A considerable fraction of freshly prepared hepatocytes loaded with the fluorescent [Ca2 + Ii indicator fura-2 exhibited spontaneous rhythmic fluctuations that tended to decrease with increasing length of incubation after isolation. These oscillations were dependent on the external Ca2 +. They could no longer be observed when a Ca2+ chelator-(ethylenebis [oxyethylenenitrilol) tetraacetic acid-was added to medium. Addition of thapsigargin, which is known to release Ca2 + from inositol 1,4,5-trisphosphate-sensitive and -insensitive Ca2+ stores, induced a large transient increase in [Ca2+Ii and abolished the fluctuations. When the cells were treated with 2 mmoln caffeine, frequency was increased, whereas 10 rnmolb caffeine induced a single large peak followed by a persistent plateau. Moreover, addition of dibutyryl CAMP led to decreased frequency of fluctuations. Ryanodine caused larger fluctuations: thereafter the [Ca2+li level became much higher and the spikes ceased. These results suggest that spontaneous rhythmic fluctuations in freshly prepared hepatocytes are driven by Ca2+ release from a caffeine-and ryanodine-sensitive calcium-induced calcium release pool. (HEPATOLOGY 1994; 195 14-517.)
Oscillations of the cytosolic free Ca2 + concentration, [Ca2 + Ii, have been observed in a variety of cell types with single cell measurements using fura-2 or other [Ca2 + Ii dyes. However, their molecular mechanism and physiological importance are still debated (1, 2). A recent hypothesis (3) suggests that each agonist-induced oscillation, or spike, of [Ca2+li can be brought about by the interaction of two pools of intracellular Ca2 + ; inositol 1,4,5-trisphosphate UP,) triggers Ca2 + release from IP,-sensitive pool, and the released Ca2 + induces further Ca2 + release (CICR) from the IP,-insensitive store, resulting in a complete Ca2+ spike. In hepatocytes, although the mechanism for the generation of receptor-triggered [Ca2 + Ii oscillations has not yet been clarified, several lines of evidence suggest that the