Membrane transport by murine lymphocytes. II. The appearance of thymidine transport in cells from concanavalin A-stimulated mice.

We have investigated membrane transport of thymidine and adenosine by bulk murine nonadherent spleen cells from animals treated with the lectin concanavalin A. Separation of cells from radioactive medium was achieved by means of the oil microfuge technique which permits incubation periods as short as 4 sec. We were able to distinguish between the membrane-linked function of transport and uptake which includes subsequent accumulation and metabolism of the transport solute. Previously we reported that cells derived from untreated mice could not be shown to translocate thymidine in a carrier-mediated fashion. In contrast, cells from concanavalin A-treated mice showed two membrane transport systems for thymidine, a high affinity system (Km = 160 micronM) and one with low affinity (Km = 4 mM). The transport of thymidine conformed to the usual criteria for membrane carrier-linked function: increased uptake with time, substrate saturability and chemical specificity. In contrast to thymidine uptake, the transport of adenosine by cells from lectin-treated mice was similar to that shown by cells from untreated animals. Thus a specific membrane-linked activity is altered differentially in the course of mitogen-effected lymphocyte stimulation. We have found that determination of DNA synthesis by the standard method gave values which were as much as 700% too high, since the counts obtained by direct precipitation with TCA of cells incubated with radio-labeled thymidine exceed the cell-associated radiolabel obtained by the rapid sampling technique. With lectin-stimulated cells, the discrepancy observed was up to three times greater. Hence the validity of the standard assay for blastogenesis must be viewed with caution.