scholarly journals Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes

1987 ◽  
Vol 241 (3) ◽  
pp. 835-845 ◽  
Author(s):  
D E Whipps ◽  
A E Armston ◽  
H J Pryor ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Isolated Rat Hepatocytes ◽  
Guanine Nucleotide Regulatory Protein ◽  
Phosphatidylinositol 4

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.

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

Effect of glucagon on hepatic taurocholate uptake: relationship to membrane potential

1985 ◽  
Vol 249 (4) ◽  
pp. G427-G433
Author(s):  
J. W. Edmondson ◽  
B. A. Miller ◽  
L. Lumeng
Keyword(s):  
Plasma Membrane ◽  
Bile Acid ◽  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hill Coefficient ◽  
Isolated Rat Hepatocytes ◽  
Plasma Membrane Potential ◽  
Sodium Dependent

Since glucagon can hyperpolarize hepatic plasma membrane and stimulate biliary bile acid secretion in vitro, we studied the effect of glucagon on taurocholate uptake and its relationship to plasma membrane potential in isolated rat hepatocytes. [14C]taurocholate uptake was linear through 1 min and contained a saturable sodium-dependent and a nonsaturable sodium-independent component. Km of taurocholate uptake by the sodium-dependent system was 18.4 microM. Hill coefficient for Na+ was 2.59 and for taurocholate was 1.1, suggesting that the stoichiometry is 2 Na+:1 bile acid. Stimulation of taurocholate uptake by glucagon was limited to the sodium-dependent component, detected within 5 min of hormone exposure, and was maximum at 30 min. Glucagon, from 10(-8) to 10(-5) M, stimulated taurocholate uptake and hyperpolarized concurrently the plasma membrane potential. Because valinomycin produced a dose-related depolarization of plasma membrane potential, this agent was used to counteract the effects of glucagon. With 10(-6) M glucagon, valinomycin (10(-10) M) depolarized membrane potential from -35.50 to -28.00 mV and inhibited taurocholate uptake from 60% above the control rate to 5% below. These data strongly suggest that taurocholate uptake by isolated hepatocytes is an electrogenic process, and its stimulation by glucagon may be mediated by changes in plasma membrane potential.

scholarly journals The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes

1986 ◽  
Vol 235 (1) ◽  
pp. 145-149 ◽  
Author(s):  
C M Heyworth ◽  
A M Grey ◽  
S R Wilson ◽  
E Hanski ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Guanine Nucleotide Regulatory Protein

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and ‘dense-vesicle’ cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.

scholarly journals Protein kinase A regulates the disposition of Ca2+ which enters the cytoplasmic space through store-activated Ca2+ channels in rat hepatocytes by diverting inflowing Ca2+ to mitochondria

1998 ◽  
Vol 330 (3) ◽  
pp. 1179-1187 ◽  
Author(s):  
C. Kekulu FERNANDO ◽  
B. Roland GREGORY ◽  
J. Greg BARRITT
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Ruthenium Red ◽  
Maximal Effect ◽  
Isolated Rat Hepatocytes ◽  
Carbonyl Cyanide ◽  
Ca2 Channels ◽  
Kinase A

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5ʹ-[γ-thio]-triphosphate (GTP[S]) and AlF4- inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3ʹ,5ʹ-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

scholarly journals Preparation of plasma-membrane subfractions from isolated rat hepatocytes

1977 ◽  
Vol 164 (2) ◽  
pp. 415-422 ◽  
Author(s):  
M H Wisher ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Liver Tissue ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Electrophoretic Analysis ◽  
Isolated Hepatocytes ◽  
Marker Enzyme ◽  
Isolated Rat Hepatocytes ◽  
Tissue Dissociation ◽  
Enzyme Distribution

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two ‘light’ vesicular and one ‘heavy’ junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.

The Hep G2 Cell Line as a Possible Alternative to Isolated Hepatocytes in Cytotoxicity Studies

1988 ◽  
Vol 16 (1) ◽  
pp. 16-22
Author(s):  
Marina Marinovich ◽  
Jose L. Lorenzo ◽  
Liliana M. Flaminio ◽  
Agnese Granata ◽  
Corrado L. Galli
Keyword(s):  
Cell Line ◽  
Rat Hepatocytes ◽  
Prostaglandin E ◽  
Human Hepatoma ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Isolated Rat Hepatocytes ◽  
G2 Cells ◽  
Isolated Rat

The hepatotoxicity of carbon tetrachloride (CC14) was evaluated in vitro in freshly isolated rat hepatocytes and in the human hepatoma cell line, Hep G2. Toxicity was assessed by the leakage of cytosolic enzymes (lactate dehydrogenase and aspartate aminotransferase) and cell viability (trypan blue exclusion). The established human cells were less sensitive to CCl4-induced injury; higher doses of the toxic agent and longer incubation times were necessary to elicit cell damage. Micromolar concentrations of prostaglandin E2 significantly decreased enzyme leakage in both Hep G2 cells and rat hepatocytes challenged with CC14; a stable derivative of prostacyclin (ZK 36374) was ineffective. These results suggest that human hepatoma Hep G2 cells may represent a valid alternative to isolated rat hepatocytes for an initial approach to the in vitro evaluation of cell toxicity.

Acellular liver matrix improves the survival and functions of isolated rat hepatocytes cultured in vitro.

2004 ◽  
Author(s):  
Patrizia Burra ◽  
Silvia Tomat ◽  
Maria Teresa Conconi ◽  
Carlo Macchi ◽  
Francesco P Russo ◽  
...  
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

1982 ◽  
Vol 202 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Clive J. Dix ◽  
Matthias Schumacher ◽  
Brian A. Cooke
Keyword(s):  
Cyclic Amp ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Linear Increase ◽  
Guanine Nucleotide ◽  
Production Rates ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Guanine Nucleotide Binding ◽  
Guanine Nucleotide Regulatory Protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

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