EXTRACELLULAR CALCIUM MODULATES PREREPLICATIVE CYCLIC-AMP SURGES IN EGF-STIMULATED PRIMARY NEONATAL RAT HEPATOCYTES

The cells in nearly pure (96-98%) primary cultures of hepatocytes from neonatal rat liver in high (1.0 mM)-Ca2+, serum-free, synthetic HiWo( 5),Ba-2000 medium initiated DNA synthesis and entered mitosis between 11 and 30 h after the addition of 10 ng/ml EGF. During the 10-h prerep licative period, the cultured hepatocytes, like regenerating rat liver cells, generated two large cyclic AMP transients, one peaking between 30 min and 2 h and the other around 6 h. Hepatocytes stimulated by th e same concentration of EGF in low (0.02 mM)-Ca2+ medium increased cyc lic AMP synthesis as much as the EGF-treated hepatocytes in high-Ca2medium, but they released the additional cyclic AMP into the medium an d could not generate prereplicative internal cyclic AMP surges, initia te DNA replication, or enter mitosis. These results suggest that one o f the ways external Ca2+ controls prereplicative development of hepato cytes is to restrain the release of cyclic AMP and thus enable the cel l to accumulate enough internal cyclic AMP to stimulate events require d to initiate DNA replication.