Confocal Fluorescence Microscopy for Studying the Effects of a Tyrosine Kinase Inhibitor on the Nuclear Calcium Signals in B Cells

Abstract

We have studied the effects of a tyrosine kinase inhibitor, herbimycin A, on the nuclear calcium signals in antigen-specific B cells (TP67.21). After antigen stimulation (trinitrophenol conjugated ovalbumin, TNP₂₁-OVA), we measured first the intracellular free calcium ion concentration, [Ca²⁺]_i, in fura-2-loaded B cells with a fluorescence spectrophotometer. Herbimycin A decreased the [Ca²⁺]_i in antigen-stimulated B cells in a dose-dependent manner. Then we measured the effects of herbimycin A on the calcium signals in individual B cells with a confocal fluorescence microscope. We found that antigen-induced calcium signals in the cytoplasm and the nucleus of B cells were completely inhibited by preincubation of the cells with 5—10 μM herbimycin A for 14 hr. On the other hand pretreatment of the cells with a lower concentration of herbimycin A (0.5—1 μM) partly inhibited calcium signals where the intracellular free calcium ion concentrations ([Ca²⁺]_i) showed rapid rises and falls over much shorter periods during the time course of the ([Ca²⁺]_i) in the drug-untreated cells. This indicated that at low concentrations (0.5—1 μM), herbimycin A inhibited mainly the calcium entry from the external medium. In all the experiments it seemed that nuclear calcium signals were controlled by the elevated level of the [Ca²⁺]_i in the cytoplasm of B cells.