Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells

2008 ◽  
Vol 294 (1) ◽  
pp. F38-F46 ◽  
Author(s):  
Oleh Pochynyuk ◽  
Vladislav Bugaj ◽  
Alain Vandewalle ◽  
James D. Stockand
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Purinergic Receptors ◽  
Apical Membrane ◽  
Purinergic Signaling ◽  
Apical Plasma Membrane ◽  
Resting Cells ◽  
Open Probability ◽  
Renal Epithelial Cells ◽  
Principal Cells

Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in renal epithelial cells.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Polarization of gelsolin and actin binding protein in kidney epithelial cells.

1990 ◽  
Vol 38 (8) ◽  
pp. 1145-1153 ◽  
Author(s):  
J H Hartwig ◽  
D Brown ◽  
D A Ausiello ◽  
T P Stossel ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Water Permeability ◽  
Collecting Duct ◽  
Regulatory Proteins ◽  
Actin Binding ◽  
Apical Plasma Membrane ◽  
Actin Binding Protein ◽  
Principal Cells ◽  
Actin Regulatory Proteins

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.

Antidiuretic hormone moves membranes

1988 ◽  
Vol 255 (3) ◽  
pp. F375-F382 ◽  
Author(s):  
J. S. Handler
Keyword(s):  
Plasma Membrane ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Toad Bladder ◽  
Cell Swelling ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Particle Aggregates

This review focuses on events at the apical plasma membrane of toad urinary bladder and mammalian collecting duct as their permeability to water changes in response to antidiuretic hormone (ADH) and to its withdrawal. The major marker of the permeability change is observed in freeze-fracture electron microscopy of the apical plasma membrane and consists of a dramatic increase in membrane particle aggregates and, in toad bladder but not in collecting duct, in fused vesicles (aggrephores) that contain particle aggregates in their limiting membranes. Withdrawal of ADH is accompanied by endocytosis at the apical membrane, reflecting retrieval of water-permeable, particle aggregate-containing membrane. Covalent labeling of the external surface of the apical membrane of toad bladder identifies specific proteins that are present in the apical membrane only during the response to ADH. Proteins of the same molecular weights are also present in the retrieved membrane when ADH is withdrawn. Several controversial areas are considered, including the extent of cell swelling as water flows across the epithelium from dilute apical solution to isotonic basal solution, whether only principal cells or principal cells and intercalated cells participate in the water permeability response of the collecting duct, the role of the cytoskeleton in the water permeability response, and the proposed second water permeability barrier that is affected by ADH, but not by adenosine 3',5'-cyclic monophosphate.

scholarly journals Evidence for Apical Endocytosis in Polarized Hepatic Cells: Phosphoinositide 3-Kinase Inhibitors Lead to the Lysosomal Accumulation of Resident Apical Plasma Membrane Proteins

1999 ◽  
Vol 145 (5) ◽  
pp. 1089-1102 ◽  
Author(s):  
Pamela L. Tuma ◽  
Catherine M. Finnegan ◽  
Ji-Hyun Yi ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Kinase Inhibitors ◽  
Apical Membrane ◽  
Membrane Dynamics ◽  
Endocytic Pathway ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Phosphoinositide 3 Kinase

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5′-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

scholarly journals Hypertonicity Is Involved in Redirecting the Aquaporin-2 Water Channel into the Basolateral, Instead of the Apical, Plasma Membrane of Renal Epithelial Cells

2002 ◽  
Vol 278 (2) ◽  
pp. 1101-1107 ◽  
Author(s):  
Bas W. M. van Balkom ◽  
Marcel van Raak ◽  
Sylvie Breton ◽  
Nuria Pastor-Soler ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Renal Epithelial Cells ◽  
Aquaporin 2

Differential localization of human nongastric H+-K+-ATPase ATP1AL1 in polarized renal epithelial cells

2000 ◽  
Vol 279 (3) ◽  
pp. F417-F425 ◽  
Author(s):  
Jürgen Reinhardt ◽  
Alexander V. Grishin ◽  
Hans Oberleithner ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Ion Pump ◽  
Β Subunit ◽  
Ion Pumps ◽  
Α Subunit ◽  
Renal Epithelial Cells

The human H+-K+-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K+-transporting ATPases. In concert with the structurally related gastric H+-K+-ATPase, it plays a major role in K+ reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK1 renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na+-K+-ATPase β-subunit or the gastric H+-K+-ATPase β-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the α-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na+-K+-ATPase β-subunit. Cotransfection of ATP1AL1 with the gastric H+-K+-ATPase β-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by 86Rb+uptake measurements. In contrast, cotransfected LLC-PK1cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the α-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

scholarly journals Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells

2007 ◽  
Vol 18 (7) ◽  
pp. 2707-2715 ◽  
Author(s):  
Sai Prasad Ramnarayanan ◽  
Christina A. Cheng ◽  
Maria Bastaki ◽  
Pamela L. Tuma
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Epithelial Cells ◽  
Transmembrane Domain ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Trans Golgi Network ◽  
Direct Pathway ◽  
Golgi Network

Unlike simple epithelial cells that directly target newly synthesized glycophosphatidylinositol (GPI)-anchored and single transmembrane domain (TMD) proteins from the trans-Golgi network to the apical membrane, hepatocytes use an indirect pathway: proteins are delivered to the basolateral domain and then selectively internalized and transcytosed to the apical plasma membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been identified as regulators of direct and indirect apical delivery, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway.

Ultrastructure of Amphiuma distal nephron: evidence for cellular heterogeneity

1989 ◽  
Vol 256 (4) ◽  
pp. C849-C857 ◽  
Author(s):  
D. Biemesderfer ◽  
B. Stanton ◽  
J. B. Wade ◽  
M. Kashgarian ◽  
G. Giebisch
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Distal Tubule ◽  
Apical Plasma Membrane ◽  
Distal Nephron ◽  
Principal Cells ◽  
Intramembranous Particles ◽  
Collecting Tubule

To obtain more information on the ultrastructure of the distal nephron of the salamander, Amphiuma, we conducted freeze-fracture electron microscopy and morphometric experiments. In the early distal tubule, the organization of the tight junction is variable, containing from one to two strands in the proximal region and four strands in distal regions. The length density of the tight junction in this segment varies from greater than 60 m/cm2 of apical membrane surface to less than 10/cm2 of apical membrane surface. These observations agree with a previous study demonstrating that the junction of this segment exhibits considerable axial heterogeneity. The junctions of the late distal tubule and collecting tubule are more complex. In the late distal tubule, the tight junction is composed of 6-8 strands, whereas the tight junction of the collecting tubule is composed of 8-12 strands. The collecting tubule contains principal cells and two types of intercalated cells: alpha and beta. The alpha-cells contain a high density of rod-shaped particles in the apical plasma membrane and in membranes of apical cytoplasmic vesicles. The beta-cells contain rod-shaped particles only in the basolateral membrane. In principal cells, we observed a novel organization of intramembranous particles within the apical plasma membrane. A model describing the relationship of the two types of intramembranous particles within the membrane is presented. This study demonstrates that the amphibian and mammalian distal nephron share many morphological characteristics including cellular and axial heterogeneity.

scholarly journals Modulation of the expression of an apical plasma membrane protein of Madin-Darby canine kidney epithelial cells: cell-cell interactions control the appearance of a novel intracellular storage compartment.

1987 ◽  
Vol 104 (5) ◽  
pp. 1249-1259 ◽  
Author(s):  
D E Vega-Salas ◽  
P J Salas ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Storage Compartment ◽  
Intracellular Storage ◽  
Darby Canine Kidney ◽  
Canine Kidney

Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 microM Ca++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 micron) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apically targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia.

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