Epidermal Growth Factor Induces Dose–Dependent Calcium Oscillations in Single Fura–2-Loaded Hepatocytes

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Abstract
Digital imaging fluorescence microscopy has been used to investigate epidermal growth factor-induced calcium responses of fura–2-loaded hepatocytes in primary culture at the single–cell level. Epidermal growth factor induced oscillations in cytosolic free calcium ([Ca2+]i) consisting of a periodic train of spikes unlike the monophasic elevation in cell suspensions reported previously. In this study, 79% of the cells in the microscopic field responded to 0.1 nmol/L epidermal growth factor, and 78% of the responsive cells displayed oscillations. However, the frequency of oscillations differed considerably from cell to cell. [Ca2+]i measurement in a cell population was simulated using these data, but only a slightly biphasic pattern was obtained, indicating the significance of single–cell measurement of [Ca2+]i. Because considerable heterogeneity existed in the sensitivity to epidermal growth factor between the cells, single hepatocytes were stimulated sequentially with increasing concentrations of epidermal growth factor to investigate the dose dependence of the oscillations. The frequency of the oscillations increased with increasing epidermal growth factor concentration, but the amplitude was similar for all concentrations, suggesting the existence of frequency–encoded information even in the pathway through tyrosine kinase for epidermal growth factor signaling. The pattern of the oscillations with epidermal growth factor, especially the latency, was considerably different from that with phenylephrine, which is known to use the phosphatidylinositol pathway, possibly because of the difference in the pathway toward phosphatidylinositol turnover between these agonists. Additional stimulation with phenylephrine of epidermal growth factor-stimulated hepatocytes increased the frequency of oscillations induced by epidermal growth factor, suggesting that phenylephrine, known as a comitogen for hepatocytes, enhanced the epidermal growth factor-induced calcium signaling by increasing the frequency of [Ca2+]i oscillations. (Hepatology 1992;16:479-486.)