Abstract
Although protein kinase C(PKC) activation has been shown to inhibit Ca 2+ influx in T lymphocytes, the role of PKC on Ca 2+ sequestration or extrusion processes has not been full explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca 2+ (Ca2+i) extrusion and 45Ca 2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca 2+i both in the presence and absence of extracellular Ca 2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca 2+ i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca 2+i to basal levels by 17–53%. PKC stimulation of the Ca 2+i decline was not enhanced by the presence of Na +, indicating that PKC activators increase Ca 2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca +i decline in TG-treated cells antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca 2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca 2+i extrusion were further explored. PMA significantly increased the rate of Ca 2+ extrusion in TG-treated cells from 0.28 ± 0.02 to 0.35 ± 0.03 s −1 (mean ± SEM) and stimulated the initial rate of 45Ca 2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca 2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca 2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca 2+ extrusion mechanism likely to be the Ca 2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca 2+i not only by inhibiting Ca 2+ influx but also by stimulating plasma membrane Ca 2+i extrusion.
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