Alpha-aminoisobutyric acid transport in human leukemic lymphocytes: in vitro characteristics and inhibition by cortisol and cycloheximide

Abstract

We have studied the transport of alpha-aminoisobutyric acid (AIB)-3-¹⁴C and its response to cortisol and cycloheximide in vitro in blood lymphocytes from untreated patients with chronic lymphocytic leukemia. The accumulation of AIB-3-¹⁴C increased in a linear fashion for 60 min, and reached an apparent steady state in 120 min. The initial rate of AIB accumulation (V_o) varied from 1.1 to 10.2 μmoles/kg cell H₂O per min in cells from 16 different patients; however, V_o was reproducible in cells from five of six patients which were studied repeatedly over 1-9 months, and correlated positively with the lymphocyte count (r = 0.51, P = < 0.01). Virtually total inhibition of protein synthesis with cycloheximide was found to decrease the accumulation of AIB in cells from four patients which had high rates of AIB transport, but had no effect on transport in cells from four patients which accumulated AIB more slowly. These results indicate that active transport depends, in part, upon the presence of labile protein with a turnover rate which varies among different cell populations. Treatment with 10 μM cortisol for 240 min in vitro reduced the initial rate of AIB-3-¹⁴C accumulation (V_o) by 43.4±4.1% (SE) (range, 9-66%) in cells from 16 patients. The degree of inhibition did not vary appreciably over a 9 month period in four of five patients. The effect of cortisol was proportional to its starting concentration, and developed at low concentrations (0.1-1.0 μM). Cortisol appears to decrease AIB accumulation by inhibiting active uptake, since it neither enhanced the exodus of AIB, nor inhibited apparently nonsaturable transport. Inhibition was noncompetitive in type, suggesting that cortisol decreases the total capacity of the active transport mechanism. Cortisol inhibited AIB transport indirectly by a process which involved de novo protein synthesis, since inhibition (a) appeared only after 60 min of treatment, (b) was present in treated cells which were subsequently incubated for 60 min in cortisol-free medium, and (c) failed to develop during simultaneous blockade of protein synthesis with cycloheximide, even when cycloheximide itself did not decrease AIB transport.