Glutamine Transport
Glutamine, as mentioned previously, is the second major component of cell culture media and is the source of most of the ammonium ions generated. It is important to mention that, under the normal conditions used for cell culture (37°C), glutamine is partially decomposed in a spontaneous way at a rate of 0.2-0.6 mM/day, forming pyrrolidine-carboxylic acid and ammonium ions (39). The rest of the glutamine is incorporated into the cell by means of the different amino acid transport systems, which are not specific for glutamine and also mediate the movement of other amino acids. Due to the critical role of glutamine in mammalian cell metabolism, different transporters have been identified, although their specific mechanisms and tissue-specific regulations still require further investigation. These transporters have been recently reviewed by Bode (40). In general, they can be classified into two main categories: Na+ dependent and Na+ independent. The first type utilizes the Na+ electrochemical gradient, maintained by the Na+/K+-ATPase, to cotransport glutamine and Na+ against their concentration gradient. The Na+-dependent transporters identified include systems ACS (or B0), B0,+, y+L, A, and N. The second type facilitates the selective movement of amino acids across the plasma membrane independent of the Na+ gradient. The Na+-independent transporters identified include systems L, b0,+, and n. It is important to emphasize that glutamine is the source of intracellular glutamate and aspartate, but these charged amino acids cannot be transported into the cells from the culture at a rate that does not limit cell growth. Indeed, in proliferating cells, the activity of system XAg—, responsible specifically for the transport of glutamate and aspartate, is low, while that of the transport system A, responsible for the transport of glutamine, is high. Therefore, in rapidly proliferating cells, glutamine transport and further internal generation of glutamate and aspartate seem to be the way to obtain these amino acids. However, in some cases this different uptake rate has been used to obtain a low growth rate in cells and/or low ammonium generation in the medium, as further discussed in the section on redistribution of cell metabolism.
Another transport mechanism is the one around the mitochondria, since most of the glutamine metabolism is mitochondrial. The transport mechanism of gluta-mine into the mitochondria is still not well elucidated, although it does not appear to be rate limiting for glutamine metabolism (3). There is also no definitive understanding of the localization of the first enzyme of glutamine metabolism, phosphate-activated glutaminase (PAG), which could be located either in the mitochondrial intramembrane space or the mitochondrial matrix. This aspect was discussed in detail by Haggstrom (3), and the experimental evidence obtained so far indicated that a percentage of the glutamate-derived glutamine might be exported out of the mitochondria, while the rest could be used directly in the mitochondria either for its incorporation into the TCA or by TA reactions (discussed in the next section). The glutamate obtained by the deamidation of glutamine and released outside the mitochondria can be either used directly in the cytosol or transported back into the mitochondria. This can be done by two different transport mechanisms: the glu-tamate-aspartate exchanger and the proton symport system. The first of these mechanisms is directly related to the mitochondrial aspartate TA pathway for aspar-tate formation, otherwise its use would lead to a depletion of aspartate in the mitochondria of the cell (41).
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