CAMP STIMULATES FLUORESCENT BILE-ACID UPTAKE INTO HEPATOCYTES BY MEMBRANE HYPERPOLARIZATION

Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acid s. The purpose of this study was to determine to what extent these two phenomena are linked. Fluorescent bile acid analogues (FBA) were used to probe bile acid transport into whole cell patch-clamped hepatocyte s. Na+-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysi ne (C-NBD-L), an FBA with a net charge of -1, was shown to be electrog enic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA w ith a net charge of -2, was neutral. Incubation of hepatocytes with 8- bromo-cAMP (8-BrcAMP; 100 mu M) increased the uptake rate of the elect rogenically transported FBA by 25% (P = 0.002), but had no effect on t he uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepato cytes by 6-8 mV. To determine if hyperpolarization is responsible for the cAMP-induced increase in uptake rate, cAMP was directly introduced into hepatocytes during whole cell patch clamp under voltage-clamp co nditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was term inated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP ha d no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-m V hyperpolarization increased the uptake rate of C-NBD-L by 23%. We th erefore conclude that short-term cAMP-induced stimulation of fluoresce nt bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.