Regulation of A system
amino acid transport was studied in primary cultures of the R3230AC mammary
adenocarcinoma. Higher rates of carrier-mediated Na+-dependent
proline transport, vc, was decreased and was attributed to a two-fold decrease in Vmax and a two-fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (
alanine,
serine,
glycine, and
proline) demonstrated adaptive decreases in
proline transport; the decrease was due to two-fold reduction in Vmax, with no change in Km for
proline. Even in the presence of preferred substrates for the A system, a density-dependent decrease in
proline transport was manifested. Both fast- and slow-growing cultures maintained in MEM exhibited rapid increases in
proline transport when switched to
buffers devoid of
amino acids; two-fold increases in Vmax were seen within 4 hr, but Km was unchanged. This
starvation-induced adaptation was completely prevented by inclusion in the
buffer of 10 mM
proline, 0.1 mM alpha-(methylamino)-
isobutyric acid (MetAIB) or 10 mM
serine, whereas inclusion of the poorer A system substrate,
phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent
starvation-induced increases in
proline transport were dose-related, rapid, and reversible.
Amino acid starvation-induced increases in
proline transport were partially blocked by
cycloheximide or
actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [
proline] and decreasing vc for
proline uptake. In addition, efflux of
proline from preloaded cells preceded the increase in initial rates of
proline entry. Taken together, we concluded that: 1) A system transport in primary cultures of this mammary
adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and 2)
starvation-induced enhancement of
proline transport appears to be due to release from transinhibition, but may also involve a derepression-repression type of mechanism.