title: Coordination and reversibility of signals for proliferative activation and interleukin-2 mRNA production in resting human T lymphocytes by phorbol ester and calcium ionophore.
abstract: Sequential stimulation and washout procedures were employed to examine the kinetics and reversibility of pharmacologically manipulated second messenger signals mediating phenotypic changes and proliferative activation of resting human T lymphocytes. Phorbol dibutyrate (PDBu) was used to stimulate protein kinase C (Ca2+/phospholipid-dependent enzyme) while ionomycin was used to manipulate intracellular Ca2+ levels. Stimulation by PDBu alone induced phosphorylation of several endogenous substrates and altered expression of phenotypic markers, downregulating expression of CD4 and CD3 while increasing expression of CD2 and the interleukin 2 (IL-2) receptor. Stimulation with ionomycin alone caused an increase in intracellular Ca2+ levels but did not induce proliferation or cause major changes in the expression of phenotypic markers (CD2, CD3, CD4, CD8, IL-2, and transferrin receptors). Analysis of endogenous PDBu stimulated phosphosubstrates indicated that some substrates (pp92, pp82, pp55) underwent dephosphorylation, returning to base-line levels following PDBu removal while others (pp61, pp65) showed only partial dephosphorylation, while one (pp28) remained phosphorylated. Washing ionomycin-stimulated cells resulted in an approximately 75% reduction of intracellular Ca2+. Ionomycin exposure did not alter the affinity (KD = 22.3 +/- 7.4 nM) or number of receptors (53,497 +/- 8,291 receptors/cell) for [3H]PDBu. These data suggest that signals induced by PDBu or ionomycin are reversible following removal of the stimulating agents with respect to proliferative activation of T lymphocytes. Furthermore, a transcriptional mechanism regulating the production of IL-2 mRNA requires simultaneous activation of protein kinase C and elevation of intracellular Ca2+.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
authors.forename: C W
authors.initials: CW
authors.lastname: McCrady
authors.forename: C M
authors.initials: CM
authors.lastname: Ely
authors.forename: E
authors.initials: E
authors.lastname: Westin
authors.forename: R A
authors.initials: RA
authors.lastname: Carchman
mesh_terms: D015152:Blotting, Northern; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D015759:Ionomycin; D008213:Lymphocyte Activation / Q000187:drug effects*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism
chemical_list: D004987:Ethers; D007376:Interleukin-2; D010750:Phosphoproteins; D012333:RNA, Messenger; D015240:Phorbol 12,13-Dibutyrate; D015759:Ionomycin; D002118:Calcium