Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Abstract

Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and a-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of a-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino aciduptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicle system which should prove useful for studies of growth control. For more than two decades the cell surface membrane has been a focus for the study of differences between tumor cells and normal cells. Studies relating growth regulation to nutrient transport (1-6) have shown that the rate of cellular uptake of certain amino acids, sugars, and nucleosides decreases when growing, untransformed cells reach a confluent, "contact-inhibited" monolayer. Uptake rates have also been shown to increase when the cells are virally transformed. Pardee and coworkers (7,8) and Holley (9) have suggested that these transport variations observed in whole cells reflect primary alterations in the cell membrane, and that malignant growth is made possible by the resulting increase in nutrient supply. However, transport studies on whole cells present the problem of distinguishing between membrane-localized alterations and changes in intracellular metabolism.Attempts have been made to separate uptake from subsequent metabolism by the use of nonmetabolizable analogs.

Sodium-stimulated alpha-aminoisobutyric acid transport by membrane vesicles from simian virus-transformed mouse cells.

“…This implies that the energy of a Na+-electrochemical gradient is used to drive AIB uptake. These findings are in agreement with those of Quinlan et al (18). Both reports describe the Na+-dependent amino acid uptake into membrane vesicles derived from tissue-culture cells.…”

Calcium-Dependent Regulation of NEMO Nuclear Export in Response to Genotoxic Stimuli

“…Changes in pH, intracellular and extracellular calcium, osmolarity, and temperature appear to modulate NF-B activation by DNA-damaging agents. Interestingly, some of these changes can be observed with neoplastic transformation, within tumor microenvironments in various cancer types, and during radiation therapy and chemotherapy (8,33,43,64,71,92). Therefore, understanding the mechanisms responsible for enhancement (e.g., low pH, higher temperature) and the decrease (e.g., high osmolarity) of NF-B activation may provide novel strategies to prevent NF-B activation and to increase the efficacy of anticancer therapy.…”

Role of Na + in α-aminoisobutyric acid uptake by membrane vesicles from mouse fibroblasts transformed by simian virus 40

“…Several laboratories have reported that Na+ stimulates the uptake of amino acids, such as L-leucine, a-aminoisobutyric acid (AIB), and L-alanine, in membrane vesicles from virally transformed and nontransformed mouse fibroblasts (2)(3)(4)(5)(6)(7)(8)(9). Studies in this laboratory have demonstrated that mediated uptake of L-leucine and AIB by membrane vesicles from mouse fibroblasts transformed by simian virus 40 is stimulated by a NaCl gradient (external > internal) (2,5). In those studies it was shown that this enhancement is Na+ specific and includes a prominent "overshoot," namely, a transient intravesicular accumulation above the equilibrium level (i.e., active transport) during the initial uptake phase.…”