Camp ◽  
Apical Surface ◽  
C Terminus ◽  
Confocal Immunofluorescence ◽  
Small Intestinal Villus

cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport.

scholarly journals Cell surface expression of murine, rat, and human Fc receptors by Xenopus oocytes.

1984 ◽  
Vol 160 (2) ◽  
pp. 606-611 ◽  
Author(s):  
E Pure ◽  
A D Luster ◽  
J C Unkeless
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Surface Expression ◽  
Gamma Interferon ◽  
Membrane Receptors ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
High Avidity ◽  
U937 Cells ◽  
Mouse Igg2a

We report that Xenopus laevis oocytes can efficiently translate and insert heterologous membrane receptors into the oocyte plasma membrane, where they can be detected by the binding of either monoclonal antibodies or ligands. Thus, oocytes injected with mRNA from the mouse J774 macrophage-like cell line, the rat RBL-1 basophilic leukemia, and the U937 promonocyte cell line, bound 2.4G2 Fab, rat IgE, and mouse IgG2a, respectively. The increase in the high avidity Fc gamma R observed after gamma-interferon induction of U937 cells was also observed after injection of mRNA from gamma-interferon-induced U937 cells into oocytes. This suggests either much greater message stability or a greater rate of transcription of Fc gamma Rhi mRNA in the gamma-interferon-induced cells. The assay affords a sensitive method for the detection of rare mRNA species that code for plasma membrane proteins.

Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females

1992 ◽  
Vol 263 (3) ◽  
pp. G380-G385 ◽  
Author(s):  
D. Sorrentino ◽  
S. L. Zhou ◽  
E. Kokkotou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Transport System ◽  
Fatty Acid Uptake ◽  
Surface Expression ◽  
Female Rats ◽  
Acid Uptake ◽  
Fatty Acid Binding ◽  
Male And Female Rats

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.

In vivo shedding of apical plasma membrane in the thyroid follicle cells of the mouse

1984 ◽  
Vol 236 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Mikael Nilsson ◽  
Torsten �fverholm ◽  
LarsE. Ericson
Keyword(s):  
Plasma Membrane ◽  
Follicle Cells ◽  
Apical Plasma Membrane ◽  
Thyroid Follicle

Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the alpha IIb beta 3-integrin in melanoma cells

1995 ◽  
Vol 108 (6) ◽  
pp. 2175-2186 ◽  
Author(s):  
J. Timar ◽  
R. Bazaz ◽  
V. Kimler ◽  
M. Haddad ◽  
D.G. Tang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Intermediate Filaments ◽  
Brefeldin A ◽  
Surface Expression ◽  
Melanoma Cells ◽  
Apical Plasma Membrane ◽  
Intracellular Pool ◽  
Soluble Ligand ◽  
Surface Receptor

In metastatic B16a murine melanoma cells, alpha IIb beta 3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, alpha IIb beta 3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and alpha IIb beta 3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC-stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the alpha IIb beta 3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells.

Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity

1999 ◽  
Vol 276 (2) ◽  
pp. F179-F190 ◽  
Author(s):  
Jørgen Frøkiaer ◽  
David Marples ◽  
Heinz Valtin ◽  
John F. Morris ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Northern Blotting ◽  
Apical Plasma Membrane ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Aquaporin 2 ◽  
Acute Response ◽  
Camp Levels

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 ± 7% (±SE) of those in control mice ( n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels ( P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.

scholarly journals Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

2010 ◽  
Vol 298 (4) ◽  
pp. F935-F940 ◽  
Author(s):  
Janet D. Klein ◽  
Mitsi A. Blount ◽  
Otto Fröhlich ◽  
Chad E. Denson ◽  
Xiaoxiao Tan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Lines ◽  
Specific Antibody ◽  
Cultured Cells ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Amino Acid Peptide ◽  
In The Wild

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by 32P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

scholarly journals Apical targeting of syntaxin 3 is essential for epithelial cell polarity

2006 ◽  
Vol 173 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Nikunj Sharma ◽  
Seng Hui Low ◽  
Saurav Misra ◽  
Bhattaram Pallavi ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Polarization ◽  
Apical Plasma Membrane ◽  
Membrane Expression ◽  
Tight Junction Formation ◽  
Necessary And Sufficient ◽  
Syntaxin 3

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P &lt; 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P &lt; 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

scholarly journals Direct Interaction with Rab11a Targets the Epithelial Ca2+ Channels TRPV5 and TRPV6 to the Plasma Membrane

2006 ◽  
Vol 26 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Stan F. J. van de Graaf ◽  
Qing Chang ◽  
Arjen R. Mensenkamp ◽  
Joost G. J. Hoenderop ◽  
René J. M. Bindels
Keyword(s):  
Plasma Membrane ◽  
Transient Receptor Potential ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Surface Expression ◽  
The Body ◽  
Cell Surface Expression ◽  
Apical Plasma Membrane ◽  
Transient Receptor

ABSTRACT TRPV5 and TRPV6 are the most Ca2+-selective members of the transient receptor potential (TRP) family of cation channels and play a pivotal role in the maintenance of Ca2+ balance in the body. However, little is known about the mechanisms controlling the plasma membrane abundance of these channels to regulate epithelial Ca2+ transport. In this study, we demonstrated the direct and specific interaction of GDP-bound Rab11a with TRPV5 and TRPV6. Rab11a colocalized with TRPV5 and TRPV6 in vesicular structures underlying the apical plasma membrane of Ca2+-transporting epithelial cells. This GTPase recognized a conserved stretch in the carboxyl terminus of TRPV5 that is essential for channel trafficking. Furthermore, coexpression of GDP-locked Rab11a with TRPV5 or TRPV6 resulted in significantly decreased Ca2+ uptake, caused by diminished channel cell surface expression. Together, our data demonstrated the important role of Rab11a in the trafficking of TRPV5 and TRPV6. Rab11a exerts this function in a novel fashion, since it operates via direct cargo interaction while in the GDP-bound configuration.

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