METHODOLOGICAL ASPECTS OF PRESSURE LOADING OF FURA-2 INTO CHARACEAN CELLS

Four different fura-2 compounds were tested for the application in Cha racean cells (fura-AM; fura-C-18; fura-K-5; fura-dextran; MW = 10 kDa) . It is demonstrated that Characean cells impose special problems when cytosolic pCa has to be measured with fluorescent ratio dyes. Fluores cence (lambda(ex) = 340 nm) from the dye which had diffused from the c ytosol to the huge central vacuole with milimolar Ca2+ concentrations overrides the signal from the cytosol and makes Ca2+-quantification di fficult. This can be avoided by pressure injection of fura-dextran. Be cause of inhomogeneities in dye concentration or in thickness of the c ytoplasmic layer, cytoplasmic streaming causes high noise or pretend o scillations in pCa if data are obtained by subsequent image grabbing. In addition, vesicles filled with high concentrations of dye may somet imes be expelled into the vacuole during the loading procedure enhance this effect. These sources of inhomogeneities can be minimized by loa ding fura-dextran via the neighbouring cell. The slow loading procedur e through the plasmodesmata takes 1-10 h. It results in a more homogen eous distribution of the dye. The operation of the new method is illus trated by the measurement of Ca2+-transients transients during action potentials, the temperature dependence of the fluorescence signal in v ivo and in vitro and the butyrate-induced elevation of [Ca2+](c). Fura -AM was found not to be well suited for use in algal cells. Fura-C-18 has toxic effects and induces clotting of the cytoplasm. In addition, some aspects of the properties of dextran-derivates are discussed.