PROTEIN-KINASE-C MODULATES CYTOSOLIC-FREE CALCIUM BY STIMULATING CALCIUM-PUMP ACTIVITY IN JURKAT T-CELLS

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca-i(2+)) extrusion and Ca-45(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-i(2+) both in the presence and absence of extracellular Ca2f, whereas inactive phorbol esters had no effect. PMC activators added at the peak of a Ca-i(2+) transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca-i(2+) to basal levels by 17-53%, PKC stimulation of the Ca-i(2+) decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2f pump activity rather than a Na+/Ca2+ exchange mechanism, As CD3 receptor activation enhanced the Ca-i(2+) decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca-i(2+) extrusion were further explored, PMA significantly increased the rate of Ca2+ 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 Ca-45(2+) efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca-i(2+) decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump, In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response, We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump, In lymphocytes, receptor/PLC-linked PKC activation modulates Ca-i(2+) not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca-i(2+) extrusion.