Kinetic analysis of receptor-mediated endocytosis of epidermal growth factor by isolated rat hepatocytes

1991 ◽  
Vol 260 (3) ◽  
pp. C457-C467 ◽  
Author(s):  
S. Yanai ◽  
Y. Sugiyama ◽  
D. C. Kim ◽  
T. Iga ◽  
T. Fuwa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Rate Constant ◽  
Egf Receptor ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Isolated Rat Hepatocytes ◽  
Surface Receptors ◽  
Internalization Rate ◽  
Epidermal Growth ◽  
Isolated Rat

The interaction of epidermal growth factor (EGF) with cell surface receptors and their subsequent endocytosis in isolated rat hepatocytes were analyzed by measuring changes in the concentrations of cell surface-bound, internalized, and degraded EGF. The kinetic model proposed by Wiley and Cunningham (Cell 25: 433-440, 1981) and Gex-Fabry and Delisi [Am. J. Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R768-R779, 1984] was basically utilized for the model analysis. The following kinetic parameters were obtained: association and dissociation rate constants for EGF-receptor interaction, internalization rate constant for EGF-receptor complex (kappa e), internalization rate constant for free receptor (kappa t), sequestration rate constant (kappa s) of the complex from shallow (exchangeable) to deep (nonexchangeable) membraneous compartment, intracellular degradation rate constant and initial cell-surface receptor density. The kappa s value, which was obtained by analyzing the time profiles of EGF association with cells, was approximately 5-10 times larger than the kappa e value determined by directly measuring internalized EGF with the acid-washing technique. This suggests the necessary presence of deep (nonexchanging) compartment of the complex in the plasma membrane. The calculated kappa e value is at least several times larger than the kappa t value, yielding the kinetic basis for the occurrence of receptor downregulation induced by excess EGF. We conclude that, in the overall receptor-mediated processing of EGF after bound to the cell surface receptors, the dissociation process is rapid [half-time (t1/2) less than 1 min], the degradation process is much slower (t1/2 approximately equal to 3 h), and the receptor internalization process is intermediate (t1/2 approximately equal to 6-7 min). In addition, two pools for EGF-receptor complex in the plasma membrane seem to be present, although their identification cannot be made.

Internalization of EGF in perfused rat liver is independent of the degree of receptor occupancy

1990 ◽  
Vol 258 (5) ◽  
pp. G682-G689
Author(s):  
H. Sato ◽  
Y. Sugiyama ◽  
Y. Sawada ◽  
T. Iga ◽  
T. Fuwa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Rat Liver ◽  
Rate Constant ◽  
Egf Receptor ◽  
Receptor Occupancy ◽  
Perfused Rat Liver ◽  
Surface Receptors ◽  
Surface Receptor ◽  
Internalization Rate ◽  
20 Nm

The internalization rate of epidermal growth factor (EGF) by the perfused rat liver was evaluated to determine whether the internalization rate constant depends on the degree of receptor occupancy. A tracer concentration of 125I-labeled EGF (30 pM) alone or 125I-EGF plus unlabeled EGF (20 nM) was infused into the liver in the single-pass perfusion system at 37 degrees C. At various times (2-20 min), the perfusion medium was switched to medium of pH 3.0, and the radioactivity of 125I-EGF emerging into the outflow (surface-bound EGF) and remaining in the liver (internalized EGF) was determined. At the tracer condition, less than 0.1% of the surface receptor was occupied by 125I-EGF for 20 min perfusion. When excess unlabeled EGF (20 nM) was present in the perfusate and the bulk of the cell surface receptors was occupied, the density of the cell surface EGF receptor after 20 min dropped to 14% of the initial value. The internalization rate constant, defined as the probability of an occupied receptor being internalized per minute, was calculated from the slope of a plot of the amount of internalized EGF vs. the integrated amount of surface receptor-bound EGF with time. The internalization rate constants calculated from the plots with and without unlabeled EGF in the perfusate were 0.21 and 0.33 min-1, respectively, and did not differ significantly (P greater than 0.1). These results indicated that the internalization rate constant of the EGF receptor complex is independent of the degree of receptor occupancy in the liver.

scholarly journals Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes

1984 ◽  
Vol 218 (2) ◽  
pp. 307-312 ◽  
Author(s):  
B Draznin ◽  
M Trowbridge ◽  
L Ferguson
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Receptor Internalization ◽  
Insulin Concentration ◽  
Receptor Complex ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.

scholarly journals Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

1982 ◽  
Vol 92 (3) ◽  
pp. 634-647 ◽  
Author(s):  
PL Zeitlin ◽  
AL Hubbard
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Rat Hepatocytes ◽  
Surface Binding ◽  
Isolated Rat Hepatocytes ◽  
Electron Microscopic Autoradiography ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Isolated Rat

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes

1997 ◽  
Vol 152 (3) ◽  
pp. 407-412 ◽  
Author(s):  
M Montiel ◽  
M C Caro ◽  
E Jiménez
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Ang Ii ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5. Journal of Endocrinology (1997) 152, 407–412

Different modulation of hepatocellular Na+/H+ exchange activity by insulin and EGF

1994 ◽  
Vol 267 (3) ◽  
pp. G364-G370 ◽  
Author(s):  
J. Haimovici ◽  
J. S. Beck ◽  
C. Molla-Hosseini ◽  
D. Vallerand ◽  
P. Haddad
Keyword(s):  
Liver Cell ◽  
Indirect Evidence ◽  
Rat Hepatocytes ◽  
Regulatory Mechanisms ◽  
Isolated Rat Hepatocytes ◽  
Removal Experiments ◽  
Epidermal Growth ◽  
Isolated Rat ◽  
Exchange Activity

Indirect evidence suggests that insulin, like epidermal growth factor (EGF), stimulates liver cell Na+/H+ exchange. We directly studied the effect of insulin on intracellular pH (pHi) and the Na+/H+ exchanger in isolated rat hepatocytes with the fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The effects of insulin were compared with those of EGF. Studies were carried out in the absence of HCO3- and in the presence of acetazolamide to isolate the Na+/H+ exchanger from other pH regulatory mechanisms. Insulin (9 nM) caused a reversible acidification of baseline pHi, whereas no significant effect was observed with EGF (30 nM). pHi was acidified by two different methods (NH4Cl pulse and external Na+ removal) to assess liver cell Na+/H+ exchange activity. In the NH4Cl pulse experiments, insulin had no significant effect on the Na+/H+ exchanger compared with the control (0.141 +/- 0.009 pH units/min, n = 14, and 0.122 +/- 0.023 pH units/min, n = 16, respectively). However, in the same conditions, EGF nearly doubled the rate of the Na+/H+ exchange activity (0.193 +/- 0.015 pH units/min, P < 0.05, n = 6). In the Na+ removal experiments, EGF again significantly increased the pHi recovery rate (0.542 +/- 0.032 pH units/min, n = 3) compared with the control (0.227 +/- 0.028 pH units/min, n = 5) and insulin (0.245 +/- 0.053 pH units/min, n = 5). Compared with control conditions, a subchronic administration of insulin (9 nM) in vitro had no significant effect on the Na+/H+ exchanger, nor did it affect baseline pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Epidermal Growth Factor (EGF) on Thyroid Function in the Sheep and in Isolated Rat Hepatocytes

1986 ◽  
pp. 359-363
Author(s):  
Jo M. Corcoran ◽  
Michael J. Waters ◽  
Jonathan Marks ◽  
Greg Jorgensen ◽  
Kay V. Waite ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Thyroid Function ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Epidermal Growth ◽  
Isolated Rat
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