Biochimica et Biophysica Acta, 693 (1982) 13-21 13 Elsevier Biomedical Press BBA71427 THE REGULATION BY CELL DENSITY OF AMINO ACID TRANSPORT SYSTEM L IN SV40 3T3 CELLS PIER GIORGIO PETRONINI, GIUSEPPE PIEDIMONTE and ANGELO F. BORGHETTI * Istituto di Patologia Generale, Universitit degli Studi di Parma, Via Gramsci 14, 43100 Parma (ltaly) (Received February 25th, 1982) (Revised manuscript received July 26th, 1982) Key words: Amino acid transport," Cell density," (SV40 3T3 cell) The rate of transport of phenylalanine by System L has been measured in SV40 3'1"3 cells at various cell densities. When the activity of the L system was determined before any cell depletion of intracellular amino acids, a density-dependent increase in transport paralleled the decrease in cell density. This regulation was lost after cell depletion but reappeared after reloading the cells with pertinent substrates of System L. The phenylalanine transport activity modulated by cell density appeared to be related to the internal level of amino acids capable of exchange upto a definite concentration, beyond which transport activity by System L did not parallel a further increase of internal substrate level. Analysis of the relationship between influx and substrate concentration suggested that two saturable components contribute to entry of phenylalanine and leucine in depleted and in reloaded cells: a low-affinity and a high-affinity component. Both kinetic parameters of the high-affinity component appeared to be modulated by the loading treatment, but only V changed markedly. Activation energies for the high-affinity component of the amino acid transport reaction were calculated from an Arrhenius plot in reloaded cells, and were found to be different for low- and high-density cultures. This result is consistent with the interpretation that cell density modulated the rates at which the amino acid-carrier complex can move within the cell membrane. Introduction In a recent paper [1] we have shown that the amino acid transport activity of the Na ÷- dependent systems A and ASC decreased markedly with the increase of cell density in 3T3 and SV40 3T3 cells, whereas the activity of the Na ÷- independent systems L and Ly ÷ remained sub- stantially unchanged when assayed under ap- propriate conditions to avoid interference from trans-effects, i.e., after extensive cell depletion. However, when the activity of the L system, as * To whom correspondence should be addressed. Abbreviation: BCH, 2-aminobicyclo(2,2,2)heptane-2-carboxylic acid. 0005-2736/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press assayed by leucine uptake in a Na ÷ -free medium, is measured before any cell depletion of intracellu- lar amino acid pool, a density-dependent increase in transport paralleled the decrease of cell density in SV40 3T3 cells [2]. The amino acid transport system L shows reactivity towards amino acids with branches or rings on the side-chain and is known to be an Na ÷-independent agency endo- wed with strong exchanging properties [3]; further- more, it has been reported that its activity is closely related to the cellular levels of those amino acids that participate in exchange [4]. Therefore the higher level of System L transport activity seen in sparse SV40 3T3 cultures before depletion could well be ascribed to a higher internal level of the amino acid pool producing a stronger trans-stimu-