Abstract
We investigated spatiotemporal changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10(-6) M) resulted in an initial transient increase in [Ca2+]i, followed by a small sustained [Ca2+]i plateau above the pre-stimulation level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an alpha-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca(2+)-free medium or by pre-treatment of cells with La3+. On the other hand, the sustained plateau was eliminated by Ca(2+)-free medium or La3+. Therefore, H-35 cells have a Ca(2+)-signaling pathway which is activated via alpha-adrenergic receptors. Mn2+ entered the cytosol after NE stimulation, as shown by quenching of fura-2. This indicates that H-35 hepatoma cells possess Mn(2+)-permeable Ca2+ channels at the plasma membrane. In addition, the Ca2+ efflux pattern from H-35 cells to the extracellular space during NE stimulation was visualized by digital imaging microscopy when free fura-2 was equilibrated between the cells and the extracellular space. The efflux of Ca2+ from H-35 begins between the initial [Ca2+]i transient and the sustained [Ca2+]i plateau.