Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acids. 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 hepatocytes. Na(+)-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysine (C-NBD-L), an FBA with a net charge of -1, was shown to be electrogenic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA with a net charge of -2, was neutral. Incubation of hepatocytes with 8-bromo-cAMP (8-BrcAMP; 100 microM) increased the uptake rate of the electrogenically transported FBA by 25% (P = 0.002), but had no effect on the uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepatocytes 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 conditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was terminated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP had no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-mV hyperpolarization increased the uptake rate of C-NBD-L by 23%. We therefore conclude that short-term cAMP-induced stimulation of fluorescent bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.
Bile Acid Uptake Transporters as Targets for Therapy
