scholarly journals Endocytic vesicles from renal papilla which retrieve the vasopressin-sensitive water channel do not contain a functional H+ ATPase.

1990 ◽  
Vol 111 (2) ◽  
pp. 379-389 ◽  
Author(s):  
W I Lencer ◽  
A S Verkman ◽  
M A Arnaout ◽  
D A Ausiello ◽  
D Brown
Keyword(s):  
Plasma Membrane ◽  
Water Permeability ◽  
Collecting Duct ◽  
Water Channel ◽  
Water Channels ◽  
Proton Pumps ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Fluorescence Imaging Microscopy ◽  
Membrane Components

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran-labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.

scholarly journals Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

1992 ◽  
Vol 119 (1) ◽  
pp. 111-122 ◽  
Author(s):  
I Sabolic ◽  
F Wuarin ◽  
L B Shi ◽  
A S Verkman ◽  
D A Ausiello ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
Transmembrane Domain ◽  
Collecting Duct ◽  
Water Channels ◽  
Proton Pumping ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Early Endosomes

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.

Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F573-F582 ◽  
Author(s):  
K. Fushimi ◽  
S. Sasaki ◽  
T. Yamamoto ◽  
M. Hayashi ◽  
T. Furukawa ◽  
...  
Keyword(s):  
Water Permeability ◽  
Expression Vector ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Functional Characterization ◽  
Protein A ◽  
Water Channel ◽  
Kidney Medulla ◽  
Principal Cells ◽  
Kidney Collecting Duct

Vasopressin-regulated water permeability of the kidney collecting duct is a key component of the urine concentration machinery. Recently, a cDNA for AQP-CD, the vasopressin-regulated water channel, initially reported as WCH-CD, has been isolated (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). AQP-CD was expressed in oocyte membrane using a Xenopus expression vector, and functional characteristics of AQP-CD were examined. Osmotic water permeability (Pf) of oocytes expressing AQP-CD was 138 +/- 19 microns/s (mean +/- SE), 12 times greater than the control (11 +/- 3 microns/s), 90% inhibited by 0.3 mM HgCl2, and weakly temperature dependent (energy of activation for Pf was 4.0 kcal/mol). Urea influx measured from 15-min [14C]urea uptake by oocytes injected with AQP-CD/expression vector 1 cRNA was 86 +/- 17% of the control. Two-electrode voltage-clamp experiments revealed insignificant ion conductance of AQP-CD. Immunoblots of membranes from rat kidney medulla and oocytes expressing AQP-CD using anti-AQP-CD COOH-terminal antibody showed a 29-kDa protein and 35- to 50-kDa high-molecular-mass forms. Immunohistochemistry showed apical and subapical localization of AQP-CD in the collecting duct principal cells. Our results indicated that AQP-CD is a 29-kDa protein, a selective water channel, distinct from a urea channel, and localized to the membranes of vasopressin-sensitive components in kidney collecting duct principal cells.

Endocytosis of water channels in rat kidney: cell specificity and correlation with in vivo antidiuresis

1990 ◽  
Vol 259 (6) ◽  
pp. C920-C932 ◽  
Author(s):  
W. I. Lencer ◽  
D. Brown ◽  
D. A. Ausiello ◽  
A. S. Verkman
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Water Channels ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Osmotic Water ◽  
Principal And Intercalated Cells

Vasopressin action in the renal collecting duct is believed to be mediated by the cycling of water channels in principal and, possibly, intercalated cells. We used 6-carboxyfluorescein (6-CF) or fluorescein-labeled dextran (FITC-dextran) to determine the location and water permeability of endocytic vesicles from papilla and inner stripe of Brattleboro rats in different states of diuresis. Fifteen minutes after FITC-dextran infusion, fluorescent vesicles were concentrated at the apical pole of principal and intercalated cells. The osmotic water permeability (Pf) of these endosomes was measured by fluorescence quenching. In papillary endosomes, Pf was high (0.04 +/- 0.004 cm/s) when rats were in physiological states of antidiuresis or after treatment with vasopressin, 1-desamino-8-D-arginine vasopressin (DDAVP), or oxytocin; endosomes isolated from these regions of untreated animals had a low Pf. The number of papillary endosomes with high Pf increased with increasing doses of DDAVP. Endosomes from the inner stripe also had a high Pf only after vasopressin treatment. Confocal microscopy of sections of papilla showed that vasopressin significantly increased endocytosis in principal cells but had no effect on intercalated cells. Our data demonstrate that the bulk of fluorescently labeled vesicles from the papilla originate from the apical membrane of principal cells and contain water channels in their limiting membrane only when the rats are in physiological states of antidiuresis. In contrast, the majority of endocytosis in intercalated cells is not involved in water channel recycling.

Membrane recycling and epithelial cell function

1989 ◽  
Vol 256 (1) ◽  
pp. F1-F12 ◽  
Author(s):  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Function ◽  
Collecting Duct ◽  
Water Channel ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Membrane Recycling ◽  
Membrane Composition

The plasma membrane composition of virtually all eucaryotic cells is established, maintained, and modified by the process of membrane recycling. Specific plasma membrane components are inserted by exocytosis of transport vesicles, and are removed by endocytosis of segments of the membrane in which particular proteins are concentrated. In the kidney collecting duct, vasopressin induces the cycling of vesicles that are thought to carry water channels to and from the apical plasma membrane of principal cells, thus modulating the water permeability of this membrane. In the intercalated cells of the collecting duct, hydrogen ion secretion is controlled by the recycling of vesicles carrying proton pumps to and from the plasma membrane. In both cell types, "coated" carrier vesicles are involved, but whereas clathrin-coated vesicles participate in water channel recycling, the vesicles in intercalated cells are coated with the cytoplasmic domains of proton pumps. Following a brief outline of membrane recycling in general, this review summarizes previous data on membrane recycling in the collecting duct and related transporting epithelia and discusses some selected points relating to the role of membrane recycling and cell-specific function in the collecting duct.

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.

Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues

1995 ◽  
Vol 108 (9) ◽  
pp. 2993-3002 ◽  
Author(s):  
A. Frigeri ◽  
M.A. Gropper ◽  
F. Umenishi ◽  
M. Kawashima ◽  
D. Brown ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Urinary Bladder ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Parietal Cells ◽  
Immunogold Electron Microscopy ◽  
Transitional Epithelium ◽  
Principal Cells ◽  
Kidney Collecting Duct

It was shown recently that water channel homologs MIWC (mercurial insensitive water channel) and GLIP (glycerol intrinsic protein) colocalized in basolateral membranes of kidney collecting duct, tracheal and colonic epithelia, and in brain pia mater. We report here an extensive immunolocalization study of MIWC and GLIP in non-epithelial and glandular epithelial tissues in rat. Immunogold electron microscopy confirmed colocalization of MIWC and GLIP in basolateral membrane of principal cells in kidney collecting duct. However, in other epithelia, MIWC but not GLIP was expressed in basolateral membrane of parietal cells in stomach, and in excretory tubules of salivary and lacrimal glands; GLIP but not MIWC was expressed in transitional epithelium of urinary bladder and skin epidermis. In the central nervous system, MIWC was strongly expressed in the ependymal layer lining the aqueductal system, and in astrocytes throughout the spinal cord and in selected regions of brain. MIWC was also expressed in a plasma membrane pattern in skeletal, but not smooth or cardiac muscle. Neither protein was expressed in small intestine, testis, liver, spleen and nerve. The tissue-specific expression of MIWC suggests a role in fluid transport and/or cell volume regulation in stomach and glandular epithelia. The functional role of MIWC expression in the neuromuscular system and of GLIP expression in skin and urinary bladder is uncertain. The specific cellular sites of MIWC expression (astrocytes, trachea, sarcolemma, gastric parietal cells and kidney principal cells) correspond exactly to sites where orthogonal arrays of particles (OAPs) have been visualized by freeze-fracture electron microscopy, suggesting that MIWC may be the OAP protein.

A rat kidney tubule suspension for the study of vasopressin-induced shuttling of AQP2 water channels

2002 ◽  
Vol 283 (5) ◽  
pp. F1160-F1166 ◽  
Author(s):  
Stephen Shaw ◽  
David Marples
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Primary Cultures ◽  
Water Channels ◽  
Apical Plasma Membrane ◽  
Cellular Mechanisms ◽  
Principal Cells ◽  
Microtubule Disruption ◽  
Osmotic Water

AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.

scholarly journals Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel

2013 ◽  
Vol 304 (1) ◽  
pp. C38-C48 ◽  
Author(s):  
Naofumi Yui ◽  
Hua A. J. Lu ◽  
Ying Chen ◽  
Naohiro Nomura ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cold Shock ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Basolateral Membranes ◽  
Principal Cells ◽  
Aquaporin 2

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

Sign in / Sign up

Export Citation Format

Share Document