title: Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells.
abstract: 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+ (Ca2+i) extrusion and 45Ca2+ 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 Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i 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 Ca2+i 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 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i 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 Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.
authors: Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, USA.
authors: M
authors: M
authors: Balasubramanyam
authors: J P
authors: JP
authors: Gardner
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D007938:Leukemia; D009281:Naphthalenes / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D019311:Staurosporine; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
chemical_list: D000470:Alkaloids; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D009281:Naphthalenes; D010703:Phorbol Esters; D013729:Terpenes; D015759:Ionomycin; D019284:Thapsigargin; D007811:Lanthanum; C080287:thymeleatoxin; D012964:Sodium; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2