Stimulation of P(i) transport in opossum kidney cells by hyposmotic media

1993 ◽  
Vol 264 (4) ◽  
pp. F585-F592
Author(s):  
M. Loghman-Adham ◽  
G. T. Motock
Keyword(s):  
Fluid Phase ◽  
Kinetic Studies ◽  
Uptake Kinetic ◽  
Opossum Kidney ◽  
Pi Transport ◽  
Opossum Kidney Cells ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake ◽  
Stimulation Of

Exposure of various cells to hyposmotic media (Hypo) results in a rapid inhibition of both receptor-mediated and fluid-phase endocytosis. We used this maneuver to investigate the role of endocytosis in regulation of Pi transport in opossum kidney (OK) cells. Following exposure to Hypo, Na(+)-dependent Pi uptake increased rapidly, reaching a maximum within 5 min, and remained elevated up to 30 min. This was associated with a simultaneous reduction of horseradish peroxidase uptake. Kinetic studies showed increased apparent Vmax for Pi (9.38 +/- 0.93 vs. 13.08 +/- 1.04 nmol.mg-1.5 min-1 for control and Hypo, respectively; P < 0.05, n = 6) with no change in apparent Km. The effect was specific for Pi with no change in the Na(+)-dependent or -independent uptake of L-proline, L-glutamine, or methyl-alpha-D-glucopyranoside. Stimulation of Pi transport persisted when control and Hypo had identical ionic compositions. Stimulation of Pi transport was rapidly reversed when cells were returned to an isosmotic medium. Preincubation with Hypo at 4 degrees C had no effect on Pi transport. Addition of cycloheximide or actinomycin D did not prevent the increased Pi uptake after exposure to Hypo. The effect also persisted after protein kinase C downregulation. Stimulation of Pi transport by Hypo is consistent with reduced endocytic retrieval of Na(+)-Pi cotransporters from brush-border membrane (BBM), resulting in an increase in their number on the BBM.

scholarly journals Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na+/H+exchanger

2001 ◽  
Vol 281 (1) ◽  
pp. R10-R18 ◽  
Author(s):  
Pedro Gomes ◽  
M. A. Vieira-Coelho ◽  
P. Soares-da-Silva
Keyword(s):  
Adenylyl Cyclase ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Kidney Cells ◽  
Primary Control ◽  
Intracellular Acidification ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Stimulation Of

The present study was aimed at evaluating the role of D1- and D2-like receptors and investigating whether inhibition of Na+ transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na+/H+ exchanger, inhibition of the basolateral Na+-K+-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D1- and D2-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC50 = 220 ± 2 nM), marked intracellular acidification (IC50 = 58 ± 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 ± 4.5% reduction) without affecting intracellular pH recovery after CO2 removal. These results agree with the view that dopamine, through the activation of D1- but not D2-like receptors, inhibits both the Na+/H+ exchanger (0.001933 ± 0.000121 vs. 0.000887 ± 0.000073 pH unit/s) and Na+-K+-ATPase without interfering with the Na+-independent HCO[Formula: see text] transporter. It is concluded that dopamine, through the action of D1-like receptors, inhibits both the Na+/H+ exchanger and Na+-K+-ATPase, but its marked acidifying effects result from inhibition of the Na+/H+exchanger only, without interfering with the Na+-independent HCO[Formula: see text] transporter and Na+-K+-ATPase.

Synaptojanin family members are implicated in endocytic membrane traffic in yeast

1998 ◽  
Vol 111 (22) ◽  
pp. 3347-3356 ◽  
Author(s):  
B. Singer-Kruger ◽  
Y. Nemoto ◽  
L. Daniell ◽  
S. Ferro-Novick ◽  
P. De Camilli
Keyword(s):  
Synaptic Vesicles ◽  
Direct Evidence ◽  
Family Members ◽  
Fluid Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Close Link ◽  
Fluid Phase Endocytosis ◽  
Synaptojanin 1

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

scholarly journals Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins

1992 ◽  
Vol 281 (3) ◽  
pp. 809-817 ◽  
Author(s):  
J Yang ◽  
A E Clark ◽  
R Harrison ◽  
I J Kozka ◽  
G D Holman
Keyword(s):  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Fluid Phase ◽  
Transport Activity ◽  
Phenylarsine Oxide ◽  
Fluid Phase Endocytosis ◽  
Surface Labelling ◽  
Slow Dissociation ◽  
Stimulation Of

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins

1993 ◽  
Vol 264 (5) ◽  
pp. G880-G885 ◽  
Author(s):  
Y. Yamaguchi ◽  
E. Dalle-Molle ◽  
W. G. Hardison
Keyword(s):  
Horseradish Peroxidase ◽  
Parenchymal Cell ◽  
Fluid Phase ◽  
Mannose Receptor ◽  
Isolated Hepatocytes ◽  
Cell Counting ◽  
Cell Fractionation ◽  
Morphological Studies ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

2004 ◽  
Vol 15 (2) ◽  
pp. 861-869 ◽  
Author(s):  
Yaya Lefkir ◽  
Marilyne Malbouyres ◽  
Daniel Gotthardt ◽  
Adrian Ozinsky ◽  
Sophie Cornillon ◽  
...  
Keyword(s):  
Early Stage ◽  
Fluid Phase ◽  
Small Particles ◽  
Medium Chain ◽  
Murine Macrophages ◽  
Fluid Phase Endocytosis ◽  
Phagocytic Cups ◽  
Golgi Network ◽  
Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

scholarly journals Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4421-4431 ◽  
Author(s):  
Kan Chen ◽  
Hiroshi Nishi ◽  
Richard Travers ◽  
Naotake Tsuboi ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Fluid Phase ◽  
Neutrophil Extracellular Traps ◽  
Humoral Factors ◽  
Extracellular Traps ◽  
Fluid Phase Endocytosis ◽  
Soluble Immune Complexes ◽  
The Individual

Abstract Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)–linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.

scholarly journals Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

1999 ◽  
Vol 10 (9) ◽  
pp. 2847-2859 ◽  
Author(s):  
Eric Féraille ◽  
Maria Luisa Carranza ◽  
Sandrine Gonin ◽  
Pascal Béguin ◽  
Carlos Pedemonte ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Tyrosine Phosphorylation ◽  
Site Directed Mutagenesis ◽  
Common Mechanism ◽  
Sodium Reabsorption ◽  
Kidney Cells ◽  
Α Subunit ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Stimulation Of

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

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