Hydration and urinary alkalinization are used with high doses of methotrexate (MTX) to prevent precipitation of the drug in the renal tubules and consequential nephrotoxicity. The quantitative effect of these measures on the renal clearance of MTX was studied in 8 patients with normal renal function, and in 3 patients with reduced renal function. Multiple regression analysis indicated an influence of both factors on the ratio of the renal clearances of MTX and creatinine. In the eleven patients there was a linear correlation between this ratio and urine pH (p less than 0.001); the ratio increased from 0.88 at pH 5.5 to 2.62 at pH 8.4. The pH effect on this ratio was similar in the patients with normal and reduced kidney function. An increase in urine flow did not significantly increase the ratio between renal clearance of MTX and creatinine. The effect of urinary alkalinization on renal MTX clearance could be clinically exploited in patients with delayed elimination of MTX. The probable modifying effect of alkalinization of urine on the intentionally high plasma concentration after high dose MTX infusions should be further evaluated, particularly in patients with normal renal function.

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...

Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (GI exit) followed first-order kinetics both in untreated and hormone-stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate constant for the S phase entry increased fiveto sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at t h e time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a onelog shift of the dose-effect curve), t h e rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6-7 h vs. 26 h).

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.

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.