INHIBITION OF CALCIUM SIGNALING IN MURINE SPLENOCYTES BY POLYAMINES -DIFFERENTIAL-EFFECTS ON CD4 AND CD8 T-CELLS

Transmembrane Ca2+ influx is recognized as a universal second messenge r that transduces T-cell activation signals to cytoplasm and nucleus, thereby stimulating transcription and cell division. To examine the ro le of endogenous factors that regulate mitogenic Ca2+ signalling of T- cells, we measured the concanavalin (Con) A-induced increase in cytopl asmic free calcium ([Ca2+]i) in spleen cells of BALB/c mice, using flo w cytometry with an indicator dye, Indo-I acetoxymethyl ester (Indo-1/ AM). Con A is a polyclonal activator of T-cells. Unstimulated splenocy tes had a [Ca2+]i of 100 nM. [Ca2+]i increased with Con A in a dose-de pendent manner up to a concentration of 50 mug/ml. In the presence of 50 mug/ml Con A, [Ca2+]i was 350 nM. Natural polyamines (putrescine, s permidine and spermine) inhibited Con-A-induced Ca2+ influx in a dose- dependent manner. Putrescine was the most effective polyamine in dense nsitizing the Ca2+ signal, and decreased [Ca2+]i from 350 nM in the ab sence of putrescine to 250 nM in the presence of 100 muM putrescine. T his effect was not mimicked by structurally related homologues or inor ganic cations, suggesting a specific structural effect of the polyamin e. H.p.l.c. analysis showed that polyamines were internalized during i ncubation of cells in vitro. In experiments using monoclonal anti-CD4 and anti-CD8 antibodies, we found a differential effect of putrescine on Ca2+ influx in CD4 and CD8 subpopulations of T cells. For CD4+ cell s, [Ca2+]i decreased from 625 nM to 420 nM in the presence of 500 muM putrescine, whereas [Ca2+]i was not affected by putrescine in CD8+ cel ls. These data suggest that natural polyamines have cell-specific effe cts on mitogen-stimulated Ca2+-influx in T-cell subsets.