ATP depletion in rat cholangiocytes leads to marked internalization of membrane proteins

Intrahepatic bile ducts (BD) are a critical target of injury in the postisc hemic liver, Decreased vascular perfusion causes characteristic changes in the morphology of the ductular epithelia including a loss of secondary memb rane structures and a decrease in plasma membrane surface area. Using adeno sine triphosphate (ATP) depletion of cultured normal rat cholangiocytes (NR C) to model ischemic ducts, the present studies examined the fate of apical membrane proteins to determine whether membrane recycling might contribute to rapid functional recovery. Apical proteins, including gamma-glutamyl tr anspeptidase (GGT), Na+-glucose cotransporter (SGLT1), and apically biotiny lated proteins, were not shed into the luminal space during ATP depletion. Instead, labeling of surface proteins after ATP depletion showed a signific ant decrease in GGT and SGLTI, consistent with membrane internalization. Si milarly, z-axis confocal microscopy of biotinylated apical proteins also sh owed protein internalization. During ATP recovery, SGLT1 transport activity remained profoundly depressed even after 24 hours of recovery, indicating that the function of the internalized apical proteins is not rapidly recove red. These studies suggest that the membrane internalization in ATP-deplete d cholangiocytes is a unidirectional process that contributes to prolonged functional deficits after restoration of normal cellular ATP levels. This s ustained decrease in transport capacity may contribute to the development o f ductular injury in postischemic livers.