scholarly journals Cellular and lysosomal uptake of methylamine in isolated rat hepatocytes

1983 ◽  
Vol 210 (3) ◽  
pp. 929-936 ◽  
Author(s):  
A E Solheim ◽  
P O Seglen
Keyword(s):  
Rat Hepatocytes ◽  
Proton Pumping ◽  
Temperature Sensitive ◽  
Facilitated Diffusion ◽  
High Concentration ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Total Cell Volume ◽  
Energy Inhibitors ◽  
Energy Dependent

Upon addition of methylamine to intact cells, this lysosomotropic weak base accumulates intracellularly as the result of at least two different mechanisms: (1) facilitated diffusion across the plasma membrane, i.e. a process which is carrier-mediated and subject to both trans-stimulation (accelerative exchange) and cis-inhibition (competition) by other amines (e.g. ammonia, methylamine and triethylamine); this transport process is furthermore non-concentrative, energy-independent, and (although moderately temperature-sensitive) operative even at 0 degrees C; (2) active uptake, i.e. an energy-dependent concentrative process which is inhibited by anoxia and energy inhibitors. With time, methylamine accumulates in lysosomes and gives rise to a lysosomal swelling which is easily visible by optical microscopy, and which causes the cells to appear coarsely granular. After a 1h incubation with 10mM-methylamine, the total cell volume is increased by about 12%. Under anoxic conditions or in the presence of energy inhibitors, lysosomal swelling is abolished regardless of there being a high concentration of methylamine intracellularly (taken up by facilitated diffusion). The continuous accumulation of methylamine in lysosomes therefore seems to depend on an energy-requiring process (such as continuous proton pumping), and not only on trapping by Donnan-equilibrium-generated protons.

scholarly journals The glucagon-induced activation of pyruvate dehydrogenase in hepatocytes is diminished by 4β-phorbol 12-myristate 13-acetate. A role for cytoplasmic Ca2+ in dehydrogenase regulation

1987 ◽  
Vol 241 (3) ◽  
pp. 729-735 ◽  
Author(s):  
J M Staddon ◽  
R G Hansford
Keyword(s):  
Dehydrogenase Activity ◽  
Phorbol Ester ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Rat Hepatocytes ◽  
Pyruvate Kinase Activity ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Subsequent Addition ◽  
Isolated Rat

Phenylephrine, vasopressin and glucagon each increased the amount of active (dephospho) pyruvate dehydrogenase (PDHa) in isolated rat hepatocytes. Treatment with 4 beta-phorbol 12-myristate 13-acetate (PMA) opposed the increase in PDHa caused by both phenylephrine and glucagon, but had no effect on the response to vasopressin: PMA alone had no effect on PDHa. As PMA is known to prevent the phenylephrine-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and to diminish the increase [Ca2+]c caused by glucagon, while having no effect on the ability of vasopressin to increase [Ca2+]c, these data are consistent with the notion that in intact cells an increase in [Ca2+]c results in an increase in the mitochondrial free Ca2+ concentration, which in turn leads to the activation of PDH. In the presence of 2.5 mM-Ca2+, glucagon caused an increase in NAD(P)H fluorescence in hepatocytes. This increase is taken to reflect an enhanced activity of mitochondrial dehydrogenases. PMA alone had no effect on NAD(P)H fluorescence; it did, however, compromise the increase produced by glucagon. When the extracellular free [Ca2+] was decreased to 0.2 microM, glucagon could still increase NAD(P)H fluorescence. Vasopressin also increased fluorescence under these conditions; however, if vasopressin was added after glucagon, no further increase in fluorescence was observed. Treatment of the cells with PMA resulted in a smaller increase in NAD(P)H fluorescence on addition of glucagon: the subsequent addition of vasopressin now caused a further increase in fluorescence. Changes in [Ca2+]c corresponding to the changes in NAD(P)H fluorescence were observed, again supporting the idea that [Ca2+]c indirectly regulates intramitochondrial dehydrogenase activity in intact cells. PMA alone had no effect on pyruvate kinase activity, and the phorbol ester did not prevent the inactivation caused by glucagon. The latter emphasizes the different mechanisms by which the hormone influences mitochondrial and cytoplasmic metabolism.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent

1982 ◽  
Vol 208 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Roelof Docter ◽  
Eric P. Krenning ◽  
Greetje Bos ◽  
Durk F. Fekkes ◽  
George Hennemann
Keyword(s):  
Contrast Agents ◽  
Rat Hepatocytes ◽  
Human Erythrocytes ◽  
Uptake System ◽  
Erythrocyte Ghosts ◽  
Intact Cells ◽  
X Ray ◽  
Dose Dependent Fashion ◽  
Uptake Studies ◽  
Energy Dependent

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by ‘ghosts’ prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with Km 128±19nm (mean±s.e.m.; n=7) and Vmax. 4.6±0.7pmol of tri-iodothyronine/min per 6×107 cells. After 10min incubation Km 100±16nm (n=10) was found with Vmax. 7.7±1.2pmol of tri-iodothyronine/10min per 6×107 cells. At 0°C the uptake system is still active, with Km 132±26nm and Vmax. 1.8±0.3pmol of tri-iodothyronine/10min per 6×107 cells. The Vmax. with intact cells is 5-fold greater than the Vmax. with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na++K+-dependent adenosine triphosphatase. We have prepared erythrocyte ‘ghosts‘, resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte ‘ghosts’ were verified on the basis of an ATP-concentration-dependent functioning of the Ca2+ pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte ‘ghosts’ with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na+ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett.91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett.119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett.140, 229–233].

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 Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump

1987 ◽  
Vol 248 (3) ◽  
pp. 807-813 ◽  
Author(s):  
C J Lynch ◽  
S B Bocckino ◽  
P F Blackmore ◽  
J H Exton
Keyword(s):  
Cyclic Amp ◽  
Rat Hepatocytes ◽  
Previous Exposure ◽  
High Concentration ◽  
Isolated Rat Hepatocytes ◽  
Pump Activity ◽  
Heterologous Desensitization ◽  
Tumour Promoters ◽  
High Concentrations ◽  
Stimulation Of

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.

scholarly journals Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

2006 ◽  
Vol 395 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Yongmin Ma ◽  
Herbert de Groot ◽  
Zudong Liu ◽  
Robert C. Hider ◽  
Frank Petrat
Keyword(s):  
Time Course ◽  
Iron Chelation ◽  
Rat Hepatocytes ◽  
Calibration Method ◽  
Ex Situ ◽  
Iron Chelators ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Iron Pool ◽  
Intracellular Fluorescence

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline–iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4±1.3 μM.

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells

2001 ◽  
Vol 356 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Frank PETRAT ◽  
Herbert de GROOT ◽  
Ursula RAUEN
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Laser Scanning ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Liver Endothelial Cells ◽  
Isolated Rat

The pool of cellular chelatable iron (‘free iron’, ‘low-molecular-weight iron’, the ‘labile iron pool’) is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8±2.6μM within the cytosol and of at least 4.8μM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6±2.9μM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3±2.6μM; nucleus, 11.8±3.9μM; mitochondria, 9.2±2.7μM); in addition, chelatable iron (approx. 16±4μM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

Effect of mitochondrial clustering on O2 supply in hepatocytes

1984 ◽  
Vol 247 (1) ◽  
pp. C83-C89 ◽  
Author(s):  
D. P. Jones
Keyword(s):  
Diffusion Coefficient ◽  
Rat Hepatocytes ◽  
Dependence Analysis ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Intracellular Diffusion ◽  
O2 Supply ◽  
Intermediate Value ◽  
Isolated Rat

Isolated rat hepatocytes were treated with digitonin to selectively disrupt the plasma membrane and allow study of the O2 dependence of mitochondria within the cell cytoskeleton, but without cytosol. Half-maximal oxidation of cytochrome c occurred at 2.0 microM O2 in treated cells incubated under State 3 conditions, whereas in intact cells it was 6.0 microM and in isolated mitochondria (State 3) 0.69 microM. the intermediate value for treated cells indicates that both the geometry of mitochondrial packing and the intracellular diffusion coefficient are important in determining intracellular mitochondrial O2 dependence. Analysis of intracellular diffusion, assuming that the mitochondrial clustering increases the effective mitochondrial radius, indicates that an intracellular diffusion coefficient of between 10(-6) and 4 X 10(-6) cm2 . s-1 and an effective mitochondrial radius of approximately 2 micron would account for the observed intracellular O2 dependence of mitochondrial function.

Inhibition of intestinal citrulline synthesis causes severe growth retardation in rats

1985 ◽  
Vol 249 (6) ◽  
pp. G792-G799 ◽  
Author(s):  
N. Hoogenraad ◽  
N. Totino ◽  
H. Elmer ◽  
C. Wraight ◽  
P. Alewood ◽  
...  
Keyword(s):  
Drinking Water ◽  
Specific Inhibitor ◽  
Rat Hepatocytes ◽  
Deficient Diet ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Rat Pups ◽  
Inhibition Of Growth ◽  
Citrulline Synthesis ◽  
Serum Ammonia

delta-N-(phosphonacetyl)-L-ornithine (PALO) is a powerful and specific inhibitor of ornithine transcarbamylase (1), but it does not readily enter intact cells and therefore does not inhibit citrulline synthesis in intact liver or intestine. We have used the glycylglycine derivative of PALO (Gly-Gly-PALO) to evaluate the importance of intestinal citrulline synthesis in supplying arginine for growth. We have shown that the peptide derivative of PALO is selectively taken up by gut cells via the peptide permease and is released intracellularly as the free inhibitor. When administered in drinking water to 6-wk-old rat pups on an arginine-deficient diet, serum citrulline was reduced from 85 +/- 4.2 to 44 +/- 2.2 microM and arginine from 240.1 +/- 19.0 to 52.1 +/- 4.1 microM. Ornithine increased from 100 +/- 6.2 to 273.5 +/- 21.3 microM. Addition of 0.1 mM Gly-Gly-PALO to drinking water caused a rapid and complete inhibition of growth in rats on arginine-deficient diets, and this growth inhibition could be partially prevented by simultaneous administration of 1% (wt/wt) arginine to the diet and completely prevented with 1% (wt/wt) citrulline. The specificity of the effects of Gly-Gly-PALO on intestinal citrulline synthesis was shown by the inability of the drug to be taken up or to inhibit citrulline synthesis in isolated rat hepatocytes, and the oral administration of the drug had no effect on serum ammonia concentrations. The relative importance of endogenous synthesis of arginine compared with dietary arginine for growth was shown by the ability of Gly-Gly-PALO to inhibit the growth of rats maintained on standard laboratory chow containing normal levels of arginine.

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