Involvement of Protein Tyrosine Kinase in Osmoregulation of Na + Transport and Membrane Capacitance in Renal A6 Cells

Niisato, Naomi; Driessche, Willy Van; Liu, Mingyao; Marunaka, Yoshinori

doi:10.1007/s002320001055

Cited by 43 publications

(46 citation statements)

References 54 publications

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“…No differences were discernible between cells from different passages. Cells were maintained in plastic tissue culture flasks at 27°C in a humidified incubator with 1% CO 2 in air in a culture medium, which contained 75% (volume / volume (v/v)) NCTC-109 medium (GIBCO, Grand Island, NY, U.S.A.), 15% (v/v) distilled water and 10% (v/v) fetal bovine serum (GIBCO) (17,20,23,24). The normal solution contained (in mM) 120 NaCl, 3.5 KCl, 1 CaCl 2 , 1 MgCl 2 , and 10 HEPES adjusted to pH 7.4 with NaOH.…”

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confidence: 99%

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

Yamada¹,

Niisato²,

Marunaka³

2009

Biomed. Res.

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The epithelial Na + channel (ENaC) contributes to control of blood pressure by reabsorbing Na + in the cortical collecting duct of the kidney. The luminal Cl − concentration in the duct varies under physiological conditions. As the body Na + content is lower, the luminal Cl − concentration in the duct becomes lower. Thus, we hypothesized that the extracellular Cl − elevates ENaC activity in AVT-stimulated renal epithelial A6 cells (a model cell line of the cortical collecting duct) leading to recovery from a low body Na + content. To clarify this point, we studied effects of extracellular Cl − concentration on ENaC activity using cell-attached patch clamp technique. We found that ENaC had a single-channel conductance of 4.6 ± 0.1 pS (mean ± SE) and channel activity (open probability, Po) of 0.30 ± 0.02 at a pipette potential of 60 mV. Lowering pipette Cl − concentration diminished Po to 0.23 ± 0.02 associated with a significant decrease in open time from 0.78 ± 0.03 to 0.61 ± 0.02 s with no significant change in closed time, and shifted the current-voltage relationship leftward. These results suggest that the extracellular Cl − regulates the ENaC-mediated Na + reabsorption by affecting ENaC properties in AVT-stimulated renal epithelial cells.The epithelial sodium channel (ENaC) is expressed in the apical membrane of Na + -transporting epithelial cells such as collecting ducts of the kidney and alveolar epithelia of the lung. ENaC consists of three conserved subunits, α, β, and γ. Each subunit has two transmembrane-spanning domains (TM1 and TM2), a large extracellular domain between TM1 and TM2, and NH 2 and COOH termini within the cytoplasm (2). ENaC is tightly regulated by hormones including aldosterone and antidiuretic hormone (arginine vasotocin; AVT) (5,19). It is well known that AVT stimulates Na + absorption by increasing the channel density of ENaC on the apical membrane of A6 cells, a model cell line of the cortical collecting duct of the kidney (11). ENaC is also regulated by extracellular cations such as Na + , H + and divalent cations. For example, the extracellular Na + at a high concentration decreases ENaC activity through Na + self-inhibition, which serves as a negative feedback mechanism to regulate Na + transport (5, 22). The extracellular H + modulates ENaC current and acidic pH increases ENaC current, whereas alkaline pH decreases the current (22). Divalent cations including Zn 2+ and Ni 2+ alter ENaC activity by binding to the extracellular domain (3, 29). Several findings suggest that the extracellular domain would be a sensor for changes in the extracellular ionic environments to modulate ENaC activity. ENaC plays a key role in control of blood pressure by reabsorbing Na + in the cortical collecting duct of the kidney. Body ionic conditions vary the luminal Cl − concentration in the cortical collect-

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confidence: 99%

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

Yamada¹,

Niisato²,

Marunaka³

2009

Biomed. Res.

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“…In A6 cell monolayers, hypotonic stress induces Na ϩ transport immediately (within 10 min) after application of the stress (18,24,26,27,29,37). Several studies revealed that hypotonic stress has a genomic and a nongenomic impact on Na ϩ reabsorption (26,29,33,37).…”

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confidence: 99%

“…Several studies revealed that hypotonic stress has a genomic and a nongenomic impact on Na ϩ reabsorption (26,29,33,37). We previously reported that the rapid, nongenomic effect of hypotonic stress on Na ϩ reabsorption is mediated through ligand-independent activation of receptortype tyrosine kinases (RTKs), such as epidermal growth factor receptor and platelet-derived growth factor receptor (37).…”

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confidence: 99%

Intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of SGK1 and ENaC in renal epithelial A6 cells

Taruno

Niisato²,

Marunaka³

2008

American Journal of Physiology-Renal Physiology

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Taruno A, Niisato N, Marunaka Y. Intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of SGK1 and ENaC in renal epithelial A6 cells. Am J Physiol Renal Physiol 294: F177-F186, 2008. First published October 24, 2007 doi:10.1152/ajprenal.00250.2007.-In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloridesensitive Na ϩ transport. Hypotonic action on Na ϩ transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na ϩ transport-related genes, such as serum-and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial Na ϩ channel (ENaC), increasing Na ϩ transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca 2ϩ in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca 2ϩ concentration in A6 cells, we hypothesized that Ca 2ϩ -dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca 2ϩ -dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na ϩ transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca 2ϩ plays a role as the second messenger in hypotonic stress-induced Na ϩ transport by stimulating transcription of SGK1 and ENaC subunits. sodium transport; calmodulin; W7

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“…In general, reports of increases in ␣-ENaC protein abundance are associated with elevations in ␣-ENaC transcript (1,14,15). However, in the A6 model of the renal collecting duct, hypotonicty is known to 1) increase transepithelial sodium transport without an increase the ENaC protein levels (20) and 2) increase the abundance of ␣-ENaC transcript (19). These effects of hypotonicity on ENaC regulation may be particularly relevant in the context of mouse models with chronic concentrating defects.…”

Section: Discussionmentioning

confidence: 99%

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

Morris¹,

Uchida²,

Brooks³

et al. 2005

American Journal of Physiology-Renal Physiology

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. Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice. Am J Physiol Renal Physiol 289: F194 -F199, 2005. First published February 15, 2005 doi:10.1152/ajprenal.00121.2004.-Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 Ϫ/Ϫ) mice. Here, we have investigated the longterm consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (Ϫ/Ϫ) mice vs. heterozygotes [AQP1 (ϩ/Ϫ)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (Ϫ/Ϫ) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits ␤-ENaC and ␥-ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, ␤-ENaC, ␥-ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (Ϫ/Ϫ) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (Ϫ/Ϫ) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (Ϫ/Ϫ) mice showed decreased expression of UT-A1 and increased expression of ␤-ENaC and ␥-ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 Ϫ/Ϫ mice and this manifestation is related to the associated concentrating defect.ClC-nK1; urea THE RENAL COLLECTING DUCT (CD) exhibits considerable physiological and histological heterogeneity along its length. The solute transport process and cellular composition of the CD change as the segment descends from the renal cortex, through the hypertonic medullary interstitium, down to the papillary tip. The phenotypic heterogeneity along the CD is readily appreciated by contrasting the cortical CD (CCD) with the inner medullary CD (IMCD). The principal cells of the rat, rabbit, and mouse CCD and IMCD have distinct transport characteristics that are a reflection of the differential expression of transporter proteins in these segments. For example, the CCD is well...

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Involvement of Protein Tyrosine Kinase in Osmoregulation of Na + Transport and Membrane Capacitance in Renal A6 Cells

Cited by 43 publications

References 54 publications

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

Intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of SGK1 and ENaC in renal epithelial A6 cells

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

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