Intracellular Calcium Measurements

To measure intracellular calcium concentration, cells were incubated with 2 μM Fura-2-AM in Krebs-Ringer solution buffered with HEPES (125 mM NaCI, 5 mM KCI, 1.2 mM MgSO₄, 2 mM CaCl₂, 10 mM glucose, and 25 mM HEPES/NaOH, pH 7.4) for 40 min at 37°C. Then the cells were washed twice with pre-warmed Krebs-Ringer solution and transferred to the recording chamber of an inverted microscope (Axiovert 100, Zeiss, Germany) equipped with a Ca²⁺ imaging unit. Polychrome IV (TILL Photonics, Germany) was used as a light source. Fura-2 fluorescence images were collected with a PCO Super VGA SensiCam (Axon Instruments, CA, USA) and analyzed with Axon Imaging Workbench 6.2 software (Axon Instruments). Intracellular calcium dynamics of the cells after exposure to 50 nM KCI, 1 mM ATP or field electrical stimulation (40 action potentials at 20 Hz) was recorded. Single cell 340/380 nm fluorescence ratios, acquired at 1-4/s, were analyzed with an Origin 6.0 software (Microcal Software Inc., MA, USA).