HYPOCALCEMIA DECREASES THE EARLY AND LATE RESPONSES TO EPIDERMAL GROWTH-FACTOR IN RAT HEPATOCYTES

Extreme variations in extracellular Ca2+ concentrations ([Ca2+](e)) modify the signaling generated by many hormones and growth factors, However, the influence of physiological changes in [Ca2+](e) on the response to hepatic mitogens remains largely unknown, To study the influence of [Ca2+](e) on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+](e) similar to those observed in normocalcemia or hypocalcemia, To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+](e) prevailing in vivo, Intracellular Ca2+ concentrations ([Ca2+](i)) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+](e) strongly influenced the [Ca2+](i) response to EGF with significantly smaller [Ca2+](i) increases in hepatocytes of normal rats kept in low [Ca2+](e) compared with those kept in normal [Ca2+](e). In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+](e). In normal [Ca2+](e), the EGF-induced increases in [Ca2+](i) were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+](e) significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+](e). Furthermore, the blunted responses in [Ca2+](i) mobilization and DNA synthesis associated with hypocalcemia could not be over-come by increasing concentrations of EGF nor by normalization of [Ca2+](e) in vitro, These data demonstrate that [Ca2+](e) within the physiological concentration range can strongly influence the hepatocyte response to EGF. At [Ca2+](e) comparable to that of hypocalcemia in vivo our data point to the appearance of a phenomenon of hepatocellular resistance to the early (increases in [Ca2+](i)) and late (DNA synthesis) cellular responses to EGF.