Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

1995 ◽  
Vol 147 (2) ◽  
Author(s):  
P.R. Smith ◽  
L.C. Stoner ◽  
S.C. Viggiano ◽  
K.J. Angelides ◽  
D.J. Benos
Keyword(s):  
Epithelial Cells ◽  
Na Channels ◽  
Lateral Mobility ◽  
Renal Epithelial Cells ◽  
Epithelial Na Channels

scholarly journals Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model

2017 ◽  
Vol 41 (5) ◽  
pp. 1865-1880 ◽  
Author(s):  
Kouhei Sasamoto ◽  
Rie Marunaka ◽  
Naomi Niisato ◽  
Hongxin Sun ◽  
Akiyuki Taruno ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Epithelial Cells ◽  
Protease Inhibitor ◽  
Apical Membrane ◽  
Recycling Rate ◽  
Na Channels ◽  
Extracellular Loop ◽  
Na Transport ◽  
Renal Epithelial Cells ◽  
Epithelial Na Channels

Background/Aim: Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. Methods: We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. Results: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. Conclusion: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.

Localization of amiloride-sensitive sodium channels in A6 cells by atomic force microscopy

1997 ◽  
Vol 272 (4) ◽  
pp. C1295-C1298 ◽  
Author(s):  
P. R. Smith ◽  
A. L. Bradford ◽  
S. Schneider ◽  
D. J. Benos ◽  
J. P. Geibel
Keyword(s):  
Na Channel ◽  
High Resolution Imaging ◽  
Na Channels ◽  
Renal Epithelial Cells ◽  
Gold Particles ◽  
Force Microscopy ◽  
Atomic Force ◽  
Epithelial Na Channels ◽  
Resolution Imaging ◽  
Epithelial Na Channel

Atomic force microscopy (AFM) was used for high-resolution imaging of the apical distribution of epithelial Na+ channels in A6 renal epithelial cells. A6 cells grown on coverslips were labeled with antibodies generated against an amiloride-sensitive epithelial Na+ channel complex purified from bovine renal medulla that had been conjugated to 8-nm colloidal gold particles before preparation for AFM. AFM revealed that there was a marked increase in the height of the microvilli in cells labeled with the anti-epithelial Na+ channel antibodies compared with unlabeled cells or cells labeled with rabbit nonimmune immunoglobulin G conjugated to colloidal gold particles. We interpret this apparent increase in microvillar height to be due to anti-epithelial Na+ channel antibody binding to the apical microvilli. These data demonstrate that epithelial Na+ channels are restricted to the apical microvilli in Na+-transporting renal epithelial cells. Furthermore, they demonstrate the applicability of using AFM for high-resolution imaging of the cell surface distribution of epithelial ion channels.

scholarly journals Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model

2018 ◽  
Vol 50 (1) ◽  
pp. 363-377 ◽  
Author(s):  
Rie Marunaka ◽  
Akiyuki Taruno ◽  
Toshiro Yamamoto ◽  
Narisato Kanamura ◽  
Yoshinori Marunaka
Keyword(s):  
Mathematical Model ◽  
Epithelial Cells ◽  
Intracellular Trafficking ◽  
Degradation Rate ◽  
Apical Membrane ◽  
Recycling Rate ◽  
Na Channels ◽  
Protein Tyrosine ◽  
Epithelial Na Channels ◽  
Hypotonic Condition

Background/Aims: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. Methods: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. Results: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. Conclusion: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.

scholarly journals Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells.

1991 ◽  
Vol 114 (1) ◽  
pp. 53-60 ◽  
Author(s):  
D A Jans ◽  
R Peters ◽  
P Jans ◽  
F Fahrenholz
Keyword(s):  
Signal Transduction ◽  
Epithelial Cells ◽  
Lateral Diffusion ◽  
Camp Production ◽  
Lateral Mobility ◽  
Renal Epithelial Cells ◽  
Transduction Mechanisms ◽  
Mobile Fraction ◽  
Temperature Pretreatment

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.

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