Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 2. S1P stimulates phospholipase D (PLD) activity in ZG cells. ZG cells were labeled with 1 mCi/ml [3H]myristate in DMEM containing 0.2% BSA for 3 h. The cells were then washed three times with this same medium, but in the absence of label, and incubated further in BSA-free DMEM for 2.5 h. Cells were stim- ulated with 5 mM S1P for various times (upper panel) or with increasing concentrations of S1P for 30 min, as indicated (lower panel), in the presence of 1% ethanol. [3H]phosphatidylethanol formation was determined as indicated in Materials and Methods. Results were calculated as a percentage of the radioactivity pres- ent in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to incubations with vehicle. Values are means 6 SEM of four independent experi- ments performed in triplicate, except for the value at 5 mM S1P, which is the mean 6 SEM of 15 experiments.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activity Assay, Labeling, Incubation, Radioactivity

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 3. Effect of S1P and angiotensin II on aldosterone secretion and PLD activation in ZG cells. Cells were incubated for 2 h with S1P (5 mM) in the presence (closed bars) or absence (open bars) of angiotensin II (10 nM). Upper panel: Aldosterone secretion was determined as described in Materials and Methods. Results are calculated as described for Fig. 1 and are expressed as means 6 SEM of three independent experiments performed in triplicate. Lower panel: For determination of PLD activity, ethanol (1%) was added 5 min before cell stimulation, and incubations were con- tinued further for 30 min. The enzyme activity was determined by measuring the accumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioactivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stim- ulation relative to control (CTRL) incubations. Values are means 6 SEM of five independent experiments performed in triplicate.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Incubation, Activity Assay, Cell Stimulation, Radioactivity, Control

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 5. Effect of pertussis toxin (PTX) on aldosterone secretion and PLD activation by S1P in ZG cells. Upper panel: Cells were preincubated in the absence (open bars) or presence (closed bars) of 1 mg/ml PTX for 16 h. S1P (5 mM) or vehicle [control (CTRL)] was then added, as indicated, and the incubations were continued further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are calculated and expressed as described for Fig. 1 and are means 6 SEM of three independent experiments performed in triplicate. Lower panel: Cells were preincubated in the absence (open bars) or presence (closed bars) of 1 mg/ml PTX for 16 h. S1P (5 mM) or vehicle [control (CTRL)] was then added, as indicated. Ethanol (1%) was added 5 min before stimulation with S1P, and incubations continued further for 30 min. PLD activity was determined by measuring the accumula- tion of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioac- tivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to control incubations. Values are means 6 SEM of three independent experiments performed in triplicate. ** P , 0.01; * P , 0.05.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Control, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 8. Involvement of extracellular Ca21 in S1P-induced aldoste- rone secretion and PLD activation. Upper panel: Cells were treated as described for Fig. 1 and preincubated for 30 min with 5 mM EGTA (closed bars), 5 mM BAPTA-AM (gray bars), 5 mM EGTA 1 5 mM BAPTA-AM (hatched bars), or vehicle (open bars). They were then stimulated with vehicle (CTRL) or S1P (5 mM) as indicated and incubated further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are expressed as described for Fig. 1 and are means 6 SEM of three independent experiments. Lower panel: Cells were treated and labeled as described for Fig. 2 and preincubated for 30 min with 5 mM EGTA (closed bars), 5 mM BAPTA-AM (gray bars), 5 mM EGTA 1 5 mM BAPTA-AM (hatched bars), or vehicle (open bars), as indicated. Ethanol (1%) was added 5 min before stimulation with vehicle or S1P (5 mM), and incubations were continued fur- ther for 30 min. PLD activity was determined by measuring the ac- cumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated and expressed as described for Fig. 2 and are means 6 SEM of three independent experiments. * P , 0.05.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Incubation, Labeling, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 9. Involvement of PKC in PLD activation by S1P in ZG cells. Upper panel: Cells were preincubated with vehicle (open bars) or 2 mM PMA for 48 h (closed bars) to downregulate PKC. Ethanol (1%) was added 5 min before stimulation with S1P (5 mM), PMA (2 mM), or vehicle, and incubations were continued further for 30 min. PLD activity was determined by measuring the accumula- tion of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioac- tivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to con- trol (CTRL) incubations. Values are means 6 SEM of three inde- pendent experiments performed in triplicate. Lower panel: Cells were preincubated for 30 min with vehicle (CTRL), 2 mM hispidine (HISP), 0.5 mM dequalinium (DEQ), 5 mM rottlerin (ROT), or 0.5 mM dequalinium 1 5 mM rottlerin (DEQ1ROT), as indicated. Ethanol (1%) was added 5 min before stimulation with 5 mM S1P or vehicle, and incubations were continued further for 30 min. Results were calculated and expressed as for the upper panel and are means 6 SEM of three independent experiments performed in triplicate. ** P , 0.01; * P , 0.05;1 P , 0.05, DEQ1ROT versus DEQ or ROT.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 11. Inhibition of S1P-stimulated PLD and aldosterone secre- tion by N-hexanoylsphingosine (C6-ceramide) in ZG cells. Upper panel: Cells were treated and labeled as described for Fig. 3. They were then preincubated in the absence (open bars) or presence (closed bars) of 10 mM C6-ceramide for 2 h in BSA-free DMEM. S1P (5 mM) was added, and incubations were continued further for 30 min in the presence of 1% ethanol. PLD activity was determined by measuring the accumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioactivity present in [3H]phosphatidylethanol compared with that in total lipids and expressed as fold stimula- tion relative to control (CTRL) incubations. Values are means 6 SEM of four independent experiments performed in triplicate. Lower panel: Cells were preincubated with vehicle (open circles) or with the indicated concentrations of C6-ceramide (closed circles) for 2 h in DMEM supplemented with 0.2% BSA. S1P (5 mM) was then added, and incubations were continued further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are expressed as fold stimulation relative to incubations with vehicle and are means 6 SEM of four indepen- dent experiments performed in triplicate. ** P , 0.01.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Inhibition, Labeling, Activity Assay, Radioactivity, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells

doi: 10.1007/s00018-023-04813-9

Figure Lengend Snippet: Binding of anti-GM3 antibody (GMR6) to GM3/partner lipid is dependent on incubation temperatures and phase transition temperatures of partner lipids. A Binding of 0.37 µg/mL of anti-GM3 antibody to indicated lipids (0.5 nmol/well) was measured by ELISA. GM1, ganglioside GM1; GM2, ganglioside GM2; GM3, ganglioside GM3; GlcCer, glucosylceramide; LacCer, lactosylceramide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; pSM, palmitoyl sphingomyelin;Cer, ceramide; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); TOCL, 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol; CerPE, N-acyl-sphingosylphosphorylethanolamine. B Binding of 0.37 µg/mL of anti-GM3 antibody to GM3/DOPC (1:9), GM3/POPC (1:9), GM3/DMPC (1:9) or GM3/DPPC (1:9) (0.5 nmol GM3/well) at indicated temperature measured by ELISA. (C) Binding of 0.37 µg/mL of anti-GM3 antibody to GM3/pSM (1:9), GM3/GlcCer (1:9), GM3/GM1 (1:9) or GM3 alone (0.5 nmol GM3/well) at indicated temperatures measured by ELISA. Data in A–C are means ± SD of three experiments. D Binding of anti-GM3 antibody (10 ng/mL) to GM3/DOPC (1:5) (orange) or GM3/DPPC (1:5) (green) liposomes measured by surface plasmon resonance (SPR). SPR was performed as described in Methods. E Binding of anti-GM3 antibody to DPPC/GM3 (5:2) (orange) or DPPC/GM3/Chol (5:2:5) (green) liposomes. F Binding of different concentration of anti-GM3 antibody to GM3/DPPC (1:9) liposomes

Article Snippet: GM2 (GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1’-ceramide) from bovine brain was from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Binding Assay, Incubation, Sublimation, Enzyme-linked Immunosorbent Assay, Liposomes, SPR Assay, Concentration Assay