4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Leukemia--Lymphoid

The transport system responsible for glutamine, alanine and glutamate in MOLT4 human T4 leukemia cell line were characterized. Kinetic studies of sodium-dependent glutamine and alanine transport exhibited a single saturable high-affinity carrier with a Michaelis constant of 152 +/- 26 microm and 203 +/- 36 microm and a maximal transport velocity of 960 +/- 165 and 1096 +/- 208 nmol/10(9)cells/min, respectively. Glutamate uptake was less than one-tenth of glutamine and alanine, and linearly increased with glutamate concentration which was mediated by diffusion. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), known as anion channel blockers, inhibited the sodium-dependent glutamine and alanine transport by 40% at 10 microm. Cellular contents of these amino acids in MOLT4 cells revealed glutamate to be the highest among them despite low glutamate influx. A glutamine metabolism study using whole cells indicated this high conversion rate from glutamine to glutamate, but no conversion to another amino acid. Based on these results, the high glutamate concentration in MOLT4 was speculated to be synthesized from transported glutamine by active glutaminase.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Alanine; Amino Acid Transport System ASC; Biological Transport; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Glutaminase; Glutamine; Humans; Kinetics; Leukemia, Lymphoid

Outwardly rectifying Cl- channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1-6 min at +80 mV, 20 degrees C). The single-channel current at +80 mV was 4.5 +/- 0.3 pA and at -80 mV, it was 1.0 +/- 0.4 pA. After activation, the probability of being open (Po) for the lymphocyte channel was voltage independent. Activation of the Cl- channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20 degrees C exhibited Cl- channel activity. In contrast, 49% of recordings made at 30 degrees C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30 degrees C than at 20 degrees C. There was no marked change in the single-channel conductance at 30 degrees C. Open-channel conductance was blocked by 200 microM indanyloxyacetic acid (IAA) or 1 mM SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl- channels thought to be involved in fluid secretion.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Chloride Channels; Chlorides; Electrophysiology; Humans; Indans; Ion Channels; Leukemia, Lymphoid; Membrane Proteins; Regression Analysis; T-Lymphocytes; Temperature; Tumor Cells, Cultured