The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation

1983 ◽  
Vol 56 (2) ◽  
Author(s):  
Anders Fjose ◽  
IanF. Pryme ◽  
JohanR. Lillehaug
Keyword(s):  
Endoplasmic Reticulum ◽  
Phorbol Ester ◽  
Subcellular Fractions ◽  
In Vitro Incubation ◽  
Choline Incorporation ◽  
Stimulation Of

scholarly journals In vitro effects of L-arginine on lysosomal cysteine proteases activity in isolated experiment and in the state of oxidative stress

2015 ◽  
Vol 96 (5) ◽  
pp. 876-882
Author(s):  
M A Fomina ◽  
A M Kudlaeva
Keyword(s):  
Oxidative Stress ◽  
Sucrose Solution ◽  
Membrane Damage ◽  
Cysteine Proteases ◽  
Lysosomal Membranes ◽  
In Vitro Incubation ◽  
Cathepsin H ◽  
Lysosomal Cysteine Proteases ◽  
Stimulation Of

Aim. Assessment of direct influence of arginine on lysosomal cysteine proteases activity in vitro, in isolation as well as the stimulation of oxidative stress. Methods. The study was conducted on the 72 female conventional mature Wistar rats 280-320 g divided into 6 series of 12 rats each. Lysosome slurries were isolated from the liver of intact animals with a subsequent in vitro incubation in a sucrose solution, in the presence of L-arginine, as well as in the presence of L-arginine accompanied by the stimulation of oxidative stress. Samples of control groups were exposed in vitro with the addition of isolate and oxidant, respectively. Each batch was reproduced three times, incubation was performed at 37 °C in a water bath for 1, 2 and 4 hours. The activity of cathepsins B, L and H was studied using spectrofluorimetric method in two fractions - intra- and extralysosomal. Acid phosphatase activity was used as the main marker of membrane labialization. Results. One hour Incubation with 5 mM arginine in vitro led to inhibition of the cathepsin H activity and lysosomal membrane damage, however, further increase in incubation time led to its stabilization. In vitro exposure to 5 mM H2O2 caused an increase in activity of cathepsines B and L and the drop in the cathepsin H activity without obvious changes in the distribution of enzymes between extra and intralysosomal fractions. In a state of oxidative stress 2-hour in vitro incubation with 5 mM arginine reduced the permeability of lysosomal membranes for cathepsines B, H and L; while 4-hour incubation led to the destabilization of lysosomal membranes. Conclusion. The direct effect of arginine at a concentration of 5 mM within the 1,2 and 4-hour time intervals leads to a distinct change as a lysosomal cysteine protease activity and stability of lysosomal membranes.

scholarly journals A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation

1978 ◽  
Vol 176 (1) ◽  
pp. 9-14 ◽  
Author(s):  
D N Palmer ◽  
B R Rabin ◽  
D J Williams
Keyword(s):  
Lipid Peroxidation ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Chemical Carcinogen ◽  
Centrifugal Forces ◽  
In Vitro Incubation ◽  
Liver Membrane ◽  
Membrane Bound

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.

scholarly journals The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fractions in vitro. General features of the systems used

1971 ◽  
Vol 123 (5) ◽  
pp. 805-814 ◽  
Author(s):  
T. F. Slater ◽  
B. C. Sawyer
Keyword(s):  
Lipid Peroxidation ◽  
Endoplasmic Reticulum ◽  
Carbon Tetrachloride ◽  
Rat Liver ◽  
Lipid Peroxides ◽  
Square Root ◽  
Microsomal Membranes ◽  
Stimulation Of ◽  
Homolytic Dissociation

1. The general features of the reaction by which carbon tetrachloride stimulates lipid peroxidation have been elucidated in rat liver microsomal suspensions and in mixtures of microsomes plus cell sap. The production of lipid peroxides has been correlated with malonaldehyde production in the systems used. 2. The stimulation of malonaldehyde production by carbon tetrachloride requires a source of reduced NADP+ and is dependent on the extent of the endogenous peroxidation of the microsomal membranes: if extensive endogenous peroxidation occurs during incubation then no stimulation by carbon tetrachloride is apparent. 3. The stimulation of malonaldehyde production by carbon tetrachloride has been shown to be proportional to the square root of the carbon tetrachloride concentration in the incubation mixture. It is concluded that the stimulation of malonaldehyde production by carbon tetrachloride results from an initiation process that is itself dependent on the homolytic dissociation of carbon tetrachloride to free-radical products. 4. The increased production of malonaldehyde due to carbon tetrachloride is accompanied by a decreased activity of glucose 6-phosphatase in rat liver microsomal suspensions. 5. The relative activities of bromotrichloromethane, fluorotrichloromethane and chloroform have been evaluated in comparison with the effects of carbon tetrachloride in increasing malonaldehyde production and in decreasing glucose 6-phosphatase activity. Bromotrichloromethane was more effective, and fluorotrichloromethane and chloroform were less effective, than carbon tetrachloride in producing these two effects. It is concluded that homolytic bond fission of the halogenomethanes is a requisite for the occurrence of the two effects observed in the endoplasmic reticulum.

scholarly journals The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

1975 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
G Ragnotti ◽  
M G Aletti
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Protein Biosynthesis ◽  
Liver Weight ◽  
Subcellular Fractions ◽  
Intrinsic Defect ◽  
Preliminary Treatment ◽  
Membrane Bound ◽  
Stimulation Of

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.

scholarly journals Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the ‘de novo’ pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake

1993 ◽  
Vol 293 (3) ◽  
pp. 739-744 ◽  
Author(s):  
H Tronchère ◽  
F Tercé ◽  
M Record ◽  
H Chap
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Phorbol Ester ◽  
De Novo ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Choline Uptake ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Choline Incorporation ◽  
Unique Effect

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the ‘de novo’ pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the ‘de novo’ pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).

Stimulation of bovine luteal oxytocin secretion in vitro by a phorbol ester and calcium ionophore.

1990 ◽  
Vol 68 (8) ◽  
pp. 2465 ◽  
Author(s):  
C A Cosola-Smith ◽  
L H Appell ◽  
S E Abdelgadir ◽  
F Stormshak
Keyword(s):  
Phorbol Ester ◽  
Calcium Ionophore ◽  
Stimulation Of

scholarly journals Carrier-mediated transport of uridine diphosphoglucuronic acid across the endoplasmic reticulum membrane is a prerequisite for UDP-glucuronosyltransferase activity in rat liver

1997 ◽  
Vol 323 (3) ◽  
pp. 645-648 ◽  
Author(s):  
Xavier BOSSUYT ◽  
Norbert BLANCKAERT
Keyword(s):  
Endoplasmic Reticulum ◽  
Permeability Barrier ◽  
Endoplasmic Reticulum Membrane ◽  
Carrier Mediated Transport ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Stimulation Of ◽  
Er Membrane

UDP-glucuronosyltransferases (EC 2.4.1.17) is an isoenzyme family located primarily in the hepatic endoplasmic reticulum (ER) that displays latency of activity both in vitro and in vivo, as assessed respectively in microsomes and in isolated liver. The postulated luminal location of the active site of UDP-glucuronosyltransferases (UGTs) creates a permeability barrier to aglycone and UDP-GlcA access to the enzyme and implies a requirement for the transport of substrates across the ER membrane. The present study shows that the recently demonstrated carrier-mediated transport of UDP-GlcA across the ER membrane is required and rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes. We found that in both microsomes and permeabilized hepatocytes a gradual inhibition by N-ethylmaleimide (NEM) of UDP-GlcA transport into the ER produced a correspondingly increasing inhibition of 4-methylumbelliferone glucuronidation. That NEM selectively inhibited the UDP-GlcA transporter, without affecting intrinsic UGT activity, was demonstrated by showing that NEM had no effect on glucuronidation in microsomes or hepatocytes with permeabilized ER membrane. Additional evidence that UDP-GlcA transport is rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes was obtained by showing that gradual selective trans-stimulation of UDP-GlcA transport by UDP-GlcNAc, UDP-Xyl or UDP-Glc in each case produced correspondingly enhanced glucuronidation. Such stimulation of transport and glucuronidation was inhibited completely by NEM, which selectively inhibited UDP-GlcA transport.

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