Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed : Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak z 6 h) and a subsequent decrease in activity of both high-& and low-K, cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dosedependent (1 pM-1 pM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (K, x 1 and x 10 pM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or P-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system. Hepatocytes maintained in vitro as primary cultures [I -101 offer an attractive experimental tool, not only due to the potential usefulness of these cells in investigations of the biology and pharmacology of liver [ll-131, but also because relatively few other model systems exist for long term studies of differentiated epithelial cells in vitro under controlled conditions.We have investigated the cyclic AMP system and its regulation in primary monolayer cultures of adult rat hepatocytes. The studies first intended to explore if the hepatocytes in culture maintain normal formation and degradation of cyclic AMP. Hepatocytes in monolayer possess the enzymes involved in cyclic AMP metabolism [5,, but relatively few details are known. Our results indicate that the cells in culture retain hormone sensitivity, but al...

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE2 and PGF2 alpha have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGF alpha]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE2, PGF2 alpha, PGD2, and the synthetic analog dimethyl-PGE2 markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE2 and PGF2 alpha on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE2 and PGF2 alpha showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest a) that PGE2 and PGF2 alpha facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine.

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.

Abstract. Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dosedependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24–30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nm and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 μm insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5–15 nm. When added in combination with 1.7 nm EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nm. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.