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.

Characterization of glucagon and catecholamine effects on isolated sheep hepatocytes

1988 ◽  
Vol 255 (4) ◽  
pp. R539-R546
Author(s):  
C. Morand ◽  
C. Yacoub ◽  
C. Remesy ◽  
C. Demigne
Keyword(s):  
Rat Liver ◽  
Glycogen Phosphorylase ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cyclic Monophosphate ◽  
Sheep Liver ◽  
Beta Agonists ◽  
Rat Liver Cells

The purpose of this study was to characterize the glycogenolytic response to catecholamines and glucagon in isolated sheep hepatocytes. In this species, epinephrine appeared to exert its action on hepatic glycogenolysis by altering the cytosolic concentrations of both adenosine 3',5'-cyclic monophosphate (cAMP) and Ca2+. In contrast to results obtained in rat hepatocytes, glucagon failed to induce a rise in free cytosolic Ca2+ in sheep liver. Experiments on isolated hepatocytes or on liver plasma membranes showed that in sheep, glucagon was more efficient than epinephrine in promoting the production of cAMP. In the presence of glucagon or epinephrine, the activation of the glycogen phosphorylase a always appeared greater in sheep than in rat liver cells, whereas the variations in cellular cAMP were quite limited in sheep. The alpha 1- and beta-agonists (phenylephrine and isoproterenol) were alone as efficient as epinephrine in promoting phosphorylase a activation in sheep hepatocytes. All these results indicate the existence in sheep liver of a glycogen phosphorylase highly responsive to hormones.

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)

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

Characterization of the Binding of Bovine Thrombin to Isolated Rat Hepatocytes

1988 ◽  
Vol 60 (03) ◽  
pp. 419-427 ◽  
Author(s):  
Britta Weyer ◽  
Torben E Petersen ◽  
Ole Sonne
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Prolonged Incubation ◽  
Receptor Assay ◽  
Isolated Rat

SummaryIsolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 × 105 low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37¸ C and 7 min at 4¸ C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1—2 × 10−2 s−1 and 3—4 × 10−4 s−1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just aswell in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation ofthe ligand.

Characterization of Somatogenic and Lactogenic Binding Sites in Isolated Rat Hepatocytes

Endocrinology ◽  
1976 ◽  
Vol 99 (4) ◽  
pp. 1033-1045 ◽  
Author(s):  
MICHAEL B. RANKE ◽  
CHARLES A. STANLEY ◽  
ALFRED TENORE ◽  
DAVID RODBARD ◽  
ALFRED M. BONGIOVANNI ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

scholarly journals Effects of bile salts on the plasma membranes of isolated rat hepatocytes

1980 ◽  
Vol 188 (2) ◽  
pp. 321-327 ◽  
Author(s):  
D Billington ◽  
C E Evans ◽  
P P Godfrey ◽  
R Coleman
Keyword(s):  
Alkaline Phosphatase ◽  
Bile Salt ◽  
Bile Salts ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Soluble Form ◽  
Isolated Rat Hepatocytes ◽  
Low Concentrations

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20–30% of the plasma-membrane enzymes 5′-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10–20% of the 5′-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5–2.0mM) than either glycocholate or taurocholate (12–16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly ‘soluble’ form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.

scholarly journals Quantitative analysis of tubulin and microtubule compartments in isolated rat hepatocytes.

1977 ◽  
Vol 75 (3) ◽  
pp. 731-742 ◽  
Author(s):  
E P Reaven ◽  
Y Cheng ◽  
M D Miller
Keyword(s):  
Binding Sites ◽  
High Capacity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Scatchard Analysis ◽  
Isolated Rat Hepatocytes ◽  
Actual Length ◽  
High Affinity ◽  
Biochemical Approach ◽  
Two Populations

A combined morphometric and biochemical approach has been used to identify and quantitate microtubules and tubulin in isolated hepatocytes. The total soluble pool of microtubule protein was estimated by specific high affinity binding to radiolabeled colchicine. Scatchard analysis of the data identified two populations of binding sites: high affinity-low capacity sites resembling tubulin and low affinity-high capacity sites believed to represent nonspecific colchicine-binding sites. Data from these studies indicate that tubulin represents 1% of the soluble protein of the cell, that 9.0 X 10(-14) dimers of tubulin are present per microgram soluble hepatocyte protein, and that the average hepatocyte contains 3.1 X 10(7) tubulin dimers. Our calculations suggest that this amount of tubulin would form a microtubule 1.9 cm in length if totally assembled. However, stereological measurements indicate that the actual length of microtubules in the cytosolic compartment of the average hepatocyte is only 0.28 cm. Thus, these experiments suggest that only 15% of the available tubulin in hepatocytes of postabsorptive rats is assembled in the form of microtubules.

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