Differential Ca2+ signaling in neonatal and adult rat hepatocyte doublets

Background/Aims: Intracellular Ca2+ ([Ca2+]i) is important in various cellular functions, including cellular proliferation and differentiation. To elucidate the relationship between [Ca2+]i oscillations and physiological hepatocyte proliferation, phenylephrine-evoked [Ca2+]i responses were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. Methods/Results: DNA synthesis in hepatocytes, determined by BrdU incorporation, was 20% in 1-week-old rats, and decreased to <1% as the rats aged. Correspondingly, [Ca2+]i responses evoked by 10 μmol/l phenylephrine in hepatocyte doublets shifted from transient to sinusoidal-type [Ca2+]i oscillations and then to a sustained increase in [Ca2+]i, followed by a gradual return to baseline. The incidence of [Ca2+]i oscillations was 100±0.0%, 83.3±16.7%, 38.7±0.6% and 5.5±5.0% in 1-, 3-, 6- and 8-week-old rats, respectively. Removal of extracellular Ca2+ did not abolish [Ca2+]i oscillations, indicating that [Ca2+]i oscillations were caused primarily by Ca2+ mobilization from internal sites of the cells. The [Ca2+]i level in each of the adjacent cells was synchronous in sustained increase in [Ca2+]i, but asynchronous in [Ca2+]i oscillations. In proliferating doublets obtained from 1-week-old rats, the frequency of oscillations increased in a dose-dependent manner for phenylephrine concentrations of 1 to 100 μmol/l. Conclusions: Phenylephrine-evoked [Ca2+]i oscillations were directly related to hepatocyte proliferation and were mediated by frequency modulation. These results suggest that phenylephrine-evoked [Ca2+]i oscillations may contribute to cell-cycle progression of hepatocytes in physiological liver growth.