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 (original) (raw)
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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...
The relationship between sodium-dependent transport of anionic amino acids and cell proliferation
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
The relationship between the transport of anionic amino acids and the proliferative status of the cell population has been studied in NIH-3T3 cells. Proliferative quiescence, verified by determinations of growth-rate quotient and incorporation of thymidine, is associated with a marked increase of the influx of L-aspartate. After 7-10 days of serum starvation, the initial influx of L-aspartate increases by 8-10-times with respect to the transport activity determined in growing cells. The operational properties of the influx of L-aspartate are similar in growing and quiescent cells; in particular, the influx of the anionic amino acid is mostly Na+-dependent and completely suppressed by an excess of L-glutamate and D-aspartate, but not of D-glutamate. These features suggest that, in both cases, aspartate uptake occurs through system XxG. The quiescence-related increase in aspartate transport is gradual, sensitive to the inhibition of protein synthesis and referable to the enhanced maximal capacity of transport system XxG. Restoration of serum concentration in the culture medium of serum-starved cells causes a decrease in aspartate transport that is maximal in correspondence to late G~/S phases. It is concluded that the XXG system for anionic amino-acid uptake is sensitive to the proliferative status of the cell population and that, in particular, its transport activity is stimulated by the establishment of proliferative quiescence.
The role of the neutral amino acid transporter SNAT2 in cell volume regulation
Acta Physiologica, 2006
Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.
Basolateral potassium membrane permeability of A6 cells and cell volume regulation
The Journal of Membrane Biology, 1994
The K + permeabilities (86Rb(K) transport) of the basolateral membranes (Jb K) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation. Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca (max < 1 min) and cell swelling (max at 3-5 rain) followed by a regulatory volume decrease (5-30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the S6Rb(K) transport and the SCC were partially blocked by Ba 2+, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3-6 rain) stimulation of the 86Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca2+-free media or quinidine addition did not affect the initial osmotic stimulation of Jb K but prevented its "secondary regulation," whereas TEA, glibenclamide and DIDS completely blocked the initial Jb K increase. Under hypo-osmotic conditions, the initial Jb K increase was enhanced by the presence of 1 mM of barium and delayed with higher concentrations (5 ram). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media. We propose that a TEA-and glibenclamide-sensitive but quinidine-insensitive increase in K + permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K + permeability mediated by the observed calcium rise.
The Na+/K+/Cl cotransport in C6 glioma cells. Properties and role in volume regulation
European Journal of Biochemistry, 1988
The role of the Naf/K+/CI-cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide > bumetanide > piretanide > furosemide.
Journal of Biological Chemistry, 1980
The rate of transport of a-aminoisobutyric acid (AIB) has been measured in Swiss 3T3 cells at different cell densities in the range lo3 to 5 X lo4 cells/cm2. There is a pronounced increase in the rate of Na'-dependent AIB uptake below 4 X lo3 cells/cm*, which can be observed both in growing cells (10% serum) or in cells arrested early in the GI (Go) portion of the cell cycle in medium containing plasma-derived serum. The r a t e of AIB transport of cells arrested in Go can be inhibited by the addition of a plasma membrane-enriched fraction prepared from 3T3 cells. The results are interpreted in terms of two elements that control the rate of AIB transport, one dependent on the position in the cell cycle and the other on cell contact. Preliminary observations suggest that BALB 3T3 cells behave in a similar way.
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.
American Journal of Physiology-Cell Physiology, 1999
The response to chronic hypertonic stress has been studied in human endothelial cells derived from saphenous veins. In complete growth medium the full recovery of cell volume requires several hours and is neither associated with an increase in cell K+ nor hindered by bumetanide but depends on an increased intracellular pool of amino acids. The highest increase is exhibited by neutral amino acid substrates of transport system A, such as glutamine and proline, and by the anionic amino acid glutamate. Transport system A is markedly stimulated on hypertonic stress, with an increase in activity roughly proportional to the extent and the duration of the osmotic shrinkage. Cycloheximide prevents the increase in transport activity of system A and the recovery of cell volume. It is concluded that human endothelial cells counteract hypertonic stress through the stimulation of transport system A and the consequent expansion of the intracellular amino acid pool.