thromboxane-b2 and 1-2-dioctanoylglycerol

We studied the regulation of thromboxane (TX) synthesis in promyelocytic leukemia cells during macrophage differentiation. Cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) showed rates of TXB2 synthesis from exogenous arachidonic acid that exceeded that of control cells by a factor of up to 81. Cells treated with sn-1,2-dioctanoylglycerol (diC8) showed similarly high TXB2 synthesis rates when diC8 was added concomitantly with a subthreshold concentration of TPA or when given in multiple doses. These activities depended on de novo synthesis of prostaglandin H (PGH) synthase because: microsomal PGH synthase activity showed large increases in Vmax values, and mass measurements of PGH synthase revealed the presence of PGH synthase in differentiating cells whereas the enzyme was undetectable in control cells. These results indicate that macrophage differentiation is associated with stimulation of TXB2 synthesis that requires both activation of protein kinase C and de novo synthesis of PGH synthase.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Differentiation; Cell Line; Diglycerides; Glycerides; Humans; Kinetics; Leukemia, Myeloid; Macrophages; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2; Trifluoperazine

1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro) and 1-oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets. 2. Pre-treatment (10-300 s) with Oco2Gro (15-60 microM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62-125 microM) had no effect on thrombin-induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3. Collagen (20 micrograms/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10-300 s) with Oco2Gro (15-60 microM), PMA (16 nM) or OleAcGro (62 microM) before collagen addition or addition of these agents 30-60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2--2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations (less than 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5-15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 microM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with less than 2-min incubations) at sub-maximal agonist concentrations. 5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while Oco2Gro (60 microM) and PMA (16 nM) caused a 4--5-fold increase

Topics: Blood Platelets; Blood Proteins; Calcium; Collagen; Diglycerides; Ethers; Glycerides; Humans; Ionomycin; Kinetics; Male; Molecular Weight; Phosphorylation; Tetradecanoylphorbol Acetate; Thrombin; Thromboxane B2