Nuclear calcium gradients in cultured rat hepatocytes

Ca²⁺ concentrations ([Ca²⁺]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca²⁺] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca²⁺ (210 ± 15 nM) was 1.6- to 2-fold higher than nuclear Ca²⁺ (128 ± 12 nM). This difference in Ca²⁺ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca²⁺; however, the nuclear-to-cytosolic Ca²⁺ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca²⁺ permeability barriers and Ca²⁺ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca²⁺ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca²⁺-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.