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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K-562 promyelocytic leukemia cells,and HL-60 human leukemia cells, also adhere to tissue culture flasks when serum is withdrawn, and albuminexerts an inhibitory effect on cell adhesion by those cells, implying that this inhibition is a common phenomenonin hematopoietic cells.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.

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.

Previously, we reported on the distinct effects of bioactive lysophospholipids, including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and sphingosylphosphorylcholine (SPC), on membrane potentials in rat C6 glioma cells. In the present report we have tested lysophosphatidylserine (LPS), another bioactive lysophospholipid, on membrane potentials in the same cell line. Membrane potentials were estimated by measuring the fluorescence changes of DiBAC-loaded glioma cells. LPS largely increased membrane potentials in a concentration-dependent manner. The LPS-induced membrane potential increases were not affected by treatment with pertussis toxin, implying no involvement of Gi/o proteins. In contrast to other lysophospholipids, the LPS-induced membrane potential increase was not diminished by a Na(+)-free media but was enhanced by suramin. Furthermore, this change was blunted by EIPA, an inhibitor of Na(+)/H(+) exchanger, but not by SITS, a specific inhibitor of bicarbonate transporter. Our observations suggest that LPS acts on membrane potentials in a unique manner in the C6 glioma cells, although the precise action mechanism requires additional investigation.

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.

Ginseng has an anti-cancer effect in several cancer models. As a mechanism study of ginsenoside-induced growth inhibition in cancer cells, we measured change of membrane potential in prostate cancer and glioma cells by ginsenosides, active constituents of ginseng. Membrane potential was estimated by measuring fluorescence change of DiBAC-loaded cells. Among 11 ginsenosides tested, ginsenosides Rb₂, Rg₃, and Rh₂ increased significantly and robustly the membrane potential in a concentration-dependent manner in prostate cancer and glioma cells. Ginsenosides Rc, Ro, and Rb₁ slightly increased membrane potential. The ginsenoside-induced membrane potential increase was not affected by treatment with pertussis toxin or U73122. The ginsenoside-induced membrane potential increase was not diminished in Na?-free or HCO₃?-free media. Furthermore, the ginsenoside-induced increase of membrane potential was not changed by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), SITS (4-acetoamido-4’-isothiocyanostilbene-2,2’-disulfonic acid), and omeprazole. In summary, ginsenosides Rb₂, Rg₃, and Rh₂ increased membrane potential in prostate cancer and glioma cells in a GPCR-independent and Na? independent manner.

Dodecylbenzene sulfonate (DBS) is a component of linear alkylbenzene sulfonate (LAS), an anionic surfactant, mainly used in household detergents. Due to the large quantity of DBS in use, there is concern over adverse environmental effects. This work examined the toxicokinetics and toxicity of the 2-phenyl isomer of dodecylbenzene sulfonate in 4-d, 10-d, and partial life-cycle tests on the midge, Chironomus riparius, exposed to aqueous solutions. Toxicokinetics were determined in 10-d uptake and 5-d elimination tests. The toxicokinetics were based on parent compound concentration in water and yielded an uptake coefficient (k u ) of 17.5 (14.87-20.20) ml/g/h, an elimination rate constant (k c ) of 0.073 (0.062-0.085) per h, a bioconcentration factor (BCF) of 56 to 240, and a half-life (t 1/2 ) of 9.5 (8.0-11.0) h. Biotransformation measurements did not reveal evidence for DBS metabolism. Thus, body residues, determined in the toxicity study, represent parent compound. In toxicity tests, 4- and 10-d LR50s (the body residue required to cause 50% mortality) in live midges were 0.72 (0.65-0.79) and 0.18 (0.08-0.42) mmol/kg, respectively. Thirty-day LR50s were 0.18 (0.09-1.64) and 0.21 (0.15-0.39) mmol/kg in duplicate studies. Of the sublethal endpoints, only developmental time increase was significant, with the lowest-observed-effect residues of 0.085 (0.067-0.105) and 0.100 (0.087-0.114) mmol/kg for male and female midges, respectively. Deformities in surviving larvae were also observed as chronic responses for body residues exceeding the 30-d LR50. The body residues required for mortality suggest that DBS acts like a polar narcotic in the midge.

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.The membrane potential was estimated by measuring the fluorescence change in DiBAC-loaded glioma cells.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 dioxybiphenyl-2,2'-dicarboxylate, a synthetic drug derived from dibenzocyclooctadiene lignans. We found no involvement of G(i/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-induced 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.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.

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.

Lysophosphatidylserine (LPS) can be generated following phosphatidylserine-specific phospholipase A2 activation. The effects of LPS on cellular activities and the identities of its target molecules, however, have not been fully elucidated. In this study, we observed that LPS stimulated intracellular calcium increased in mouse bone marrow-derived mast cells (BMMC), and rat C6 glioma and human HCT116 colon cancer cells and compared the LPS-induced Ca2+ increases with the response by lysophosphatidic acid (LPA), a structurally related bioactive lysolipid. In order to test involvement of signaling molecules in the LPS-induced Ca2+ signaling, we used pertussis toxin (PTX), U73122, and 2-APB, which are specific inhibitors for G proteins, phospholipase C (PLC), and IP3 receptors, respectively. The increases due to LPS and LPA were inhibited by PTX, U-73122 and 2-APB, suggesting that both lipids stimulate calcium signaling via G proteins (Gi/o types), PLC activation, and subsequent IP3 production, although the sensitivity to pharmacological inhibitors varied from complete inhibition to partial inhibition depending on cell type and lysolipid. Furthermore, we observed that Ki16425 completely inhibited an LPS-induced Ca2+ response in three cell types, but that the effect of VPC32183 varied from complete inhibition in BMMC and C6 glioma cells to partial inhibition in HCT116 cells. Therefore, we conclude that LPS increases [Ca2+]i through Ki16425/VPC32183-sensitive G protein-coupled receptors (GPCR), G protein, PLC, and IP3 in mouse BMMC, rat C6, and human HCT116 cells.

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.

Due to the long time course required to achieve steady state with highly lipophilic contaminants such as PCBs (polychlorinated biphenyls), data derived from short-term toxicity tests may lead to an erroneous interpretation of hazard. In addition, PCBs bioaccumulated over time can cause sublethal impairments in organisms at concentrations much lower than required for mortality. Here, the body residues of 1,1-dichloro-2,2-bis-p-chlorophenyl ethane (DDE) and select PCB congeners associated with a spectrum of chronic effects in the midge, Chironomus riparius, were evaluated. The route of exposure was ingestion of the PCB-contaminated alga, Chlorella vulgarus, and trout chow loaded with the selected test compound. Two separate exposures of midges were performed. In the first experiment, midges were exposed from the second instar to the pupal stage. In the second exposure, midges were exposed from the second instar to the adult stage. A variety of sublethal endpoints was monitored, including developmental time within a stadium, body weight, and fecundity for the female adult. The dose was assessed as the whole body residue concentration of the contaminant. Overall, the midge concentration increased with increasing exposure concentration in algae and trout chow. Body weight at the end of each stadium was the assessment parameter that was least significantly affected among the test endpoints monitored. In contrast, a significant increase in development time was the endpoint that was most frequently observed in response to contaminant exposure. Reduction in fecundity was found only for DDE-exposed midges. These data, in which chronic endpoints are related to body residues, suggest that body residues will be useful in defining sublethal hazards of DDE and some PCB congeners.

Dodecylbenzene sulfonate (DBS) is a component of linear alkylbenzene sulfonate (LAS), an anionic surfactant, mainly used in household detergents. Due to the large quantity of DBS in use, there is concern over adverse environmental effects. This work examined the toxicokinetics and toxicity of the 2-phenyl isomer of dodecylbenzene sulfonate in 4-d, 10-d, and partial life-cycle tests on the midge, Chironomus riparius, exposed to aqueous solutions. Toxicokinetics were determined in 10-d uptake and 5-d elimination tests. The toxicokinetics were based on parent compound concentration in water and yielded an uptake coefficient (ku) of 17.5 (14.87-20.20) ml/g/h, an elimination rate constant (ke) of 0.073 (0.062-0.085) per h, a bioconcentration factor (BCF) of 56 to 240, and a half-life (t 1/2) of 9.5 (8.0-11.0) h. Biotransformation measurements did not reveal evidence for DBS metabolism. Thus, body residues, determined in the toxicity study, represent parent compound. In toxicity tests, 4- and 10-d LR50s (the body residue required to cause 50% mortality) in live midges were 0.72 (0.65-0.79) and 0.18 (0.08-0.42) mmol/kg, respectively. Thirty-day LR50s were 0.18 (0.09-1.64) and 0.21 (0.15-0.39) mmol/kg in duplicate studies. Of the sublethal endpoints, only developmental time increase was significant, with the lowest-observed-effect residues of 0.085 (0.067-0.105) and 0.100 (0.087-0.114) mmol/kg for male and female midges, respectively. Deformities in surviving larvae were also observed as chronic responses for body residues exceeding the 30-d LR50. The body residues required for mortality suggest that DBS acts like a polar narcotic in the midge.