Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of increasing cholesterol (CHS) concentrations on OCTN1 mediated transport of TEA and acetylcholine. The recombinant OCTN1 was reconstituted in liposomes as described in . except that ( a and b ) proteoliposomes batches used for each time course experiment were reconstituted in the presence of 0 (○), 2.1 (●), 4.2 (□), 8.3 (■) % CHS (corresponding to 0, 21, 42 and 83 µg CHS/mg total lipids, respectively). ( a ) Transport was started adding 0.1 mM [ 14 C]-TEA ( b ) or 0.1 mM [ 3 H]-acetylcholine, at time zero to proteoliposomes and stopped at the indicated times as described in . Time course data were interpolated in a first order rate equation. The values are means ± SD from three different experiments. ( c ) Transport rates of [ 14 C]-TEA (○) or [ 3 H]-acetylcholine (●) uptake, calculated as K x transport at equilibrium from ( a , b ), reported as function of the % CHS.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of CHS on regulation of OCTN1 by ATP or NaCl. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the presence of 8.3% CHS corresponding to 83 µg CHS/mg total lipids and ( a ) in the presence of the indicated ATP concentrations. Transport was started adding 0.1 mM [ 14 C]-TEA (white column, ○) or [ 3 H]-acetylcholine (grey column, □) at time zero to proteoliposomes. ( b ) Increasing NaCl extraliposomal concentration were added together to the radioactive substrates. The transport reaction was stopped at 20 min. Percent residual activity with respect to the control data were interpolated in an IC 50 equation (Dose-response curves). The values are means ± SD from three experiments. Significantly different for * p < 0.01 as calculated from Student’s t test analysis.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes, Concentration Assay, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of hemicholinium and TEBA on the OCTN1 activity measured in the presence or absence of CHS. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the absence (○) or in the presence (●) of 8.3% CHS corresponding to 83 µg CHS/mg total lipids. ( a ) Transport was started adding 0.1 mM [ 3 H]-acetylcholine at time zero to proteoliposomes in the presence of increasing external hemicholinium concentrations (0-0.1-0.3-0.5-0.8-1-1.5-2.6 mM) or ( b ) adding 0.1 mM [ 14 C]-TEA in the presence of increasing external TEBA concentrations (0-0.5-1-2.5-5-10-20 mM). The transport reaction was stopped at 20 min. Percent residual activity with respect to the control data were interpolated in an IC 50 equation (Dose-response curves). The values are means ± SD from three experiments.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Activity Assay, Recombinant, Liposomes, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of CHS on OCTN1 transport kinetics. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the absence (○) or in the presence (●) of 8.3% CHS corresponding to 83 µg CHS/mg total lipids. The transport rate was measured adding [ 14 C]-TEA ( a ) or [ 3 H]-acetylcholine ( b ) at the indicated increasing concentrations to proteoliposomes. The transport reaction was stopped at 10 min. Data were plotted according to Lineweaver–Burk as reciprocal transport rate vs reciprocal TEA ( a ) or acetylcholine ( b ) concentration. The values are means ± SD from three experiments.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes, Concentration Assay