Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

Abstract

Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.

Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na+/K+)‐ATpase activity

“…The increased System A activity induced by ouabain was dependent on protein synthesis as indicated by sensitivity to cycloheximide (Leister et al, 1988). In contrast to this result, the increase in ( N a + / K + ) pump activity following exposure to ouabain for 18 h was not cycloheximide-sensitive.…”

“…The kinetic constants obtained in Figures 1-3 Table 1. A further characterization of this increase in System A activity induced by ouabain treatment is described in the accompanying paper (Leister et al, 1988).…”

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

“…Both carriers, the A system and the Na',K+-ATPase, are thus related mechanistically by sharing Na' as a substrate. It is therefore not surprising that the activities of both these systems are closely interrelated (16)(17)(18)(19)(20)(21)(22). Besides the concentration of Na' affecting the activity of the Na',K+-ATPase, under certain conditions that lead to increases in Na+ content there is an associated increase in the number of Na+,K+-ATPase molecules on the plasma membrane (16,(23)(24)(25)(26)(27)(28)(29)(30) and an increase in the abundance of their corresponding mRNA (29,30).…”

Altered sensitivity of system A amino acid transport to ouabain in normal and transformed C3H-10T1/2 cells during the cell cycle.

“…Cells that were treated with ouabain required de novo protein synthesis to express the enhanced system A transport activity (20). When cells were treated with ouabain in the presence of cycloheximide, which inhibits de novo protein synthesis, they showed no increase in system A transport yet still showed about a 50% sensitivity to ouabain (unpublished observations).…”

“…It thus appears that, in some cells, Ali as well as the Na' chemical gradient potential can contribute to the uptake of Aib, but the conditions that induce changes in the mechanisms have not been clearly defined (19,20). We show in this communication that cells that are in a certain phase in the cell cycle, transformed, or exposed to ouabain for prolonged periods respond with an increased system A transporter activity (presumably due to an increased synthesis of transporter) and a bioenergetically altered mechanism of transport that is coupled to the Na', K+-ATPase pump.…”