Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 μmol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca2+‐mobilizing agent, 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (25 μmol/L) on cytosolic Ca2+ in isolated hepatooytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single‐pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca2+‐sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% ± 10% (mean ± S. D.) decrease in bile flow and a sustained 271% ± 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 μmol/L) and the protein kinase C inhibitor H‐7 (50 μmol/L) abolished the pressure in crease but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 μmol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% ± 4% decrease in bile flow and a sustained 7% ± 4% increase in perfusion pressure. Pretreatment with H‐7 alone corrected the vasopressin‐induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two‐thirds without affecting the increase in perfusion pressure. 2,5′‐di(tert‐butyl)‐1,4‐benzohydroquinone produced a transient 65% ± 20% decrease in bile flow and a transient 56% ± 15% increase in perfusion pressure. In isolated hepatocytes, bromo‐A23187, the nonfluorescent form of the ionophore, produced a sustained 56% ± 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% ± 75% increase and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone resulted in a sustained 149% ± 66% increase. The ionophoreinduced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin‐induced increase was reduced by 38%. These results indicate that agents that increase cytosolic Ca2+ in isolated hepatocytes from either internal or external sources also inhibit bile secretion in the isolated perfused liver independently of hemodynamic or protein kinase C effects. Furthermore, conditions in which the Ca2+ rise is inhibited in isolated hepatocytes lead to decreased inhibition of bile secretion in the perfused liver. These observations, along with the temporal relationship between changes in cytosolic Ca2+ in isolated hepatocytes and decreased bile flow in isolated perfused rat livers, suggest that increased cytosolic Ca2+ may play an inhibitory role in the regulation of bile secretion. (HEPATOLOGY 1992;15:107‐116).