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− Previously, we identified albumin as an inhibitory factor in serum for cell adhesion of T cells such ashuman Jurkat T and primary cultured human T cells. In the present study, we found that other hematopoieticcell lines including U-937 human monocytes, THP-1 human monocytes, K-562 promyelocytic leukemia cells,and HL-60 human leukemia cells, also adhere to tissue culture flasks when serum is withdrawn, and albuminexerts an inhibitory effect on cell adhesion by those cells, implying that this inhibition is a common phenomenonin hematopoietic cells. Furthermore, we found that cell adhesion is inhibited by antioxidants such as (-)-epigal-locatechin-3-gallate (EGCG), morin, and a-tocopherol. Our results suggest that albumin may inhibit basal celladhesion of hematopoietic cells and that the oxidative balance in the plasma may be important for cell adhe-sion of hematopoietic cells in vivo . Keywords : Monocytes, Leukocytes, Albumin, Adhesion, Hematopoietic cells, Oxidative signaling

Aim: To study the effects of dibenzocyclooctadiene lignans isolated from Schisandra chinensis , such as wuweizisu C, gomisin N, gomisin A, and schisandrin, on the membrane potential in C6 glioma cells. Methods: The membrane potential was estimated by measuring the fluorescence change in DiBAC-loaded glioma cells. Results: Wuweizisu C decreased the membrane potential in a concentration-dependent manner. Gomisin N and gomisin A, however, showed differential modulation and no change was induced by schisandrin or dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybipheny 1-2,2′-dicarboxylate, a synthetic drug derived from dibenzocyclooctadiene lignans. We found no involvement of Gi/o proteins, phospholipase C, and extracellular Na + on the wuweizisu C-induced decrease of the membrane potential. Wuweizisu C by itself did not change the intracellular Ca 2+ [Ca 2+ ]i concentration, but decreased the ATP-indu-ced Ca 2+ increase in C6 glioma cells. The 4 lignans at all concentrations used in this study did not induce any effect on cell viability. Furthermore, we found a similar decrease of the membrane potential by wuweizisu C in PC12 neuronal cells. Conclusion: Our results suggest that the decrease in the membrane potential and the modulation of [Ca 2+ ]i concentration by wuweizisu C could be important action mechanisms of wuweizisu C.

Previously, we studied N,N-dimethyl- d -erythro-sphingosine (DMS)-induced cell death and signaling in U937 human monocytes; we found that DMS-induced sphingosine kinase- and PKC-independent apoptosis. In the present study, we studied apoptotic responses by three N-methyl derivatives of sphingosine: N-monomethyl- d -erythro-sphingosine (MMS), N,N,N-trimethyl- d -erythro-sphingosine (TMS), and d -erythro-sphingosine (SPH). The potency order in the apoptotic response was DMS ≥ MMS > TMS > SPH. We compared cellular responses to the derivatives in terms of activities of MAPK signaling molecules, mitochondrial membrane potential (ΔΨm), and reactive oxygen species (ROS) generation. Our results suggest that the degree of N-methylation affects the apoptosis-inducing capacity and other related responses including MAPK modulation, ΔΨm, and ROS generation. Dimethylation and monomethylation on the C2 amine of sphingosine enhance the apoptotic response; however, trimethylation induces differential modulation of signaling molecules and less cytotoxicity. Our investigation will be useful for understanding the actions of sphingolipids in apoptosis and for developing chemotherapeutics based on DMS structure.

We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular concentration () in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased in both HCT116 and HT29 cell lines. Increases of by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for receptors, an involvement of endogenous LPA receptors in the responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase in human colon cancer cells, probably via endogenous LPA receptors, G proteins and receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

N,N-Dimethyl-D-ribo-phytosphingosine (DMPH) is an N-methyl derivative of sphingosine. In the present paper, we studied effects of DMPH on intracellular Ca concentration, pH, glutamate uptake, and cell viability in human 1321N1 astrocytes. DMPH increased intracellular Ca concentration and cytosolic pH significantly in a dose-dependent manner. DMPH also inhibited glutamate uptake by 1321N1 astrocytes. Finally, treatment of cells with DMPH for 24 h reduced viability of cells largely and concentration-dependently. In summary, DMPH increased intracellular Ca concentration and pH, inhibited glutamate uptake and evoked cytotoxicity in 1321N1 astrocytes. Our observations with DMPH in the 1321N1 astrocytes would enhance understanding of DMPH actions in the brain.

In the present study, we studied N , N -dimethyl- d - erythro -sphingosine (DMS)-induced cell death and its signaling mechanism in U937 human monocytes. We found that DMS induced cell death in a concentration-dependent manner, while sphingosine 1-phoshate did not. DMS also induced DNA fragmentation, nuclear disruption, and cytochrome c release from mitochondria in a concentration- and time-dependent manner, implying apoptotic cell death. DMS was found to increase mitochondrial membrane potential (MMP) immediately after addition of DMS and to decrease MMP at 2 h after addition. However, sphingosine kinase inhibitors and PKC inhibitors did not induce cell death in U937 cells, a result that appears to exclude sphingosine kinase and PKC as target molecules of DMS in the cell death induction process. Furthermore, DMS modulated the activity of several signaling molecules. DMS induced activation of JNK and p38 MAP kinase, while it decreased the activity of ERK and Akt kinase. However, decrease of MMP, inhibition of JNK, p38 MAP kinase, ERK, or Akt with specific inhibitors could not mimic the DMS-induced cell death, implying multiple concerted processes are involved in DMS-induced cell death. In summary, DMS induced apoptotic cell death via modulation of MMP, JNK, p38 MAP kinase, ERK, and Akt kinase, but not through inhibition of sphingosine kinase or PKC in U937 cells.

Treatment with isoprenaline led to a change in the cell morphology of rat C6 glioma cells. This morphological change was reverted by the addition of sphingosine 1-phosphate (S1P). Using this morphological change as a response marker we determined that DS-SG-44 ((2S,3R)-2-amino-3-hydroxy-4-(4-octylphenyl)butyl phosphoric acid) was an agonist of S1P receptors. The DS-SG-44-induced morphological reversion was not observed with such structurally related molecules as DS-SG-45 ((2S,3R)-2-amino-3-hydroxy-4-(3-octylphenyl)butyl phosphoric acid) and DS-SG-12 ((2S,3R)-2-amino-4-(4-octylphenyl)butane-1,3-diol). The S1P- and DS-SG-44-induced shape changes were nseither reproduced with the S1P1/S1P3 receptor agonist VPC24191 nor inhibited by the S1P1/S1P3 receptor antagonist, VPC23019. Transfection with small interfering RNA (siRNA) for the S1P2 receptor greatly inhibited the DS-SG-44-induced shape change, and in part an S1P-induced response. In the presence of VPC23019, siRNA transfection for the S1P2 receptor almost completely blocked the S1P- and DS-SG-44-induced shape changes. Our results suggested that DS-SG-44, a newly-synthesized S1P analogue, acted as an S1P receptor agonist and that the S1P-induced shape change in rat C6 glioma cells was mediated mainly through the S1P2 receptor, and cooperatively through the S1P1/S1P3 receptors.

Phosphatidic acid (PA) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in C6 rat glioma and L2071 mouse fibroblast cells. Dioleoyl PA (PA, 18:1) was the most efficacious, followed by dipalmitoyl PA (16:0 PA) and dimyristoyl PA (14:0 PA). Lysophosphatidic acid (LPA) also increased the [Ca(2+)](i) in the both cells. PA desensitized LPA-induced Ca(2+) response completely in C6 cells, but partly in L2071 cells. Treatment of pertussis toxin (PTX), a specific inhibitor of G(i/o)-type G proteins, completely ameliorated LPA- and PA-induced Ca(2+) response in C6 cells. However, in L2071 cells, PTX inhibited PA-induced Ca(2+) increase by 80% and LPA-induced one by 20%. Ki16425, a specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited both LPA- and PA-induced Ca(2+) responses in C6 cells. On the other hand, in L2071 cells, Ki16425 completely inhibited PA-induced Ca(2+) response, but partly LPA-induced one. VPC32183, another specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited LPA- and PA-induced Ca(2+) responses in both C6 and L2071 cells. Therefore, PA and LPA appear to increase [Ca(2+)](i) through Ki16425/VPC32183-sensitive LPA receptor coupled to PTX-sensitive G proteins in C6 cells. In L2071 cells, however, LPA increases [Ca(2+)](i) through Ki16425-insensitive LPA receptor coupled to PTX-insensitive G proteins and Ki16425-sensitive LPA receptor coupled to PTX-sensitive G protein, whereas PA utilized only the latter pathway. Our results suggest that PA acts as a partial agonist on endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-sensitive G protein, but not on LPA receptors, which are not sensitive to Ki16425 and coupled to PTX-insensitive G protein.