Purinoceptor P 2U identification and function in human intrahepatic biliary epithelial cell lines

Abstract

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have deveveloped were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P 2u-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P 2u-purinoceptor stimulation which is consistent with the opening of a Ca 2+-sensitive Cl − channel. The physiological significance of P 2u-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.