Astrocytes from Na+-K+-Cl−cotransporter-null mice exhibit absence of swelling and decrease in EAA release

2002 ◽  
Vol 282 (5) ◽  
pp. C1147-C1160 ◽  
Author(s):  
Gui Su ◽  
Douglas B. Kintner ◽  
Michael Flagella ◽  
Gary E. Shull ◽  
Dandan Sun
Keyword(s):  
Cell Volume ◽  
Glutamate Release ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
A Value ◽  
Cortical Astrocytes ◽  
High K ◽  
Stimulation Of

We reported previously that inhibition of Na+-K+-Cl− cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K+concentration ([K+]o)-induced swelling and intracellular Cl− accumulation in rat cortical astrocytes. In this report, we extended our study by using cortical astrocytes from NKCC1-deficient (NKCC1−/−) mice. NKCC1 protein and activity were absent in NKCC1−/− astrocytes. [K+]o of 75 mM increased NKCC1 activity approximately fourfold in NKCC1+/+ cells ( P< 0.05) but had no effect in NKCC1−/− astrocytes. Intracellular Cl− was increased by 70% in NKCC1+/+ astrocytes under 75 mM [K+]o ( P < 0.05) but remained unchanged in NKCC1−/− astrocytes. Baseline intracellular Na+ concentration ([Na+]i) in NKCC1+/+ astrocytes was 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM [Na+]i in NKCC1−/− astrocytes ( P < 0.05). Relative cell volume of NKCC1+/+ astrocytes increased by 13 ± 2% in 75 mM [K+]o, compared with a value of 1.0 ± 0.5% in NKCC1−/− astrocytes ( P < 0.05). Regulatory volume increase after hypertonic shrinkage was completely impaired in NKCC1−/− astrocytes. High-[K+]o-induced 14C-labeledd-aspartate release was reduced by ∼30% in NKCC1−/− astrocytes. Our study suggests that stimulation of NKCC1 is required for high-[K+]o-induced swelling, which contributes to glutamate release from astrocytes under high [K+]o.

Contribution of Na+-K+-Cl−cotransporter to high-[K+]o- induced swelling and EAA release in astrocytes

2002 ◽  
Vol 282 (5) ◽  
pp. C1136-C1146 ◽  
Author(s):  
Gui Su ◽  
Douglas B. Kintner ◽  
Dandan Sun
Keyword(s):  
Cell Volume ◽  
Recovery Rate ◽  
Glutamate Release ◽  
Volume Changes ◽  
Control Value ◽  
Astrocyte Swelling ◽  
High K ◽  
Stimulation Of

We hypothesized that high extracellular K+ concentration ([K+]o)-mediated stimulation of Na+-K+-Cl− cotransporter isoform 1 (NKCC1) may result in a net gain of K+ and Cl−and thus lead to high-[K+]o-induced swelling and glutamate release. In the current study, relative cell volume changes were determined in astrocytes. Under 75 mM [K+]o, astrocytes swelled by 20.2 ± 4.9%. This high-[K+]o-mediated swelling was abolished by the NKCC1 inhibitor bumetanide (10 μM, 1.0 ± 3.1%; P < 0.05). Intracellular36Cl− accumulation was increased from a control value of 0.39 ± 0.06 to 0.68 ± 0.05 μmol/mg protein in response to 75 mM [K+]o. This increase was significantly reduced by bumetanide ( P < 0.05). Basal intracellular Na+ concentration ([Na+]i) was reduced from 19.1 ± 0.8 to 16.8 ± 1.9 mM by bumetanide ( P < 0.05). [Na+]i decreased to 8.4 ± 1.0 mM under 75 mM [K+]o and was further reduced to 5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of [Na+]i on return to 5.8 mM [K+]o was decreased by 40% in the presence of bumetanide ( P < 0.05). Bumetanide inhibited high-[K+]o-induced 14C-labeledd-aspartate release by ∼50% ( P < 0.05). These results suggest that NKCC1 contributes to high-[K+]o-induced astrocyte swelling and glutamate release.

Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells

2000 ◽  
Vol 279 (1) ◽  
pp. G40-G48 ◽  
Author(s):  
Thorsten Sonnentag ◽  
Wolf-Kristian Siegel ◽  
Oliver Bachmann ◽  
Heidi Rossmann ◽  
Andreas Mack ◽  
...  
Keyword(s):  
Volume Regulation ◽  
Parietal Cells ◽  
Cell Swelling ◽  
Secretory Process ◽  
Cell Shrinkage ◽  
Rapid Loss ◽  
Volume Changes ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Stimulation Of

Concomitant Na+/H+ and Cl−/HCO3 − exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10−5 M, carbachol at 10−4 M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 ± 6, 9 ± 4, and 23 ± 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 ± 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na+/H+ exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 μM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na+/H+ and Cl−/HCO3 − exchange.

Proximal tubule volume regulation in hyperosmotic media: intracellular K+, Na+, and Cl-

1989 ◽  
Vol 257 (6) ◽  
pp. C1093-C1100 ◽  
Author(s):  
L. Rome ◽  
J. Grantham ◽  
V. Savin ◽  
J. Lohr ◽  
C. Lechene
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Volume Regulation ◽  
Electron Probe ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Cell Shrinkage ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Expected Increase

Nonperfused proximal S2 segments from rabbit kidney cortex have been shown to keep cell volume constant as medium osmolality is slowly raised but to shrink and not exhibit regulatory volume increase (RVI) if medium osmolality is abruptly elevated (J. Lohr and J. Grantham. J. Clin. Invest. 78: 1165-1172, 1986). In the current study, 0.5 mM butyrate in the medium 1) extended the range from 361 to 450 mosmol/kgH2O over which cells maintained volume constant as osmolality was gradually raised and 2) restored RVI after cell shrinkage when osmolality was rapidly raised from 295 to 400 mosmol/kgH2O. Volume regulation was associated with net increases in intracellular Na+ and Cl- but no change in K+ (measured by electron probe). The increments in Na+ and Cl- were insufficient to account for the total addition of osmolytes required for volume maintenance or restoration. The fraction of the expected increase in intracellular osmoles accounted for by the increase in [(K+)i + (Na+)i + (Cl-)i] was 52 and 21% for gradual and rapid osmotic changes, respectively. We conclude that butyrate enhances the capacity of S2 segments to regulate volume in hyperosmotic medium by promoting addition of Na+ and Cl- and by other undermined factors.

The stimulation of Na,K,Cl cotransport and of system A for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle

1996 ◽  
Vol 10 (8) ◽  
pp. 920-926 ◽  
Author(s):  
Ovidio Bussolati ◽  
Jacopo Uggeri ◽  
Silvana Belletti ◽  
Valeria Dall'Asta ◽  
Gian C. Gazzola
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Cell Volume ◽  
Amino Acid Transport ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Volume Increase ◽  
System A ◽  
Stimulation Of

Effects of apical membrane Cl(-)-formate exchange on cell volume in rabbit proximal tubule

1990 ◽  
Vol 258 (3) ◽  
pp. F530-F536 ◽  
Author(s):  
L. Schild ◽  
P. S. Aronson ◽  
G. Giebisch
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Volume Changes ◽  
Proximal Tubule Cells ◽  
Volume Increase ◽  
Tubule Lumen ◽  
Stimulation Of

We used real-time recordings of cell volume changes to test for the role of the Cl(-)-formate exchanger in mediating NaCl entry across the apical membrane of rabbit proximal tubule cells. In the absence of extracellular Cl-, 0.5 and 5 mM formate in the tubule lumen induced an increase in cell volume of 1 and 9%, respectively. Formate-induced cell swelling was reduced by alkalinizing the tubule lumen or by addition of luminal amiloride (2 mM), indicating that the increase in cell volume results from the intracellular accumulation of Na-formate via nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. The cell volume increase induced by 0.5 mM formate was potentiated (from 1 to 4%) by Cl-, as expected for a formate-mediated stimulation of NaCl uptake via parallel Cl(-)-formate exchange and Na(+)-H+ exchange across the apical membrane. By contrast, the cell volume increase induced by 5 mM formate was attenuated (from 9 to 4%) by Cl-. The attenuating effect of Cl- on formate-induced cell swelling required the operation of the apical membrane Cl(-)-formate exchanger. The effect of 1:1 Cl(-)-formate exchange to attenuate formate-induced cell swelling can be explained if the cell possesses a volume-activated anion exit pathway, most likely at the basolateral cell membrane, that is capable of mediating the efflux of Cl- but not formate from the cell.

Cell volume control in vestibular dark cells during and after a hyposmotic challenge

1994 ◽  
Vol 266 (4) ◽  
pp. C1046-C1060 ◽  
Author(s):  
P. Wangemann ◽  
N. Shiga
Keyword(s):  
Cell Volume ◽  
Volume Control ◽  
Cell Width ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Dark Cells ◽  
Cell Height ◽  
Vestibular Dark Cells ◽  
Cell Volume Control

Cell height was measured as an index of volume in a preparation of vestibular dark cells in which the perfusate had access to both sides of the epithelium. In response to a hyposmotic challenge induced by removal of 75 mM NaCl, cell height increased to 107%; however, cell width did not increase. Significantly larger increases in cell height were observed in the absence of Cl- or K+ or in the presence of ouabain, lidocaine, barium, or quinidine, at 7 degrees C, or after fixation with glutaraldehyde. However, no significantly different swelling was observed during a hyposmotic challenge in the absence of Na+ or in the presence of bumetanide or ethoxyzolamide. Subsequent return to control osmolarity caused a regulatory volume increase that was dependent on Na+, Cl-, and K+, inhibited by bumetanide, ouabain, or 7 degrees C, however not inhibited by ethoxyzolamide, barium, quinidine, or lidocaine. The data suggest that cell volume control during the hyposmotic challenge involved a mechanism dependent on cytosolic KCl and the Na(+)-K(+)-ATPase and that the Na(+)-Cl(-)-K+ cotransporter was involved in regulatory volume increase.

Unidirectional sodium and potassium flux in myogenic L6 cells: mechanisms and volume-dependent regulation

1995 ◽  
Vol 78 (1) ◽  
pp. 272-281 ◽  
Author(s):  
C. K. Sen ◽  
O. Hanninen ◽  
S. N. Orlov
Keyword(s):  
Cell Volume ◽  
Transport Systems ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
L6 Cells ◽  
Relative Contribution ◽  
Potassium Flux ◽  
86Rb Influx ◽  
Sodium And Potassium ◽  
Effect Of Inhibitors

To clarify the relative participation of particular ion transport systems in net univalent cation fluxes under basal conditions and altered volume of skeletal muscle-derived cells, the effect of inhibitors of the Na(+)-K+ pump (ouabain), univalent ion cotransporters [bumetanide, furosemide, and (dihydroindenyl)oxy alkanoic acid], and N+/H+ exchanger (ethylisopropylamiloride) on 86Rb and 22Na fluxes has been studied in L6 myoblasts incubated in isosmotic (320 mosmol/kg) and anisosmotic media. Under the isosmotic condition, the relative contribution of ouabain-inhibited and ouabain-insensitive bumetanide-inhibited component of 86Rb influx was approximately 15–20 and 60%, respectively. 22Na influx was inhibited by bumetanide and ethylisopropylamiloride by 25 and 15%, respectively. Under isosmotic conditions, an increase of L6 cell volume was observed after addition of extracellular acetylcholine, extracellular K(+)-induced depolarization, or lowering of the pH of the incubation medium. High extracellular glutathione (150 microM) did not affect the cell volume of the muscle-derived cells bathed in isosmotic medium. Results of this study suggest that the bumetanide-inhibited component of K+ influx plays a key role in the adjustment of transmembrane K+ gradient in L6 myoblasts. The Na+/H+ exchanger appears to be important in regulatory volume increase.

scholarly journals Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl−-dependent mechanism

2001 ◽  
Vol 281 (6) ◽  
pp. C1948-C1953 ◽  
Author(s):  
Gengru Jiang ◽  
Janet D. Klein ◽  
W. Charles O'Neill
Keyword(s):  
Growth Factors ◽  
Cell Volume ◽  
Lysophosphatidic Acid ◽  
Myosin Light Chain ◽  
Fibroblast Growth ◽  
Volume Increase ◽  
Aortic Endothelial Cells ◽  
Cytoskeletal Regulation ◽  
Dependent Mechanism ◽  
Stimulation Of

The Na-K-2Cl cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular Cl−. This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by growth factors and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive86Rb+ influx) was rapidly stimulated by serum, lysophosphatidic acid, and fibroblast growth factor, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a 17% increase in cell volume and a 25% increase in Cl− content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular Cl− content. When cellular Cl− was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular Cl−. This resetting of the Cl− set point of the transporter enables the cotransporter to produce a hypertrophic volume increase.

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