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  • American Physiological Society  (98)
  • 1
    Online Resource
    Online Resource
    American Physiological Society ; 2004
    In:  American Journal of Physiology-Cell Physiology Vol. 286, No. 4 ( 2004-04), p. C768-C778
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 286, No. 4 ( 2004-04), p. C768-C778
    Abstract: ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2004
    detail.hit.zdb_id: 1477334-X
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    American Physiological Society ; 2013
    In:  American Journal of Physiology-Renal Physiology Vol. 304, No. 5 ( 2013-03-01), p. F553-F564
    In: American Journal of Physiology-Renal Physiology, American Physiological Society, Vol. 304, No. 5 ( 2013-03-01), p. F553-F564
    Abstract: Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK 1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.
    Type of Medium: Online Resource
    ISSN: 1931-857X , 1522-1466
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2013
    detail.hit.zdb_id: 1477287-5
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  • 3
    Online Resource
    Online Resource
    American Physiological Society ; 1998
    In:  American Journal of Physiology-Cell Physiology Vol. 275, No. 1 ( 1998-07-01), p. C216-C226
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 275, No. 1 ( 1998-07-01), p. C216-C226
    Abstract: A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5′-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5′-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    Language: English
    Publisher: American Physiological Society
    Publication Date: 1998
    detail.hit.zdb_id: 1477334-X
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    American Physiological Society ; 2005
    In:  American Journal of Physiology-Cell Physiology Vol. 288, No. 6 ( 2005-06), p. C1390-C1401
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 288, No. 6 ( 2005-06), p. C1390-C1401
    Abstract: Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) “recycling” pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20°C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2005
    detail.hit.zdb_id: 1477334-X
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    American Physiological Society ; 2003
    In:  American Journal of Physiology-Cell Physiology Vol. 285, No. 4 ( 2003-10), p. C750-C762
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 285, No. 4 ( 2003-10), p. C750-C762
    Abstract: Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr 325 with Phe 325 prevented ligand-induced internalization of V2R determined by [ 3 H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY moti f of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2003
    detail.hit.zdb_id: 1477334-X
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    American Physiological Society ; 2011
    In:  American Journal of Physiology-Cell Physiology Vol. 300, No. 6 ( 2011-06), p. C1442-C1455
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 300, No. 6 ( 2011-06), p. C1442-C1455
    Abstract: Previously, we demonstrated that the vacuolar-type H + -ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1–a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K D = 2.84 × 10 −10 M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2011
    detail.hit.zdb_id: 1477334-X
    SSG: 12
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  • 7
    In: American Journal of Physiology-Renal Physiology, American Physiological Society, Vol. 292, No. 5 ( 2007-05), p. F1303-F1313
    Abstract: The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK 1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.
    Type of Medium: Online Resource
    ISSN: 1931-857X , 1522-1466
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2007
    detail.hit.zdb_id: 1477287-5
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  • 8
    Online Resource
    Online Resource
    American Physiological Society ; 2006
    In:  American Journal of Physiology-Heart and Circulatory Physiology Vol. 291, No. 6 ( 2006-12), p. H2816-H2824
    In: American Journal of Physiology-Heart and Circulatory Physiology, American Physiological Society, Vol. 291, No. 6 ( 2006-12), p. H2816-H2824
    Abstract: The slope of the log of power versus the log of frequency in the arterial blood pressure (BP) power spectrum is classically considered constant over the low-frequency range (i.e., “fractal” behavior), and is quantified by β in the relationship “1/ f β .” In practice, the fractal range cannot extend to indefinitely low frequencies, but factor(s) that terminate this behavior, and determine β, are unclear. We present 1) data in rats ( n = 8) that reveal an extremely low frequency spectral region (0.083–1 cycle/h), where β approaches 0 (i.e., the “shoulder”); and 2) a model that 1) predicts realistic values of β within that range of the spectrum that conforms to fractal dynamics (∼1–60 cycles/h), 2) offers an explanation for the shoulder, and 3) predicts that the “successive difference” in mean BP (mBP) is an important parameter of circulatory function. We recorded BP for up to 16 days. The absolute difference between successive mBP samples at 0.1 Hz (the successive difference, or Δ) was 1.87 ± 0.21 mmHg (means ± SD). We calculated β for three frequency ranges: 1) 0.083–1; 2) 1–6; and 3) 6–60 cycles/h. The β for all three regions differed ( P 〈 0.01). For the two higher frequency ranges, β indicated a fractal relationship (β 6–60/h = 1.27 ± 0.01; β 1–6/h = 1.80 ± 0.16). Conversely, the slope of the lowest frequency region (i.e., the shoulder) was nearly flat (β 0.083–1 /h = 0.32 ± 0.28). We simulated the BP time series as a random walk about 100 mmHg with ranges above and below of 10, 30, and 50 mmHg and with Δ from 0.5 to 2.5. The spectrum for the conditions mimicking actual BP time series (i.e., range, 85–115 mmHg; Δ, 2.00) resembled the observed spectra, with β in the lowest frequency range = 0.207 and fractal-like behavior in the two higher frequency ranges (β = 1.707 and 2.057). We suggest that the combined actions of mechanisms limiting the excursion of arterial BP produce the shoulder in the spectrum and that Δ contributes to determining β.
    Type of Medium: Online Resource
    ISSN: 0363-6135 , 1522-1539
    RVK:
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2006
    detail.hit.zdb_id: 1477308-9
    SSG: 12
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  • 9
    Online Resource
    Online Resource
    American Physiological Society ; 2017
    In:  Physiology Vol. 32, No. 6 ( 2017-11), p. 400-401
    In: Physiology, American Physiological Society, Vol. 32, No. 6 ( 2017-11), p. 400-401
    Type of Medium: Online Resource
    ISSN: 1548-9213 , 1548-9221
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2017
    detail.hit.zdb_id: 3115360-4
    detail.hit.zdb_id: 2005759-3
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    American Physiological Society ; 2018
    In:  Physiology Vol. 33, No. 6 ( 2018-11-01), p. 370-371
    In: Physiology, American Physiological Society, Vol. 33, No. 6 ( 2018-11-01), p. 370-371
    Type of Medium: Online Resource
    ISSN: 1548-9213 , 1548-9221
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2018
    detail.hit.zdb_id: 3115360-4
    detail.hit.zdb_id: 2005759-3
    SSG: 12
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