PURINOCEPTOR P-2U IDENTIFICATION AND FUNCTION IN HUMAN INTRAHEPATIC BILIARY EPITHELIAL-CELL LINES

The mechanisms that regulate ion and fluid transport by the human intr ahepatic bile duct have not been well defined, Human intrahepatic bili ary cell lines that we have developed were used to identify and charac terize purinoceptors based on increases in intracellular calcium in re sponse to ATR and other nucleotides. Intracellular free calcium was me asured in cell suspensions using the fluorescent probe Fura-2 and a fl uorescence spectrophotometer. Halide efflux was measured in single cel ls using fluorescence microscopy and the fluorescent probe SPQ. Intrac ellular calcium increases equivalently in response to ATP and UTP, pea king, 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-anal ogues. Halide efflux increases with P-2u-purinoceptor stimulation whic h is consistent with the opening of a Ca2+-sensitive Cl- channel. The physiological significance of P-2u-purinoceptor activation and its eff ect on the ionic content and flow rate of bile remains to be determine d.