Measurement of free cytosolic calcium in single cells: method and application.

Intracellular calcium [Ca2+]i acts as an important intracellular messenger system for secretion and synthesis, cell growth and differentiation. In order to demonstrate definitively that a change in [Ca2+]i is responsible for a physiological event, one has to measure [Ca2+]i directly within intact cells and correlate the time course of any [Ca2+]i changes with the biological response. Measurement of [Ca2+]i was done in a single cell preloaded with fluorescent Ca indicator fura2 using a fluorescent unit (lonoquant) consisting of an inverted microscope (Zeiss IM 35) equipped with a mercury lamp and a rotating filter wheel containing filters at wavelengths of 340 and 380 nm. Cells were alternately excited and emission signals of fura 2-loaded cells were collected by a photomultiplier and recorded on-line on a computer screen. As a model system, the rat C-cell carcinoma cell line rMTC 6-23 secreting calcitonin was used. An acute elevation of extracellular calcium resulted in an increase in [Ca2+]i within 5 sec and rapid release of preformed calcitonin. This tight linkage between extracellular calcium and [Ca2+]i is mediated via Ca influx through voltage-dependent Ca channels. These channels are modulated by intracellular cAMP, yielding a rhythmic oscillation of [Ca2+]i, as well as by extracellular somatostatin blocking the Ca channel and the increase of [Ca2+]i via a pertussis toxin sensitive Gi protein. The change in [Ca2+]i is associated with changes in calcitonin secretion, confirming the stimulus secretion coupling via voltage-dependent Ca channels in C-cells.