Receptor-mediated and fluid-phase transcytosis of horseradish peroxidase across rat hepatocytes

Abstract

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.

Figure 1

Kinetics of biliary secretion of FITC-dextran (a) and ASOR (c). Livers were isolated and following 30-minute perfusion, FITC-dextran (5 mg/mL) or ¹²⁵I-ASOR was administered in the perfusion medium (KHB) for 1 minute and 2 minutes, respectively, at 37°C (time 0) followed by a single-pass perfusion with marker-free KHB at 37°C. (a) Single bile drops were collected and the FITC-dextran concentration was determined using a calibration curve. Data were converted into secretion (ng FITC-dextran/min and g liver). In (b) the corresponding bile flow is shown (mean ± SD from two (a, b) perfusions). (c) Single bile drop were subjected to precipitation with 10% TCA. Secretion of intact ASOR (TCA precipitable counts) and degradation products (TCA soluble counts) of one typical experiment out of six are shown. (d) Corresponding bile flow.

Figure 2

Kinetics of biliary secretion of HRP in the absence of mannan in the perfused rat liver. Isolated livers were perfused with HRP (0,8 mg/mL) for 1 minute at 37°C (time 0) followed by a single pass perfusion with marker-free KHB at 37°C. The amount of HRP in single bile drops was determined and the secretion rate was calculated (a) based on the bile flow (b). Data shown are the mean ± SD five perfusions. (c) The total amount of HRP (cummulative HRP secretion) secreted into bile as a function of the perfusion time is shown.

Figure 3

Effect of mannan on biliary HRP secretion. Livers were isolated and following 30 minutes perfusion, HRP (0,8 mg/mL) plus 1.6 mg/mL mannan was administered in the perfusion medium (KHB) for 1 minute at 37°C (time 0) followed by single-pass perfusion with marker-free KHB at 37°C for 40 minutes. The amount of HRP in single bile drops was determined and the secretion rate was calculated (a) based on the bile flow (b). Data shown are the mean ± SD of four perfusions. (c) The total amount of HRP (cummulative HRP secretion) secreted into bile as a function of the perfusion time is shown.

Figure 4

Effect of mannan on cumulative HRP secretion. The cumulative secretion from the experiments shown in Figure 2 (absence of mannan) and Figure 3 (presence of mannan) was calculated for 0–10 minutes (1st peak) and 10–40 minutes (2nd peak). Data shown are the mean ± SEM from five and four perfusions, respectively. Note that only the first peak (0–10 minutes) was reduced in the presence of mannan. Asterisk indicates significant differences at P ≤ .05.

Figure 5

Scheme of potential pathways involved in HRP secretion into bile. Compartments labeled by HRP are indicated by blue color. Although HRP is also directed to lysosomes, only those endosomes involved in transcytosis are shown in blue. MRs (yellow bars) presumably recycle back to the sinusoidal plasma membrane.