Cytoskeletal-dependent activation of system A for neutral amino acid transport in osmotically stressed mammalian cells: a role for system A in the intracellular accumulation of osmolytes.
System A activity for neutral amino acid transport is increased after hypertonic shock in NBL-1 (an epithelial cell line) and CHO-K1 cells (a nonepithelial cell line) by a mechanism which is consistent with the synthesis of a regulatory protein that activates preexisting system A carrier proteins (Ruiz-Montasell et al., 1994, Proc. Natl. Acad. Sci. USA, 91,9569-9573). In this study, we have further investigated this biological response by determining the role of cytoskeletal structures in system A regulation by hypertonic stress. Using inhibitors of the microfilament and microtubule networks, we show that the increase in system A activity after hypertonic treatment requires the integrity of both cytoskeletal structures in NBL-1 cells, although the increase in system A activity triggered by amino acid starvation is completely insensitive to any of these drugs. In contrast, the enhancement of system A activity in osmotically stressed CHO-K1 cells is not sensitive to inhibitors of the microtubule network. In both cell types, the results suggest that the inhibitors block the increase of system A activity. System A transport decreases when CHO-K1 cells return to isotonic conditions by a mechanism that is insensitive to inhibitors of protein and mRNA synthesis. The increase in system A transport activity is also followed by the accumulation of neutral amino acids (fourfold for alanine), which is totally blocked by the same agents (cycloheximide and actinomycin D) that prevent the increase in system A activity after hypertonic treatment, thus indicating that system A is crucial for maintaining a high concentration of organic osmolytes inside the cell.
Gómez-Angelats
M
M
Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Spain.
López-Fontanals
M
M
Felipe
A
A
Casado
F J
FJ
Pastor-Anglada
M
M
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D006982:Hypertonic Solutions; D007700:Kinetics; D009994:Osmolar Concentration; D013312:Stress, Physiological; D013395:Sucrose / Q000494:pharmacology
D000596:Amino Acids; D006982:Hypertonic Solutions; D003571:Cytochalasin B; D013395:Sucrose; D003078:Colchicine