The γ-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity

2005 ◽  
Vol 288 (4) ◽  
pp. F650-F657 ◽  
Author(s):  
Kaarina Pihakaski-Maunsbach ◽  
Shigeki Tokonabe ◽  
Henrik Vorum ◽  
Christopher J. Rivard ◽  
Juan M. Capasso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Osmotic Shock ◽  
Collecting Duct ◽  
Choline Chloride ◽  
Basolateral Membrane ◽  
Γ Subunit ◽  
Subunit Expression

Hypertonicity mediated by chloride upregulates the expression of the γ-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428–6433, 2003). The purpose of this study was to examine the cellular locations and the time course of γ-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase γ-subunit or the γb splice variant. Cells grown in 300 mosmol/kgH2O showed no immunoreactivity for the γ-subunit, whereas cells adapted to 600 or 900 mosmol/kgH2O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH2O with sodium chloride or choline chloride showed incorporation of γ into plasma membrane 12 h after osmotic challenge and distinct membrane staining in ∼40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH2O by addition of sodium acetate did not result in expression of the γ-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase γ-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the γ-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.

Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

2007 ◽  
Vol 293 (4) ◽  
pp. F1308-F1313 ◽  
Author(s):  
Mitsi A. Blount ◽  
Janet D. Klein ◽  
Christopher F. Martin ◽  
Dmitry Tchapyjnikov ◽  
Jeff M. Sands
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Membrane Staining ◽  
Protein Protein Interaction ◽  
Medullary Collecting Duct

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

scholarly journals Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells.

1985 ◽  
Vol 33 (8) ◽  
pp. 837-839 ◽  
Author(s):  
A Messing ◽  
A Stieber ◽  
N K Gonatas
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Lateral Spread ◽  
Ciliary Ganglion ◽  
Monolayer Cultures ◽  
Indirect Immunoperoxidase ◽  
Ciliary Ganglion Neurons ◽  
Ganglion Neurons

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.

scholarly journals Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane

1978 ◽  
Vol 77 (2) ◽  
pp. 323-328 ◽  
Author(s):  
WW Franke ◽  
C Grund ◽  
E Schmid ◽  
E Mandelkow
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Membrane Surface ◽  
Hexagonal Lattice ◽  
Interparticle Distance ◽  
Macromolecular Complexes ◽  
Inner Surface ◽  
Cross Bridges ◽  
Plasma Membrane Surface

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.

Vectorial efflux of cGMP and its dependence on sodium in the cortical collecting duct

1996 ◽  
Vol 271 (6) ◽  
pp. R1676-R1681 ◽  
Author(s):  
B. A. Stoos ◽  
J. L. Garvin
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Transport Mechanisms ◽  
Cortical Collecting Duct ◽  
Phosphodiesterase Inhibition ◽  
Luminal Membrane ◽  
Sodium Dependent ◽  
Independent Mechanism ◽  
Presence And Absence

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important second messenger that regulates transport in the nephron. We propose that the transport mechanisms that remove cGMP from the cell are different in the luminal and basolateral membranes of the cortical collecting duct (CCD). We examined efflux of cGMP from cultured and isolated perfused CCDs in response to atrial natriuretic factor (ANF) and nitric oxide (NO). In the presence of phosphodiesterase inhibition, these compounds resulted in preferential efflux of cGMP across the basolateral membrane in both cultured and isolated CCDs. In the presence of ANF, efflux was five times higher across the basolateral than the luminal membrane in cultured CCD cells (n = 14). In isolated CCDs, effluxes across the basolateral and luminal membranes were 1.02 +/- 0.2 and 0.03 +/- 0.01 fmol.mm-1.min-1, respectively, in the presence of ANF (n = 6; P < 0.007) and 0.87 +/- 0.21 and 0.02 +/- 0.01 fmol.mm-1.min-1, respectively, in the presence of NO (n = 6; P < 0.011). Efflux across the basolateral membrane in the presence and absence of sodium was 37 +/- 7.3 and 19.9 +/- 5 fmol.cm-2.min-1, respectively, in cultured cells (n = 12; P < 0.044) and 1.02 +/- 0.2 (n = 6) and 0.41 +/- 0.12 (n = 5) fmol.mm-1.min-1 in isolated perfused tubules (P < 0.042). There was no difference in luminal transport in the presence and absence of sodium in either model. We conclude that there are at least two different mechanisms involved in the removal of cGMP from the cell, one sodium dependent and the other sodium independent. The basolateral membrane appears to contain both, whereas the luminal membrane contains only the sodium-independent mechanism.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

scholarly journals The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

1991 ◽  
Vol 273 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Darnanville ◽  
R Bredoux ◽  
K J Clemetson ◽  
N Kieffer ◽  
N Bourdeau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Glycoprotein Ib ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

Silencing and overexpression of the γ-subunit of Na-K-ATPase directly affect survival of IMCD3 cells in response to hypertonic stress

2006 ◽  
Vol 291 (6) ◽  
pp. F1142-F1147 ◽  
Author(s):  
Juan M. Capasso ◽  
Christopher J. Rivard ◽  
Tomas Berl
Keyword(s):  
Cell Survival ◽  
Vector Control ◽  
Collecting Duct ◽  
Critical Role ◽  
Hypertonic Stress ◽  
Empty Vector Control ◽  
Empty Vector ◽  
Γ Subunit ◽  
Subunit Expression ◽  
The Impact

The γ-subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either γ-subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed γ-subunit protein under isotonic conditions. Clones expressing the γb isoform showed enhanced survival at lethal acute hypertonicity compared with either γa isoform or empty vector (control) expressing clones. We also evaluated the loss of γ-subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable γ-subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of γ mRNA and protein was essentially undetectable. The impact of silencing γ-subunit expression resulted in a 70% reduction at 48 h ( P < 0.01) in cell survival compared with empty vector (control) clones. γ siRNA clones showed a 45% decrease in myo-inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the γ-subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.

scholarly journals AS160 Modulates Aldosterone-stimulated Epithelial Sodium Channel Forward Trafficking

2010 ◽  
Vol 21 (12) ◽  
pp. 2024-2033 ◽  
Author(s):  
Xiubin Liang ◽  
Michael B. Butterworth ◽  
Kathryn W. Peters ◽  
Raymond A. Frizzell
Keyword(s):  
Protein Expression ◽  
Sodium Channel ◽  
Time Course ◽  
Plasma Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Rab Protein ◽  
Adipocyte Plasma Membranes

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (Isc). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and Isc 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and Isc under basal conditions to ∼80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, β and ε. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3β and ε and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.

Purification of distinct plasma membranes from canine renal medulla

1978 ◽  
Vol 234 (3) ◽  
pp. F247-F254
Author(s):  
R. Iyengar ◽  
D. S. Mailman ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Collecting Duct ◽  
Gradient Centrifugation ◽  
Phospholipid Composition ◽  
Renal Medulla ◽  
Similar Distribution ◽  
Mitochondrial Atpase ◽  
Fold Enrichment

Two types of plasma membrane were purified from canine distal renal medulla by the techniques of differential and zonal density-gradient centrifugation followed by free-flow electrophoresis. One group of plasma membranes was identified as basal-laterally derived based on a 30-fold enrichment of Na-K-ATPase, a 20-fold enrichment of vasopressin-stimulated adenylate cyclase, and a 33-fold enrichment of [3H]vasopressin binding sites. The second type of plasma membrane was free of these markers, but had a cholesterol and phospholipid composition similar to them. Alkaline phosphatase also had a similar distribution in the two fractions. This lighter membrane fraction contained a membrane-bound cyclic AMP-dependent protein kinase as well as substrate for this kinase. In addition there was a 26-fold enrichment of specific activity of an anion (SO32-)-activated ATPase which was insensitive to mitochondrial ATPase inhibitor protein, in contrast to the mitochondrial fraction of the tissue. Based on the relative preponderance of collecting duct tissue in the distal medulla and the yield of membrane protein, these membranes are tentatively identified as containing apical membranes of the collecting duct.

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