Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide--adenine dinucleotide couple acting on adenosine 3':5'-cyclic monophosphate phosphodiesterase.

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.