thapsigargin and thymeleatoxin

L-type Ca(2+) channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba(2+) influx in a depolarizing medium. In the absence of extracellular Ca(2+), activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba(2+) influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca(2+)-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in a Ca(2+)-free medium from 30 to approximately 80 nM. This [Ca(2+)](i) increase declined slowly, giving rise to a modest elevation of [Ca(2+)](i) that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca(2+)-free medium. However, when the Ca(2+) ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca(2+) channel activity, suggesting that a lower amplitude and prolonged release of Ca(2+) stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 microM) and ascomycin (5 microM), inhibitors of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1mM CaCl(2) to the Tg-treated cells increased [Ca(2+)](i) to approximately 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca(2+)](i) increase elicited by passive depletion of internal Ca(2+) stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca(2+)](i) via store-dependent Ca(2+) entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca(2+) channels in a myocyte model involves a balance between the differential Ca(2+) sensitivities and opposing actions of PKC and calcineurin.

Topics: Animals; Calcineurin; Calcium; Calcium Channels, L-Type; Calcium-Transporting ATPases; Cell Line; Cyclosporine; Enzyme Inhibitors; Ion Channel Gating; Ion Transport; Myoblasts, Skeletal; Phorbol Esters; Protein Kinase C; Rats; Sarcoplasmic Reticulum; Tacrolimus; Tetradecanoylphorbol Acetate; Thapsigargin

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.

Topics: Alkaloids; Calcium; Calcium-Transporting ATPases; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Ionomycin; Ionophores; Lanthanum; Leukemia; Naphthalenes; Phorbol Esters; Protein Kinase C; Sodium; Staurosporine; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

In a mammary carcinoma cell line, the glucocorticoid induction of a stably integrated mouse mammary tumor virus-luciferase reporter gene is potentiated up to ten-fold by treatment of the cells with phorbol ester tumor promoters. This potentiation is stereospecific and is observed with a structurally unrelated tumor promoter that also activates protein kinase C. Another tumor promoter, thapsigargin, that acts via mobilization of intracellular calcium stores, fails to potentiate the hormone response. This potentiation contrasts to the inhibition or lack of effect observed in fibroblasts. Cell specific interactions of different signal transduction pathways with steroid response pathways may play an important role in determining the tissue specificity of steroid hormone action.

Topics: Animals; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Activation; Indoles; Lactams; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Phorbol Esters; Promoter Regions, Genetic; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured