Functional expression of Na, K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and alpha-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20-24h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na, K-ATPase pump during blast transformation of human lymphocytes.

The transition of phytohemagglutinin-activated human lymphocytes from resting state to proliferation is accompanied by a long-term increase in ouabain-sensitive Rh(K) influx which is closely related to a cyclosporin A-sensitive step of GO/ GI/S progression. At least two distinct phases of the up-regulation of cation pump has been revealed: the initial stage (5-20 h) which is cycloheximide-inhibitahle and actinomycin D (aamanitin)-unaffected, and the later stage (after 20 h) which is cycloheximide-and actinomycin D (a-amanitin)-inhihitable. Thus, the enhanced Na,K-ATPase pump during the cell progression from quiescence to proliferation is controlled both at translational and transcriptional levels.

Increase in Na+,K+‐ATPase mRNAs was detected in activated lymphocytes by the RT‐PCR method. α1 subunit mRNA gradualy increased with time and by 36 h was 2.4 times higher than at the start. Increase in the β1 mRNA was transient reaching a maximum in the 8 h probe and declining to the initial level in the 24 and 36 h probes. The elevation of Na+,K−‐ATPase mRNAs does not underlie a cycloheximide‐inhibited increase in cation pumping peculiar to the prereplicative period as can be judged from the fact that Act D fails to eliminate PHA‐induced enhancement of pump fluxes.

An immunosuppressant cyclosporin A (CsA) inhibits T-cell proliferation by blocking the nuclear factor of activated Tcells (NFAT) required for expression of the interleukin-2 (IL-2) gene. This work has demonstrated for the first time that in human blood lymphocytes (HBLs) activated by phytohemagglutinin (PHA), CsA at anti-proliferative doses inhibits the late sustained increase in ouabain-sensitive Rb(K) influxes, which accompanies the growth phase of G 0 /G 1 /S transition. CsA affects neither the initial, transient activation of the pump in response to PHA nor the ouabain-resistant ion fluxes during cell cycle progression. When the HBLs were rendered competent to proliferate by phorbol 12,13-dibutyrate ester and ionomycin in the presence of CsA, the exogenous IL-2 did not bypass the initial inhibitory effect of CsA on the long-term pump enhancement. When applied after the competence induction, CsA produced no effect on the sustained increase in ouabain-sensitive Rb influxes during the IL-2-induced progression phase. These results indicate that in activated HBLs, (1) IL-2 is involved in functional expression of the Na/K pump during cell transition from quiescence to proliferation, (2) the cell cycle-associated upregulation of the pump is related to a CsA-sensitive signalling pathway.z 1999 Federation of European Biochemical Societies.