scholarly journals Intraluminal calcium of the liver endoplasmic reticulum stimulates the glucuronidation of p-nitrophenol

1993 ◽  
Vol 292 (1) ◽  
pp. 99-104 ◽  
Author(s):  
G Bánhegyi ◽  
G Bellomo ◽  
R Fulceri ◽  
J Mandl ◽  
A Benedetti
Keyword(s):  
Endoplasmic Reticulum ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Transferase Activity ◽  
Physiological Concentration ◽  
Isolated Rat Hepatocytes ◽  
Endoplasmic Reticulum Calcium ◽  
Atpase Inhibitors ◽  
Udp Glucuronosyltransferase ◽  
The Relationship

The relationship between the intraluminal Ca2+ content of endoplasmic reticulum and the rate of the glucuronidation of p-nitrophenol was investigated in isolated rat hepatocytes. Different agents which decrease the Ca2+ level in the endoplasmic reticulum [calcium ionophores (A23187, ionomycin) or Ca(2+)-ATPase inhibitors(thapsigargin,2,5-di-(t-butyl)-1,4-benzohydroquinone+ ++)] inhibited the conjugation of p-nitrophenol. Depletion of intracellular Ca2+ stores by preincubation of hepatocytes in the absence of free Ca2+ (in the presence of excess EGTA) also decreased the rate of glucuronidation; Ca2+ re-admission to EGTA-treated hepatocytes restored glucuronidation. In intact liver microsomes the p-nitrophenol UDP-glucuronosyl-transferase activity was not modified by varying the external free Ca2+ concentrations within a cytosol-like range. Emptying of the Ca2+ from the lumen of microsomal vesicles by A23187, after MgATP-stimulated Ca2+ sequestration, decreased the glucuronidation of p-nitrophenol. A similar effect was observed in filipin-permeabilized hepatocytes. In native and in detergent-treated microsomes, Ca2+ (1-10 mM) increased the p-nitrophenol UDP-glucuronosyltransferase activity. It is suggested that the physiological concentration of Ca2+ in the lumen of the endoplasmic reticulum is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyltransferase; the depletion of Ca2+ decreases the activity of the enzyme.

scholarly journals Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes

1997 ◽  
Vol 328 (2) ◽  
pp. 463-471 ◽  
Author(s):  
C. Kekulu FERNANDO ◽  
B. Roland GREGORY ◽  
Frosa KATSIS ◽  
E. Bruce KEMP ◽  
J. Greg BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mass ◽  
G Protein ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Hepatocytes ◽  
Gtp Binding

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 μM intracellular) of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 μM intracellular) of GTP[S] or guanosine 5ʹ-[βγ-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 μM) prevented the inhibition of Ca2+ inflow by GTP[S] and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4- (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5)P3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

scholarly journals The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

1986 ◽  
Vol 237 (1) ◽  
pp. 33-39 ◽  
Author(s):  
E Fries ◽  
I Lindström
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Low Temperatures ◽  
Intracellular Transport ◽  
Rat Hepatocytes ◽  
Subcellular Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Different Temperatures ◽  
Lag Period ◽  
Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes

1984 ◽  
Vol 219 (3) ◽  
pp. 911-916 ◽  
Author(s):  
C Cascales ◽  
E H Mangiapane ◽  
D N Brindley
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Microsomal Fraction ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Cell Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Phosphatidate Phosphohydrolase ◽  
Isolated Rat

The incubation of hepatocytes with 1-4mM-oleate increased the total activity of phosphatidate phosphohydrolase that was measured in the presence of Mg2+ to about 2-fold. This was accompanied by an increase in the proportion of the enzyme that was isolated with the particulate fractions. Conversely, the addition of up to 4mM-oleate decreased the recovery of phosphatidate phosphohydrolase in the cytosolic fraction from about 70% to 3% when hepatocytes were lysed with digitonin. Most of the increase in the membrane-associated phosphohydrolase activity was isolated after cell fractionation in the microsomal fraction that was enriched with the endoplasmic-reticulum marker arylesterase. It is proposed that the translocation of phosphatidate phosphohydrolase facilitates the increased synthesis of triacylglycerols in the liver when it is presented with an increased supply of fatty acids.

scholarly journals Glucosamine-sensitive and -insensitive detritiation of [2-3H]glucose in isolated rat hepatocytes: a study of the contributions of glucokinase and glucose-6-phosphatase

1995 ◽  
Vol 308 (1) ◽  
pp. 23-29 ◽  
Author(s):  
E Van Schaftigen
Keyword(s):  
Exchange Reaction ◽  
Regulatory Protein ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Amino Sugar ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
In Situ Experiments ◽  
Glucose Phosphorylation ◽  
Isolated Rat

Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.

scholarly journals Inhibition of phosphatidylcholine synthesis by vasopressin and angiotensin in rat hepatocytes

1982 ◽  
Vol 208 (2) ◽  
pp. 453-457 ◽  
Author(s):  
S Alemany ◽  
I Varela ◽  
J M Mato
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Hepatocytes ◽  
Fast Transient ◽  
Phosphatidylcholine Synthesis ◽  
Isolated Rat

The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.

Stereochemical Aspects of 1,3-Butadiene Metabolism and Toxicity in Rat and Mouse Liver Microsomes and Freshly Isolated Rat Hepatocytes

1997 ◽  
Vol 10 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Joe L. Nieusma ◽  
David J. Claffey ◽  
Chris Maniglier-Poulet ◽  
Tomasz Imiolczyk ◽  
David Ross ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Freshly Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals Role of Cell Death in Toxicology: Does It Matter How Cells Die?

2019 ◽  
Vol 59 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Sten Orrenius
Keyword(s):  
Cell Death ◽  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Rat Liver Microsomes ◽  
Research Activity ◽  
Isolated Rat Hepatocytes ◽  
Cellular Defense ◽  
Toxic Injury

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

scholarly journals Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes

1999 ◽  
Vol 341 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Roland B. GREGORY ◽  
Robert A. WILCOX ◽  
Leise A. BERVEN ◽  
Nicole C. R. VAN STRATEN ◽  
Gijs A. VAN DER MAREL ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Cytoskeleton ◽  
Cytochalasin B ◽  
Rat Hepatocytes ◽  
Small Region ◽  
Cortical Actin ◽  
Isolated Rat Hepatocytes ◽  
Maximal Stimulation ◽  
Adenophostin A ◽  
Stimulation Of

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-α-D-glucopyranoside 2,3′,4′-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4,5)P3P4(5)-1-(2-nitrophenyl)ethyl ester [‘caged’ Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

Translocon pores in the endoplasmic reticulum are permeable to small anions

2006 ◽  
Vol 291 (3) ◽  
pp. 511-517 ◽  
Author(s):  
Beáta Lizák ◽  
Ibolya Czegle ◽  
Miklós Csala ◽  
Angelo Benedetti ◽  
József Mandl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Active Sites ◽  
Liver Microsomes ◽  
Endoplasmic Reticulum Membrane ◽  
Rat Liver Microsomes ◽  
Microsomal Enzymes ◽  
Active Centers ◽  
Udp Glucuronosyltransferase ◽  
Luminal Compartment

Contribution of translocon peptide channels to the permeation of low molecular mass anions was investigated in rat liver microsomes. Puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores, raised the microsomal uptake of radiolabeled UDP-glucuronic acid, while it did not increase the uptake of glucose-6-phosphate or glutathione. The role of translocon pores in the transport of small anions was also investigated by measuring the effect of puromycin on the activity of microsomal enzymes with intraluminal active sites. The mannose-6-phosphatase activity of glucose-6-phosphatase and the activity of UDP-glucuronosyltransferase were elevated upon addition of puromycin, but glucose-6-phosphatase and β-glucuronidase activities were not changed. The increase in enzyme activities was due to a better access of the substrates to the luminal compartment rather than to activation of the enzymes. Antibody against Sec61 translocon component decreased the activity of UDP-glucuronosyltransferase and antagonized the effect of puromycin. Similarly, the addition of the puromycin antagonist anisomycin or treatments of microsomes, resulting in the release of attached ribosomes, prevented the puromycin-dependent increase in the activity. Mannose-6-phosphatase and UDP-glucuronosyltransferase activities of smooth microsomal vesicles showed higher basal latencies that were not affected by puromycin. In conclusion, translationally inactive, ribosome-bound translocons allow small anions to cross the endoplasmic reticulum membrane. This pathway can contribute to the nonspecific substrate supply of enzymes with intraluminal active centers.

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