Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Abstract

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and Pi. In rat, biliary Piconcentration (0.01 mM) is ∼100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary Pi. We aimed to functionally characterize canalicular Pireabsorption in rat liver and to identify the involved Pitransport system(s). Pitransport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative Pitransporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent Piuptake. Initial Piuptake rates were saturable with increasing Piconcentrations, exhibiting an apparent Kmvalue of ∼11 μM. Pitransport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of Pifrom primary hepatic bile and thus might play an important role in the regulation of biliary Piconcentration.