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.

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.

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid

1988 ◽  
Vol 254 (6) ◽  
pp. C829-C838 ◽  
Author(s):  
A. C. Samuelson ◽  
R. J. Stockert ◽  
A. B. Novikoff ◽  
P. M. Novikoff ◽  
J. C. Saez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Ph ◽  
Culture Medium ◽  
Rat Hepatocytes ◽  
Receptor Internalization ◽  
Receptor Complex ◽  
Cytosolic Ph ◽  
Receptor Mediated Endocytosis ◽  
Co2 Diffusion ◽  
Cultured Rat Hepatocytes

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

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

scholarly journals The Effect of Thyroid Hormones on the Beta-Adrenergic Receptor Internalization in Isolated Rat Hepatocytes

1983 ◽  
Vol 59 (11) ◽  
pp. 1693-1702
Author(s):  
Kiyoshi HASHIZUME ◽  
Keishi YAMAUCHI ◽  
Mutsuhiro KOBAYASHI ◽  
Kazutaka HARAGUCHI ◽  
Kazuo ICHIKAWA
Keyword(s):  
Thyroid Hormones ◽  
Adrenergic Receptor ◽  
Rat Hepatocytes ◽  
Receptor Internalization ◽  
Beta Adrenergic Receptor ◽  
Isolated Rat Hepatocytes ◽  
Beta Adrenergic ◽  
Isolated Rat

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

1990 ◽  
Vol 68 (6) ◽  
pp. 657-662 ◽  
Author(s):  
Louise Gariepy ◽  
Daphna Fenyves ◽  
Jean-Luc Petit ◽  
Ginette Raymond ◽  
Jean-Pierre Villeneuve
Keyword(s):  
Free Fraction ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Albumin Concentration ◽  
Isolated Rat Hepatocytes ◽  
Subsequent Elimination ◽  
Rat Livers ◽  
Mediated Catalysis ◽  
Isolated Rat

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (−50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin–propranolol complexes.Key words: propranolol clearance, albumin, isolated rat hepatocytes, cirrhosis.

Angiotensin II receptor internalization and signaling in isolated rat hepatocytes

1999 ◽  
Vol 57 (10) ◽  
pp. 1125-1131 ◽  
Author(s):  
Eugenio Jiménez ◽  
Maria C Caro ◽  
Santo Marsigliante ◽  
Mercedes Montiel
Keyword(s):  
Angiotensin Ii ◽  
Rat Hepatocytes ◽  
Receptor Internalization ◽  
Angiotensin Ii Receptor ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat
Sign in / Sign up

Export Citation Format

Share Document