Metabolic rate of membrane-permeant diacylglycerol and its relation to human resting T-lymphocyte activation.

Abstract

DL-1,2-Dioctanoylglycerol (1,2-DiC8) added to human peripheral resting T lymphocytes was rapidly metabolized to produce octanoic acid and further to small molecules, probably by the action of diacylglycerol lipase and/or nonspecific esterase. Only a small portion was converted to the corresponding phosphatidic acid or was isomerized to 1,3-DiC8 before being metabolized. The uptake of 1,2-DiC8 by the cell was apparently fast, and the rate of disappearance of 1,2-DiCs was dependent on the cell densities; at a higher density of T lymphocytes 1,2-DiC8 was removed quickly, whereas at a lower cell density 1,2-DiC8 remained for a longer period oftime. With a fixed amount of 1,2-DiC8 added, the extent of interleukin 2 receptor a-subunit (IL-2Ra) expression was inversely related to the cell density and proportional to the duration of exposure of the cells to 1,2-DiCs. Repeated doses of 1,2-DiC8 potentiated IL-2Ra expression. In contrast, a single dose of phorbol 12-myristate 13-acetate caused T-lymphocyte activation to similr extents irrespective ofthe cell density, probably because the phorbol ester was not metabolized and remained in membranes. The available evidence supports a proposal made in a previous paper and indicates that the sustaind activation of protein kinase C for at least the first 3-4 hr is essential for the activation of resting T lymphocytes.Protein kinase C (PKC) is activated by diacylglycerol (DAG), which is produced physiologically as a result of the signalinduced hydrolysis of inositolphospholipids (1). Tumorpromoting phorbol esters and membrane-permeant DAGs such as 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol (DiC8) are often used to activate this enzyme within the intact cell (2, 3). Several discrepancies have been reported, however, between the responses induced by phorbol esters and those induced by the membrane-permeant DAGs (4-6). One possible explanation for the discrepancies is that the membrane-permeant DAG is metabolized quickly and activates PKC only transiently (2), whereas phorbol ester is hardly metabolized and thus activates the enzyme for a long time (7). Although the receptor-mediated hydrolysis of inositolphospholipids was once thought to be the sole mechanism leading to the activation of PKC (8), it is becoming plausible that the hydrolysis of inositolphospholipids is normally transient and insufficient to cause the sustained activation of PKC that appears to be needed for long-term responses such as cell proliferation and differentiation (9-11). For sustained activation of this enzyme, several mechanisms have been proposed, including phosphatidylcholine hydrolysis at a relatively later phase in cellular responses (12,13).In resting T lymphocytes, the synergistic action of phorbol ester and calcium ionophore has been reported to induce interleukin 2 (IL-2) secretion and IL-2 receptor a-subunit (IL-2Ra) gene expression, eventually leading to cell proliferation (for review, see ref. 14). A previous report from this laboratory (9) has shown that repeated doses of a...

Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T‐lymphocytes

“…However, the role of DGKc~ in phosphoinositide turnover and in other biochemical events preceding the expression of IL-2 receptors remains unknown. For example, exogenous short-chain DG repeatedly administered to human T-lymphocytes was phosphorylated by cellular DGK only to a negligible extent [19]. Van der Bend et al.…”

“…However, the role of DGKc~ in phosphoinositide turnover and in other biochemical events preceding the expression of IL-2 receptors remains unknown. For example, exogenous short-chain DG repeatedly administered to human T-lymphocytes was phosphorylated by cellular DGK only to a negligible extent [19]. Van der Bend et al [20] proposed a topological sequestration of cellular DGK molecules based on the finding that DG artificially generated by treating Jurkat and other cells with bacterial phospholipase C could not serve as substrate for the cellular DGK.…”

Role of lysophosphatidylcholine in T-lymphocyte activation: involvement of phospholipase A2 in signal transduction through protein kinase C.

“…On the other hand, a single dose of a membrane-permeant diacylglycerol (DAG) is normally insufficient to induce cell activation due to its rapid metabolism within the cell, and it is known that sustained activation of protein kinase C (PKC) by a large dose or repeated doses of a membrane-permeant DAG is essential to induce activation of T lymphocytes (2,3). The formation of DAG from receptor-mediated hydrolysis of inositol phospholipids is, however, normally transient, and recent evidence strongly suggests that, at relatively later phases in cellular responses, DAG is produced from signalinduced breakdown of phosphatidylcholine (PtdCho), which is initiated presumably by the activation of phospholipase D (for reviews, see refs.…”

Ionotrophic 5-hydroxytryptamine type 3 receptor activates the protein kinase C-dependent phospholipase D pathway in human T-cells

“…We also activated T-cells directly by the addition of DiC ) and ionomycin in order to by-pass the implication of the T-cell receptor in T-cell activation and to elucidate the role of PKC in 2Me-5-HT-induced T-cell proliferation. DiC ) and ionomycin have been used by several investigators to activate directly PKC and T-cell proliferation [20,21]. Interestingly, the two serotonergic agents failed to potentiate T-cell stimulation initiated by DiC ) and ionomycin.…”