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

Abstract

The effect of phenylarsine oxide (PAO) on the internalization rate of epidermal growth factor (EGF) was investigated using perfused rat liver and isolated rat hepatocytes. In perfused liver, a tracer concentration of 125I-EGF alone or with excess unlabeled EGF (20 nM) was perfused and the internalization rate constants (knJ) were measured. In the absence of PAO, kint values did not differ significandly for either dose condition. However, with the addition of PAO to the perfusate, the kint value dropped to 4% of that of the control at the low concentration of EGF, while dropping to only 40% of that of the control at the high concentration of EGF. These results suggest the existence of a PAO-insensitive internalization pathway having a ki.t value comparable with that of the other pathway. Similar EGF concentration-dependent inhibition of II-EGF internalization caused by PAO was ascertained using isolated rat hepatocytes. PAO also decreased the cellular ATP content in isolated hepatocytes. However, when we lowered the cellular ATP content with rotenone, the cell-surface binding and internalization of EGF were comparable with the control levels. We concluded that there exist dual pathways for the internalization of EGF and that excess doses of EGF lead to EGF internalization not only through a PAO-sensitive pathway but also through a PAO-insensitive pathway, whereas at a tracer dose of EGF, the internalization occurs mainly via the PAOsensitive pathway.The discovery of a diversity of neuro-and hormonal polypeptides has increased interest in exploiting peptides and their analogues as therapeutic drugs. In general, the plasma halflives of polypeptides are very short, which becomes a major stumbling block in the development of polypeptide drugs. 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 (1-9). 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). 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). In the presence of excess EGF, the binding of EGF to...

Uptake of Pullulan in Cultured Rat Liver Parenchymal Cells

“…4 and 5. The rate of internalization of pullulan into cultured rat liver parenchymal cells was estimated by the method of Kato et al 9) From the initial slope of plots shown in Fig. 2, the internalization rate of pullulan (k int ) was estimated at 0.30Ϯ0.13 min…”

“…The amount of protein was determined by the method of Lowry et al 8) Data Analysis Internalization data were analyzed by Eq. 1 according to the method described by Kato et al 9) ( 1) where I t is the amount of internalization at time t, k int is the rate constant of initial internalization, and L s is the amount bound to the cell surface. The value of k int was estimated from the initial slope of…”

Oxidant inhibition of αLβ2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage

“…7), a feature that probably accounts for previously reported complex effects of PAO on signal transduction pathway intermediates and enzymatic activities. For example, regulated fluctuations in intracellular calcium concentration and tyrosine phosphorylation of a number of different phosphoproteins have been reported to be either inhibited or enhanced by PAO, the outcome depending on the signaling pathway or protein investigated and the timing and concentration of PAO application [38,[44][45][46][47][48][49][50][51][52][53]. Thus, it seems unlikely that LFA-1 adhesion-regulatory processes are an exclusive target of PAO, but rather represent one of a number of PAO-sensitive physiological processes.…”

Antigen-Dependent Internalization Is Related to Rapid Elimination from Plasma of Humanized Anti-HM1.24 Monoclonal Antibody

“…It is known that endocytosis is involved in the internalization mediated by antigens and receptors in various biologically active proteins and therapeutic antibodies (Pulczynski et al, 1994;Kato et al, 1996Kato et al, , 1997Lammerts van Bueren et al, 2006). Indeed, internalization of 125 I-AHM by KPMM2 cells in the presence of PAO (Finbloom, 1988;Kato et al, 1992), which is an endocytosis inhibitor, was significantly decreased (i.e., 1/21 that in the absence of PAO). Consistent with our recent results (Masuyama et al, 2009), it is evident that at least some 125 I-AHM was internalized by the endocytosis mechanism.…”

“…The inhibition experiment was conducted in a manner similar to that described above except for the addition of 0.5 mM PAO to the medium, which is known to inhibit the endocytosis of protein (Finbloom, 1988;Kato et al, 1992).…”