Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Abstract

Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of '2MI-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [In (inulin/EGF) versus time] ofthe dilution curve for EGF exhibited an upward straight line initially for a short period of time (-10 sec), whereas the ratio plot [In (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer 30 jag) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kon), the dissociation rate constant of EGF from the EGF-receptor complex (ko)9 and the sequestration rate constant ofthe complex. The kinetic parameters [the dissociation constant (Kd = kl/k,) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones-cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.Many peptide hormones in the circulating blood are eliminated by the liver. Above all, for hormones taken up via receptor-mediated endocytosis, the liver is a major homeostatic regulator in controlling the concentration of peptide hormones in circulating blood (1-3).The interaction between peptide hormones and their specific receptors has been analyzed principally by in vitro studies with isolated or cultured hepatocytes (4) and liver plasma membranes (5). These systems, however, neither maintain cell polarity nor include ligand delivery by the blood flow, thus creating difficulty when the physiological significance of the peptide-receptor interaction is being experimentally evaluated. Use of the liver-perfusion method may effectively resolve this problem, because cell polarity and spatial architecture between hepatocytes and the capillary bed are maintained in this system. However, determination of the microscopic kinetic constan...

Existence of two pathways for the endocytosis of epidermal growth factor by rat liver: phenylarsine oxide-sensitive and -insensitive pathways.

“…2 and 4), indicating that the probability of an EGF-receptor complex on the cell-surface being internalized per minute is independent of the classes ofbinding sites, as we found previously (14). However, in other studies (9,15,18,20), we could not find multiple binding sites in any experimental system, although nonspecific (unsaturable) binding was observed. We estimated that the equilibrium dissociation constant, Kd, was a few nanomolar in either isolated hepatocytes (18) or isolated liver membrane vesicles (20) and was 18 nM in perfused rat liver (9 McClain and Olefsky (22) have reported that there exist two independent pathways for insulin internalization in hepatocytes and hepatoma cells.…”

“…We have been analyzing the kinetics of the hepatic handling of a model polypeptide, epidermal growth factor (EGF), in rats in vivo (1, 2, 4), in perfused liver (9,14,15), in isolated hepatocytes (18,19), and in isolated liver plasma membrane vesicles (20). We have shown that liver plays a major role in the elimination of EGF from the circulating blood (1, 2) and suggested that the liver may regulate the plasma concentration of EGF via receptor-mediated endocytosis (1, 2, 15).…”

“…To aid in the design of effective polypeptide drugs, knowledge of the factors involved in determining their disposition in vivo is essential. One of the most remarkable features of the pharmacokinetics of polypeptides is the contribution of specific binding sites (receptors) to peptide distribution and elimination in the body (1)(2)(3)(4)(5)(6)(7)(8)(9). Receptor-mediated endocytosis is now well recognized as a general mechanism employed by many cells in the uptake of biologically important polypeptide hormones (1,2,6,10,11), and the concept of "transport (or clearance) receptor" is now well established (7).…”

“…Receptor-mediated endocytosis is now well recognized as a general mechanism employed by many cells in the uptake of biologically important polypeptide hormones (1,2,6,10,11), and the concept of "transport (or clearance) receptor" is now well established (7). The liver and the kidney have been widely accepted as the most important organs in clearing polypeptides from the circulation (1)(2)(3)(4)(12)(13)(14)(15)(16)(17).We have been analyzing the kinetics of the hepatic handling of a model polypeptide, epidermal growth factor (EGF), in rats in vivo (1, 2, 4), in perfused liver (9,14,15), in isolated hepatocytes (18,19), and in isolated liver plasma membrane vesicles (20). We have shown that liver plays a major role in the elimination of EGF from the circulating blood (1, 2) and suggested that the liver may regulate the plasma concentration of EGF via receptor-mediated endocytosis (1, 2, 15).…”

“…commercial products of analytical grade. EGF (human; Wakunaga Pharmaceutical, Osaka) was radiolabeled with 125I by the chloramine-T method (9). 125I-EGF thus obtained had a specific activity of 220-330 Ci/g (1 Ci = 37 GBq).…”