The Na+/K+/Cl cotransport in C6 glioma cells. Properties and role in volume regulation (original) (raw)
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Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 Reduces Glioma Invasion
Malignant gliomas metastasize throughout the brain by infiltrative cell migration into peritumoral areas. Invading cells undergo profound changes in cell shape and volume as they navigate extracellular spaces along blood vessels and white matter tracts. Volume changes are aided by the concerted release of osmotically active ions, most notably K+ and Cl−. Their efflux through ion channels along with obligated water causes rapid cell shrinkage. Suitable ionic gradients must be established and maintained through the activity of ion transport systems. Here, we show that the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1) provides the major pathway for Cl− accumulation in glioma cells. NKCC1 localizes to the leading edge of invading processes, and pharmacologic inhibition using the loop diuretic bumetanide inhibits in vitro Transwell migration by 25% to 50%. Short hairpin RNA knockdowns of NKCC1 yielded a similar inhibition and a loss of bumetanidesensitive cell volume regulation. A loss of NKCC1 function did not affect cell motility in two-dimensional assays lacking spatial constraints but manifested only when cells had to undergo volume changes during migration. Intracranial implantation of human gliomas into severe combined immunodeficient mice showed a marked reduction in cell invasion when NKCC1 function was disrupted genetically or by twice daily injection of the Food and Drug Administration–approved NKCC1 inhibitor Bumex. These data support the consideration of Bumex as adjuvant therapy for patients with high-grade gliomas
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997
Ehrlich cells exposed to a hypertonic medium for five hours respond by an increased expression of Na q rK q r2Cl y cotransport proteins as estimated from immunoprecipitations using polyclonal anti-cotransporter antibodies. The 3.4-fold increase in cotransport expression is followed by a concomitant 2.6-fold increase in the maximal bumetanide-sensitive K q influx during regulatory volume increase, indicating a 2.6-fold increase in the number of functional cotransporters in the plasma membrane. q 1997 Elsevier Science B.V.
The FASEB Journal
It has been known for several years that the triggering of cell proliferation is associated with an increase of the activity of Na,K,Cl cotransport and of transport system A for neutral amino acids. These systems are also enhanced during the volume recovery of hypertonically shrunk cells. We demonstrate here that during the cell cycle of NIH3T3 cells, an increase in cell volume is associated with an enhanced cell content of potassium and amino acids. Bumetanide delays cell cycle progression and hampers volume increase. The nonmetabolizable analog 2-methylamino-isobutyric acid, a specific substrate of system A, can partially substitute natural amino acids accumulated during the cell cycle as intracellular osmolytes. It is therefore proposed that the stimulation of Na,K,Cl cotransport and of system A, observed in proliferating cells, causes an expansion of cell volume through an enhanced intracellular accumulation of both inorganic and organic osmolytes and the concurrent, osmotically...
Characterization of an Na+/K+/Cl− co-transport in primary cultures of rat astrocytes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
The furosemide-and bumetanide-sensitive component of the S6Rb+ uptake into primary cultures of rat astrocytes was fully dependent on the simultaneous presence of Na ÷ and CI-in the incubation mixture and is therefore most likely an Na÷/K +/CI-co-transporter. As expected for such a co-transporter, its activity is insensitive to 0.1 mM amiloride and to 4-acetamido-4'-isothiocyanostiibene-2,2'.disulfonic acid, and of the tested anions, only Br-could partly replace CI-. The K0. 5 values for K ÷, Na + and C!-activation were 2.7, 35 and 40 mM, respectively. The activity of the co-transporter was stimulated 1.5-times in hyperosmolar (500 mosM) medium.
Mechanism of high K+ and Tl+ uptake in cultured human glioma cells
Cellular and Molecular Neurobiology, 1995
1. The aim of this study was to elucidate if the K + uptake was higher in cultured human glioma cells than in cells from other malignant tumors and to analyze the importance of membrane potential and K ÷ channels for the uptake. 2. K ÷ transport properties were studied with the isotopes a2K and the Kanalogue 201T1. 3. Comparison with cultured cells from other malignant tumors showed that the specific steady-state accumulation of T1 + was significantly higher in glioma cells (U-251MG and Tp-378MO). 4. In Ringer's solution at 37°C the rates of K + and T1 ÷ uptake were both inhibited by about 55% in ouabain and 60% in furosemide, bumetanide, or Na +or Cl-free medium. This indicated that the routes for K + and T1 + uptake were similar and due to Na,K-ATPase-dependent transport and to Na-K-C1 cotransport. 5. About 10% of the uptake was neither ouabain nor bumetanide sensitive. Ba z+, which is known to block inward-rectifying K + channels and to depolarize glial cells, and other K + channel blockers (Cs + and bupivacaine), had no effect on T1 + uptake. 6. Metabolic inhibition with dinitrophenol reduced the uptake rate to 17%. 7. The washout of T1 + was unaffected by bumetanide and K + channel blockers, but dinitrophenol caused a transient increase of 75%, an effect which persisted in the presence of K + channel blockers.
Water permeability of Na+-K+-2Cl- cotransporters in mammalian epithelial cells
The Journal of Physiology, 2005
Water transport properties of the Na +-K +-2Cl − cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide-sensitive component of 86 Rb + uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (L p) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide-sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l −1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi-osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide-sensitive component of the L p was 21 kcal mol −1 , which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na + , K + and Cl −. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86 Rb + , the NKCCs of this tissue did not contribute any bumetanide-sensitive L p. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.
Journal of Membrane Biology, 1996
To examine the involvement of Na + ,K + ,2Cl − cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on 86 Rb, 36 Cl and 22 Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward 86 Rb fluxes were not different. Bumetanide decreased basal 86 Rb uptake by 70-75% with a K i of ∼0.2-0.3 M. At concentrations ranging up to 1 M, bumetanide did not affect 36 Cl influx and reduced it by 20-30% in the range from 3 to 100 M. In contrast to 86 Rb and 36 Cl influx, bumetanide did not inhibit 22 Na uptake by VSMC. BS 86 Rb uptake was completely abolished in Na +-or Cl −-free media. In contrast to 86 Rb, basal BS 36 Cl influx was not affected by Na + o and K + o. Hyperosmotic and isosmotic shrinkage of VSMC increased 86 Rb and 36 Cl influx to the same extent. Shrinkage-induced increments of 86 Rb and 36 Cl uptake were completely abolished by bumetanide with a K i or ∼0.3 M. Shrinkage did not induce BS 86 Rb and 36 Cl influx in (Na + or Cl −)-and (Na + or K +)-depleted media, respectively. In the presence of an inhibitor of Na + /H + exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated 22 Na influx. Bumetanide (1 M) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na + ,K + ,2Cl − cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K + influx is mediated by (Na + o + Cl − o)-dependent K + /K + exchange and Na + o-dependent K + ,Cl − cotransport, respectively.
Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells
The Journal of Membrane Biology, 1986
The net loss of KC1 observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K + and CI-transport pathways. RVD is accelerated when a parallel K § transport pathway is provided by addition of gramicidin, indicating that the K + conductance is rate limiting. Addition of ionophore A23187 plus Ca 2+ also activates separate K + and CItransport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K + and C1 conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the C1-conductance is rate limiting. An A23187-induced activation of 42K and 36C1 tracer fluxes is directly demonstrated, RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the CaZ+-activated K + channel. (ii) unaffected by substitution of NO2 or SCN for CI-, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K + channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the CI-conductance. The CI conductance is increased about 60-fold during RVD. The volume-induced activation of the CI-transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca:+-free media (probably by release of Ca 2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the C1-transport pathway. These findings suggest that a transient increase in free cytosolic Ca 2+ may account for the transient activation of the CI-transport pathway. The activated anion transport pathway is unselective, carrying both CI-, Br-, NO2, and SCN . The anti-calmodulin drug pimozide blocks the volume-or A23187-induced C1-transport pathway and also blocks the activation of the K + transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K + conductance estimated from 42K flux measurements at high external K +, and from net K + flux measurements suggests single-file behavior of the Ca2+-activated K + channel. The number of Ca2+-activated K + channels is estimated at about 100 per cell.