HTC cells were loaded with 1.5 pM Fura-2 AM for 45 mins at room temperature in bicarbonate-free MEM medium. Coverslips with adhered cells were transferred into a 100 pL plastic chamber placed on the stage of an inverted microscope coupled to a spectrofluo- rimeter (Deltascan RF-D4010, Photon Technology International Inc, London, Ontario). Cells were superfused at a flow rate of 2 to 3 mL/min with standard bath solution (see above) at room temperature. Excitation wavelengths were 350 and 380 nm, while fluorescence emission was measured at 505 nm. Intracellular dye calibration was performed in situ by perfusion of 3.5 pM ionomycin in a solution containing either 4 mM EGTA (Rmin, 350:380 fluorescence ratio in calcium-free solution) or 4 mM calcium chloride (Rmax, 350:380 fluorescence ratio at saturating calcium). The auto- fluorescence was determined by quenching free FURA acid with 2 mM manganese chloride. Once corrected for autofluo- rescence, the fluorescence ratios (350:380) were transformed into calcium concentrations, using the OSCAR software supplied by Photon Technology International.
To assess changes in calcium influx, two experimental approaches that were used in many studies were selected. The first experimental protocol involved perfusing cells with a solution where extracellular calcium was withdrawn (EGTA chelation) for a period after which normal external calcium concentrations were restored. The initial rapid rise in cytosolic calcium following readmission of external calcium reflects the entry of calcium from the external environment into the cell. Initial calcium influx rates were assessed by measuring the slope of the calcium-versus-time curve following readmission of external calcium (1.8 mM) after a 13.5 min, calcium-free bathing (0 mM calcium chloride, 4 mM EGTA) period, over the initial 15 s where calcium influx was apparent. The effects of insulin or ATP were determined by relating the measured influx rate to the average value obtained for daily control experiments of calcium withdrawal and readmission, alone.
The second approach used to assess calcium influx into hepatocytes took advantage of the capacity of manganese ions to quench FURA-2 fluorescence. In this case, manganese ions are thought to act as surrogates for calcium ions. For such FURA-2 fluorescence quenching studies, 50 pM manganese chloride was used and fluorescence signals were monitored at the calcium-insensitive excitation wavelength of 357 nm. The slope of signal quenching obtained (over the first 60 s) with insulin or ATP was related to the control steady-state slope obtained (over the first 60 s) with manganese chloride before their administration. When used, inhibitors were coadministered with manganese chloride and were present throughout the subsequent exposure to insulin. Beat the drug companies and Purchase Cialis online
Statistical analysis: Significant differences between group means were evaluated by ANOVA for independent measures or by paired Students’ t test as dictated by the experimental protocol.