Adult rat hepatocytes in primary culture. VI. Developmental changes in alcohol dehydrogenase activity and ethanol conversion during the growth cycle.

Proliferation-competent primary ‘monolayer’ cultures of adult rat hepatocytes that display a repertoire of differentiated functions have been characterized for growth state-dependent expression of alcohol dehydrogenase activity (ADH; EC 1.1.1.1). Following hepa-tocyte isolation and early during in vitro lag phase, ADH ‘per cell’ is equivalent to control adult liver tissue levels. Upon entry into and progression of logarithmic growth, ADH declines biphasically to about 2% of control levels (halftime rate, R[disappearance]= 3.4 and 7.4 hr; days 0-is(halftime rate, R[disappearance]= 3.4and 7.4, respectively). increases biphasically as hepatocytes enter and remain in stationary phase (R[disappear]= 3.9 and 11.1 hr; days 6–10 and 10–14, respectively). Ultimately, control ADH is restored. Similar ‘U-shaped’ curves occur when rates of conversion of [14C]ethanol into CHCI3:CH30H (2:1) extractable material are measured. By this criterion, 10- to 13-day-old hepatocyte cultures convert ethanol or [14C]acetate 10 times faster than stationary-phase 3T3 fibroblasts' (3000 or 12,000 cpm/106 cells/24 hours versus 200 or 800 cpm/106 cells/24 hr, respectively). Both conversion processes in matured hepatocyte cultures depend on extracellular glucose levels unlike the nonhepatocyte system. Furthermore, depending on the growth state, [14C]ethanol conversion in hepatocyte cultures is inhibited up to 73% by 1 mil pyrazole, a specific ADH inhibitor. At higher initial ethanol levels (50 mil), pyrazole blockade is ineffective. The results indicate that, in this long-term culture system, normal adult hepatocytes retain non-ADH ethanol-converting enzymes and pharmacologically-sensitive ADH. Changes in the latter enzyme's activity levels in culture appear to simulate normal developmental changes in hepatic ADH in the intact animal.