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...

Summary.-The antitumour agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) was found to inhibit competitively the low-Km cyclic AMP phosphodiesterase activity in an ammonium-sulphate-precipitable fraction of the 2,000g supernatant of rat liver. With substrate concentration at 0-25 [M, 150 was 790 /tM for DTIC and 350 tM for theophylline. DTIC at 2 mm more than doubled the cAMP response to glucagon in hepatocytes and to adrenaline in MH1Cj hepatoma cells, indicating that it also exerts its inhibitory effect on the phosphodiesterase in intact cells. The possible contribution of the phosphodiesterase inhibition to the growthinhibitory and cytotoxic effects of DTIC is discussed.

Twenty cadaver femoral condyles were examined with single-energy quantitated computed tomography (QCT), and the composition of the bone scanned was analyzed chemically. The calcium concentration correlated well with the QCT density ( i = 0.89,P < 0.001). The highest correlation was recorded between the total calcium content in the scanned slices and the bone mass-related measures recorded by QCT ( r

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