Santosh J. Sacket

Increase in sphingolipid catabolic enzyme activity during aging

AbstractAim:To understand the contribution of sphingolipid metabolism and its metabolites to development and aging.Methods:A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats.Results:Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic enzyme activities, sphingomyelin synthase and ceramide synthase. This suggested an accumulation of ceramide and sphingosine during development and aging. The liver showed the highest neutral-SMase activity among the tested enzymes while the kidney and brain exhibited higher neutral-SMase and ceramidase activities, indicating a high production of ceramide in liver and ceramide/sphingosine in the kidney and brain. The activities of sphingolipid metabolic enzymes were significantly elevated in all tested tissues during development and aging, although the onset of significant increase in activity varied on the tissue and enzyme type. During aging, 18 out of 21 enzyme activities were further increased on day 720 compared to day 180.Conclusion:Differential increases in sphingolipid metabolic enzyme activities suggest that sphingolipids including ceramide and sphingosine might play important and dynamic roles in proliferation, differentiation and apoptosis during development and aging.

Sphingosine 1-phosphate (S1P) induces shape change in rat C6 glioma cells through the S1P2 receptor: development of an agonist for S1P receptors

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.

Wuweizisu C from Schisandra chinensis decreases membrane potential in C6 glioma cells

AbstractAim: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 Ca2+ [Ca2+]i concentration, but decreased the ATP-indu-ced Ca2+ 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 [Ca2+]i concentration by wuweizisu C could be important action mechanisms of wuweizisu C.

Lysophosphatidylserine induces calcium signaling through Ki16425/VPC32183-sensitive GPCR in bone marrow-derived mast cells and in C6 glioma and colon cancer cells

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.

Lysophosphatidylserine increases membrane potentials in rat C6 glioma cells.

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.

Increase of Membrane Potential by Ginsenosides in Prostate Cancer and Glioma cells

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.

Characterization of N,N,-dimethyl-D-erythro-sphingosine-induced apoptosis and signaling in U937 cells: independence of sphingosine kinase inhibition.

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-phosphate 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 2h 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.

Discovery of sphingosine 1- O -methyltransferase in rat kidney and liver homogenates

AbstractAim:To characterize sphingosine methyltransferase in rat tissues.Methods:By using S-adenosyl-L-(methyl-3 H) methionine, enzymatic activity was measured in the rat liver and kidney homogenates.Results:The optimum pH and reaction time for the enzyme assay were pH 7.8 and 1 h. ZnCl2 inhibited the activity, but not MgCl2, CaCl2, CoCl2, or NiCl2. In the kidney homogenate, enzymatic activity was detectable in the cytosol and all membrane fractions from the plasma membrane and other organelles; however, in the liver homogenate, enzymatic activity was detectable in all membrane fractions, but not in the cytosol. We also tested the enzymatic activity with structurally-modified sphingosine derivatives.Conclusion:We found sphingosine 1-O-methyltransferase activity in the rat liver and kidney homogenates.