thapsigargin and Leukemia--T-Cell

Effects of sphingosine on Ca2+ mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with an ED50 of around 8 microM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca2+]i, which declined to the resting level in the absence of extracellular Ca2+. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca2+]i induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca2+]i induced by OKT3 in the presence of Ca2+. OKT3 increased IP3 formation, but sphingosine did not affect the level of IP3 by itself nor did it cause IP3 formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP3 released Ca2+ from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca2+]i and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca2+]i. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response to [Ca2+]i to sphingosine. Phosphatidic acid (PA) elevated [Ca2+]i, after which it declined to a resting level even in the presence of extracellular Ca2+. In accordance with this, PA did not stimulate 45Ca2+ uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca2+]i induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca2+. Under similar conditions, pretreatment with PA affected an elevation of [Ca2+]i induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca2+]i. In permeabilized Jurkat cells, the addition of IP3 did not release Ca2+, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca2+-ATPase inhibitor, abolished the rises of [Ca2+]i induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca2+. The present results suggest that at least two kinds of intracellular Ca2+ stores exist in Jurkat cells, both of which are IP3- and PA-sensitive, and that sphingosine mobilizes Ca2+ from both stores in an IP3-independent manner. Furthermore, the IP3- but not the PA-sensitive intracellular Ca2+ store seems to reg

Topics: Amines; Calcium; Cell Line; Ceramides; Diacylglycerol Kinase; Diglycerides; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Leukemia, T-Cell; Models, Biological; Muromonab-CD3; Phosphatidic Acids; Phosphotransferases (Alcohol Group Acceptor); Psychosine; Pyrimidinones; Sphingosine; Terpenes; Thapsigargin; Thiazoles; Tumor Cells, Cultured

The expression by T lymphocytes (T cells) of more than one of the functionally distinct subtypes of prostaglandin E2 (PGE2) receptors (Rs), designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2. The cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP3, EP4, and EP2 Rs per cell with a Kd of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9). The EP3/EP1 R-selective agonist sulprostone, EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol, and EP2 R-selective agonist butaprost but not the EP1 R-selective antagonist SC-19220 competitively inhibited the binding of [3H]PGE2 to HSB.2 cells. Stimulation of increases in the intracellular concentration of cyclic AMP ([cAMP]i) by PGE2, misoprostol, and butaprost and of increases in the intracellular concentration of calcium ([Ca2+]i) by PGE2 and sulprostone demonstrated the respective involvement of EP2/EP4 Rs and EP3 Rs in transduction of biochemical signals. Matrix metalloproteinase (MMP)-9 was identified by zymography and Western blots as the principal MMP secreted by HSB.2 cells. The cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PGF2alpha, PGD2, PGI2, or butaprost, suggesting a principal dependence on EP3 Rs. That stimulation of MMP-9 secretion by PGE2 was not diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-ATPase, suggested that MMP-9 expression by HSB.2 cells is mediated by increases in [Ca2+]i attributable to release of Ca2+ from intracellular stores. The lack of effect of dibutyryl cAMP, forskolin, and SQ 22536, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells argued against any role for cAMP-dependent mechanisms linked to EP2/EP4 Rs. Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE2 induction of MMP-9 production by HSB.2 cells. Northern analysis indicated that PGE2 and sulprostone time-dependently increased expression of MMP-9 mRNA. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE2 is attributable to [Ca2+]i-dependent EP3 R-mediation of increases in message transcription.

Topics: Adenine; Alprostadil; Blotting, Northern; Bucladesine; Calcium; Cell Line; Colforsin; Collagenases; Cyclic AMP; Cycloheximide; Dactinomycin; Dinoprostone; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, T-Cell; Matrix Metalloproteinase 9; Misoprostol; Prostaglandins E, Synthetic; Receptors, Prostaglandin E; RNA, Messenger; T-Lymphocytes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

The effects of two inhibitors of the microsomal Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, were compared in hepatocytes and in a T-cell line (JURKAT). Both compounds mobilized the same intracellular Ca2+ pool, which contained the Ins(1,4,5)P3-sensitive store, in hepatocytes and in JURKAT cells. The mobilization of the internal Ca2+ store with either compound activated Mn2+ entry in JURKAT cells, but not in hepatocytes. This suggests different properties of the bivalent-cation entry pathway between these cell types.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Cell Line; Cells, Cultured; Cytosol; Fura-2; Humans; Hydroquinones; Kinetics; Leukemia, T-Cell; Liver; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Signal Transduction; Terpenes; Thapsigargin; Vasopressins