The effect of glucagon, epinephrine, norepinephrine, dexamethasone, insulin, and. dexamethasone plus glucagon on the transport of 2-aminoisobutyric acid (AIB) and that of glucagon on the production of cyclic AMP were examined in rat hepatocyte monolayer cultures under three different culture conditions involving calcium. The hepatocytes were studied in calcium-containing medium after treatment with or without 0.033% dimethyl sulfoxide, the solvent for the calcium ionophore A23187 (calcium controls) calcium-free medium after treatment with A23187 (calciumdepleted); and calcium-containing medium after treatment with ionophore (calcium-restored). The basal and hormonally regulated rates of AIB transport for hepatocytes in calcium control and calcium-depleted cultures were comparable. The restoration of calcium in calcium-restored cultures increased the basal and the hormonally stimulated transport of AIB when compared to the other conditions. Calcium markedly enhanced the stimulation of AIB transport in cultures treated with glucagon, catecholamines, and Nexamethasone plus glucagon. The level of cyclic AMP production in response to glucagon in calcium control and calcium-depleted cultures was the same and it was conspicuously higher than the level in calcium-restored cultures. Varying the concentration of calcium in the medium used to maintain the hepatocytes in calcium control cultures did not affect the stimulation of AIB transport or cyclic AMP production by glucagon. However, in calcium-restored cultures, increasing the calcium concentration of the medium resulted in increased stimulation of AIB transport and decreased production of cyclic AMP by glucagon. In the calcium-restored cultures, calcium in the absence of glucagon enhanced AIB transport but had no effect on cyclic AMP production. Cultures maintained for 6 hr in calcium-free medium after the depletion of calcium showed a 6-to 7-fold increase in the production of cyclic AMP in response to glucagon, but no stimulation of AIB transport. We suggest that mobilization of cellular calcium by glucagon either directly or through cyclic AMP mediates its stimulation of amino acid transport.Calcium regulates a variety of cellular functions, including the hormonal regulation of gluconeogenesis (1-3). Amino acids are substrates for hepatic gluconeogenesis and their availability, particularly during fasting, could limit the rate of gluconeogenesis. Although several hormones have been shown to regulate the transport of amino acids in perfused liver (4-6), liver slices (7), or isolated hepatocytes (8-18), the mechanisms for the hormonal regulation of amino acid transport are poorly understood. The present studies were undertaken to examine the hormonal regulation (by glucagon, epinephrine, norepinephrine, dexamethasone, and insulin) of amino acid transport in hepatocyte monolayers under three different culture conditions involving cellular calcium. Because it has been suggested that glucagon effects result from alterations in the level of intracellular cyclic A...

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.