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 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.

Regulation of ANG II and AVP receptors in isolated hepatocytes of pregnant rats

1990 ◽  
Vol 258 (4) ◽  
pp. E597-E605
Author(s):  
G. Massicotte ◽  
L. Coderre ◽  
J. L. Chiasson ◽  
G. Thibault ◽  
E. L. Schiffrin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Maximum Response ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Single Class ◽  
Pregnant Rats ◽  
Isolated Rat Hepatocytes ◽  
Biological Responses

Recent evidence suggests that angiotensin II (ANG II) and vasopressin (AVP) act on the liver via specific receptors. We have examined the binding properties of these receptors in isolated rat hepatocytes and studied the regulation of the biological responses to ANG II and AVP during pregnancy in the rat. In contrast to [3H]ANG II, 125I-labeled-[Sar1-Ile8]ANG II was markedly resistant to degradation by isolated liver cells. Displacement and saturation experiments with this iodinated antagonist revealed the presence of a single class of binding sites [2 x 10(5) sites/cell, dissociation constant (KD) = 1.0 nM]. The potency of ANG II analogues to displace 125I-[Sar1-Ile8]-ANG II agrees closely with data reported for vascular smooth muscle cells. Isolated hepatocytes have approximately 8 x 10(4) [3H]AVP binding sites/cell (KD = 1.0 nM) based on saturation experiments. AVP analogues selectively displaced [3H]AVP, suggesting the presence of V1-AVP receptor subtype. The maximum response of [Sar1]ANG II-induced glycogenolysis in the cells was decreased during gestation, whereas the effective concentration producing 50% of maximum response (EC50) was significantly increased (0.15-0.28 nM) when compared with cells from nonpregnant animals. In pregnancy, receptors for 125I-[Sar1-Ile8]ANG II were not changed in affinity (KD) or in density (Bmax). The maximum response and EC50 of AVP on liver glycogenolysis were not significantly decreased during pregnancy, whereas an increased number of AVP binding sites (from 5.0 +/- 0.5 x 10(4) to 11.0 +/- 1.7 x 10(4)) with similar KD was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane fluidity in isolated rat hepatocytes: A comparative study using DPH and TMA-DPH as fluorescent probes

1989 ◽  
Vol 4 (3) ◽  
pp. 221-227 ◽  
Author(s):  
ANTONIO BENEDETTI ◽  
GIANNA FERRETTI ◽  
GIOVANNA CURATOLA ◽  
EUGENIO BRUNELLI ◽  
ANNE MARIE JÉZÉQUEL ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Comparative Study ◽  
Membrane Fluidity ◽  
Fluorescent Probes ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Plasma Membrane Fluidity ◽  
Isolated Rat

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

scholarly journals Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes

1987 ◽  
Vol 243 (3) ◽  
pp. 655-660 ◽  
Author(s):  
P B Gordon ◽  
H Høyvik ◽  
P O Seglen
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Free Sugar ◽  
Sugar Uptake ◽  
Isolated Rat Hepatocytes ◽  
Fluid Phase Endocytosis ◽  
And Diffusion ◽  
Isolated Rat

Measurements of sugar pinocytosis (fluid-phase endocytosis of radiolabelled sucrose, lactose and raffinose) in freshly isolated rat hepatocytes are disturbed by sugar diffusing into the cells through plasma-membrane blebs. Non-pinocytic entry may be even more pronounced at 0 degrees C, and is a major contributor to ‘background’ radioactivity. By electrodisruption of the plasma membrane, a distinction can be made between pinocytotically sequestered sugar and free sugar that has entered the cytosol by diffusion. Pinocytosis proceeds at a rate of 2%/h (relative to the intracellular fluid volume), whereas the rate of sucrose entry by diffusion is more than twice as high. Three pinocytotic compartments are distinguishable in isolated hepatocytes: (1) a rapidly recycling compartment, which is completely destroyed by electrodisruption, and which may represent pinocytic channels continuous with the plasma membrane; (2) a non-recycling (or very slowly recycling) electrodisruption-resistant compartment, which allows accumulation of the lysosomally hydrolysable sugar lactose, and which therefore must represent non-lysosomal vacuoles (endosomes?); (3) a lysosomal compartment (non-recycling, electrodisruption-resistant), which accumulates raffinose and sucrose, but which hydrolyses lactose. The last two compartments can be partially resolved in metrizamide/sucrose density gradients by the use of different sugar probes.

scholarly journals Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase

1986 ◽  
Vol 240 (2) ◽  
pp. 367-371 ◽  
Author(s):  
S Keppens ◽  
H De Wulf
Keyword(s):  
Binding Sites ◽  
Glycogen Phosphorylase ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Rank Order ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
P2 Purinoceptors

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5′-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

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