Transport of fluorescent compounds into hepatocytes and the resultant zonal labeling of the hepatic acinus in the rat

Abstract

The purpose of this study was to label different zones of the hepatic acinus with fluorescent compounds in order to assess the contributions of the various acinar zones to solute transport. In a separate application, this approach allowed the determination of the zonal origin of isolated hepatocytes. Livers were perfused in situ with various concentrations of: (a) a lipid-soluble substance, fluorescein diacetate; (b) two water-soluble, positively charged molecules, acridine orange and rhodamine B; and (c) a water-soluble, negatively charged substance, fluorescein isothiocyanate. Immediately after perfusion, livers were frozen and sectioned in a cryostat. The localization of the fluorescent substance within the hepatic acinus was determined qualitatively by fluorescence microscopy and quantitatively by fluorescence microphotometry. Fluorescein diacetate, perfused at concentrations up to 2 × 10−5 M, was predominantly localized within cells closer to the terminal portal venule (acinar zone 1). In parallel experiments, the retention of fluorescein diacetate in cells of acinar zone 1 was examined during and after the isolation of hepatocytes by collagenase perfusion. The acinar zone 1 to zone 3 cell gradient of fluorescence persisted during collagenase perfusion, and fluorescence in the isolated hepatocytes was detectable by means of a fluorescence activated cell sorter. This indicated that fluorescence labeling can be used as a marker to determine the zone from which hepatocytes have been isolated. Acridine orange and rhodamine B also labeled predominantly cells of acinar zone 1 at low concentrations. With increasing concentrations of rhodamine B in the perfusate, the concentration of this substance in cells of acinar zone 3 increased, and at the same time, more cells (zones 2 and 3) were recruited for transport. Thus, progressive recruitment of hepatocytes occurred before the preceding cell had reached saturation. Fluorescein isothiocyanate provided a different model. At concentrations higher than 2 × 10−5 M, fluorescein isothiocyanate was distributed predominantly in hepatocytes of acinar zone 3 (reverse gradient). This concentration of fluorescein isothiocynate in hepatocytes of zone 3 (against the predicted zone 1 to zone 3 concentration gradient) indicated that zonal labeling within the acinus cannot be predicted solely on the basis of concentration gradients in sinusoidal blood. The transport of fluorescent substances by hepatocytes thus provides an approach to study the interaction of incoming solutes with each zone of the liver acinus.