INDUCTION OF A DOSE-RELATED INCREASE IN SULFOBROMOPHTHALEIN UPTAKE VELOCITY IN FRESHLY ISOLATED RAT HEPATOCYTES BY PHENOBARBITAL

To determine whether phenobarbital affects hepatocellular bilirubin/su lfobromophthalein uptake mechanism, we administered it to male Sprague -Dawley rats, body weight 175 +/- 25 gm, at doses of 1 to 75 mg/kg bod y wt/day for 7 days. Control rats were given an equivalent volume of p hysiological saline solution. On day 8, hepatocytes were isolated by m eans of collagenase perfusion, suspended in Hanks' solution without al bumin and incubated with high specific activity (3 Ci/mmol)[S-35]sulfo bromophthalein, which was synthesized in our laboratory and purified b y means of a new reverse-phase high-pressure liquid chromatography pro cedure. The initial uptake rate of sulfobromophthalein was determined at sulfobromophthalein concentrations of 1 to 50 mu mol/L with a rapid filtration technique. The maximum uptake velocity and Michaelis const ant for sulfobromophthalein uptake at each phenobarbital dose were det ermined by means of a computer analysis. In control studies, maximum u ptake and Michaelis constant were 48.0 +/- 16.7 (mean +/- S.D.) pmol/5 0,000 cells/min and 22 +/- 4 mu mol/L, respectively. Maximum uptake ve locity increased linearly with the log of the phenobarbital dose (r = 0.98, p < 0.01), the increase achieving statistical significance at a dose of 3 mg/kg/day. Michaelis constant, however, was essentially unch anged at phenobarbital doses of 50 mg/kg/day or less. The maximal obse rved increase in maximum uptake velocity of sulfobromophthalein (to 61 9% of control values) was appreciably greater than the maximal increas e in UDP-glucuronyltransferase activity (200% of control) or immunorea ctive ligandin concentrations (260% of control) seen in earlier studie s, suggesting a direct effect on the plasma membrane transport mechani sm.