Cgp 37157

Fluorometric measurements of mitochondrial [Ca²⁺]_m, cytosolic [Ca²⁺]_c and ΔΨ_m were performed as previously described8 (link). Briefly, saponin-permeabilized hepatocytes (2 millions) or MEFs (2.4 mg) were resuspended in 1.5 ml of intracellular medium containing 120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM Tris-HEPES at pH 7.2, and supplemented with proteases inhibitors (leupetin, antipain, pepstatin, 1 mg ml⁻¹ each), 2 mM MgATP, 2 μM thapsigargin (Enzo) and maintained in a stirred thermostated cuvette at 35 °C. Assays were performed in the presence of 20 μM CGP-37157 (Enzo) and 1 mM succinate using a multiwavelength-excitation dual-wavelength-emission fluorimeter (DeltaRAM, PTI). The extramitochondrial Ca²⁺ concentration [Ca²⁺]_c was assessed using the ratiometric Ca²⁺ probe Fura2-FA (1.5 μM, Teflabs) or Fura-loAff (formerly Fura-FF; 1 μM, Teflabs). Δψm was measured with 1.5 μM TMRM (Invitrogen). Fura and TMRM fluorescence were recorded simultaneously using 340–380 nm excitation and 500 nm emission, and 545 nm excitation and 580 nm emission, respectively. Complete depolarization (maximum de-quench of TMRM fluorescence) was elicited using of the protonophore FCCP (2 μM). Calibration of the Fura signal was carried out at the end of each measurement, adding 1 mM CaCl₂, followed by 10 mM EGTA/Tris, pH 8.5.

Saponin-permeabilized HEK cells were resuspended in 1.5 ml of intracellular medium containing 120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM Tris-HEPES at pH 7.2, and supplemented with proteases inhibitors (leupeptin, antipain, pepstatin, 1 μg/ml of each), 2 mM MgATP, 2 μM thapsigargin (Enzo) and maintained in a stirred thermostated cuvette at 35 °C. Fura-2-FF (1 μM, Teflabs) was added to the suspension to monitor [Ca²⁺] in the medium, and 1.5 μM TMRM (Invitrogen) to monitor corresponding changes in the Δψ_m. Assays were performed in the presence of 20 μM CGP-37157 (Enzo) and 2 mM succinate. Fluorescence was recorded in a multi-wavelength excitation dual-wavelength emission fluorimeter (DeltaRAM, PTI) using 340–380 nm excitation and 500-nm emission for fura-2-FF and 545 nm excitation and 580 nm emission for TMRM. Five data triplets were obtained per second. Ca²⁺-clearance was calculated after the addition of 20 μM Ca²⁺ pulse and complete depolarization (maximum de-quench of TMRM fluorescence) was elicited using the protonophore FCCP (2 μM). The Fura signal was calibrated at the end of each measurement, adding 1 mM CaCl₂, followed by 10 mM EGTA/Tris, pH 8.5.