scholarly journals Evidence for acidity of prelysosomal autophagic/endocytic vacuoles (amphisomes)

1993 ◽  
Vol 291 (1) ◽  
pp. 115-121 ◽  
Author(s):  
P E Strømhaug ◽  
P O Seglen
Keyword(s):  
Rat Hepatocytes ◽  
Internal Environment ◽  
Weak Base ◽  
Isolated Rat Hepatocytes ◽  
High Concentrations ◽  
Beta Galactosidase ◽  
Isolated Rat

[14C]Lactose electroinjected into isolated rat hepatocytes is normally autophagocytosed, transferred to lysosomes and degraded by lysosomal beta-galactosidase, but at high concentrations of asparagine the transfer is inhibited and lactose accumulates in prelysosomal autophagic/endocytic vacuoles (amphisomes). The accumulation can be prevented by addition of yeast beta-galactosidase, which is transferred to the lactose-containing vacuoles by endocytosis. Propylamine, a weak base capable of neutralizing acidic vacuoles, protects autophagocytosed lactose against both endogenous and exogenous beta-galactosidase, suggesting that amphisomes, like lysosomes, have an acidic internal environment.

scholarly journals Mechanism of long-range Ca2+ signalling in the nucleus of isolated rat hepatocytes

1997 ◽  
Vol 326 (2) ◽  
pp. 491-495 ◽  
Author(s):  
Jeffrey L. FOX ◽  
Angela D. BURGSTAHLER ◽  
Michael H. NATHANSON
Keyword(s):  
High Speed ◽  
Diffusion Mechanism ◽  
Reaction Diffusion ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Simple Diffusion ◽  
Nuclear Interior ◽  
Wide Range ◽  
High Concentrations ◽  
Isolated Rat

Ca2+ regulates a wide range of cell proteins, in both the cytosol and nucleus. It enters the nucleus from stores along the nuclear envelope, but how it then spreads through the nuclear interior is unknown. Here we used high-speed confocal line-scanning microscopy to examine the propagation of Ca2+ waves across nuclei in isolated rat hepatocytes. Nuclear Ca2+ waves began at the nucleus/cytosol border as expected, then spread across the nucleus at less than half the speed of cytosolic Ca2+ waves. High concentrations of caffeine slowed Ca2+ waves in the cytosol but not in the nucleus. We developed a mathematical model based on diffusion to analyse these data, and the model was able to describe the nuclear but not cytosolic Ca2+ waves that were experimentally observed. These findings suggest that Ca2+ waves cross the nucleus by simple diffusion, which is distinct from the reaction-diffusion mechanism by which Ca2+ waves propagate across the cytosol. Since the range of messenger action for Ca2+ in the cytosol is much smaller than the distance across the nucleus, this also suggests that the unique environment and geometry of the nuclear interior may permit this simple mechanism of Ca2+ wave propagation to control Ca2+-mediated processes in a relatively large region despite Ca2+ release pools that are spatially limited.

scholarly journals Effects of protein-degradation inhibitors on the inactivation of tyrosine aminotransferase, tryptophan oxygenase and benzopyrene hydroxylase in isolated rat hepatocytes

1982 ◽  
Vol 202 (1) ◽  
pp. 191-196 ◽  
Author(s):  
B Grinde ◽  
R Jahnsen
Keyword(s):  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Tyrosine Aminotransferase ◽  
Weak Base ◽  
Isolated Rat Hepatocytes ◽  
Tryptophan Oxygenase ◽  
The Third ◽  
Lysosomal Proteinase ◽  
Isolated Rat

The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism.

scholarly journals Utilization of tryptophan, nicotinamide and nicotinic acid as precursors for nicotinamide nucleotide synthesis in isolated rat liver cells

1988 ◽  
Vol 59 (2) ◽  
pp. 279-287 ◽  
Author(s):  
David A. Bender ◽  
Ronald Olufunwa
Keyword(s):  
Nicotinic Acid ◽  
De Novo ◽  
Rat Hepatocytes ◽  
Significant Extent ◽  
Nucleotide Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Rat Liver Cells ◽  
Extrahepatic Tissues ◽  
High Concentrations ◽  
Isolated Rat

1. Incubation of isolated rat hepatocytes with nicotinamide or nicotinic acid showed that while both vitamers were taken up from the incubation medium, neither was utilized to any significant extent as a precursor of the nicotinamide nucleotide coenzymes, NAD and NADP, and neither was capable of preventing the loss of nucleotides that occurs on incubating the cells.2. Incubation of hepatocytes with tryptophan showed that de novo synthesis from tryptophan permitted replacement of the nucleotides lost during incubation; at high concentrations of tryptophan there was an increase above the initial intracellular concentration of NAD(P). Incubation of hepatocytes with tryptophan also resulted in the formation and release from the cells of a considerable amount of niacin, as well as the two principal metabolities of NAD(P), N1-methyl nicotinamide and methyl pyridone carboxamide.3. It is suggested that, in the liver, preformed niacin is not utilized for nucleotide synthesis, and indeed the function of the liver appears to be synthesis of niacin from tryptophan, and its release for use by extrahepatic tissues that lack the pathway for de novo synthesis of nicotinamide nucleotides from tryptophan.

Inhibition of gluconeogenesis by extracellular ATP in isolated rat hepatocytes

1991 ◽  
Vol 261 (6) ◽  
pp. R1522-R1526 ◽  
Author(s):  
M. Asensi ◽  
A. Lopez-Rodas ◽  
J. Sastre ◽  
J. Vina ◽  
J. M. Estrela
Keyword(s):  
Plasma Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Extracellular Atp ◽  
Structural Analogue ◽  
Isolated Rat Hepatocytes ◽  
Lactate And Pyruvate ◽  
Alpha Beta ◽  
High Concentrations ◽  
Isolated Rat

The aim of this study was to determine the effect of externally added ATP on gluconeogenesis by isolated hepatocytes from starved rats. High concentrations of extracellular ATP inhibited gluconeogenesis from lactate and pyruvate but not from glycerol or fructose. This inhibition was associated with an increase in intracellular adenosine contents. ADP, AMP, or adenosine but not guanosine 5'triphosphate, inosine 5' triphosphate, or adenine also inhibited gluconeogenesis. alpha, beta-Methylene-ATP, a nonmetabolizable structural analogue of ATP, did not affect the rate of gluconeogenesis. Intracellular ATP levels were increased by externally added ATP or adenosine, but ATP-to-ADP ratios in the cytosolic and mitochondrial compartments were diminished. Malate and phosphoenolpyruvate contents were decreased by extracellular ATP or adenosine. Our results show that inhibition of gluconeogenesis by high levels of extracellular ATP may be mediated by adenosine derived from ATP catabolism at the plasma membrane.

scholarly journals Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes

1989 ◽  
Vol 262 (1) ◽  
pp. 277-284 ◽  
Author(s):  
B L Clarke ◽  
P H Weigel
Keyword(s):  
Rat Hepatocytes ◽  
Rapid Onset ◽  
Degradation Pathway ◽  
The State ◽  
Differential Sensitivity ◽  
Isolated Rat Hepatocytes ◽  
High Concentrations ◽  
State 1 ◽  
Very High ◽  
Isolated Rat

We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.

scholarly journals Lysosomal triacylglycerol lipase and lipolysis in isolated rat hepatocytes.

1979 ◽  
Vol 254 (18) ◽  
pp. 8841-8846
Author(s):  
L.J. Debeer ◽  
J. Thomas ◽  
P.J. De Schepper ◽  
G.P. Mannaerts
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Triacylglycerol Lipase ◽  
Isolated Rat
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