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-mu-mol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca2+-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25-mu-mol/L) on cytosolic Ca2+ in isolated hepatocytes 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-mu-mol/L) and the protein kinase C inhibitor H-7 (50-mu-mol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25-mu-mol/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 ionophore-induced 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.