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.

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.

Hepatotoxic effect of (+)usnic acid from Usnea siamensis Wainio in rats, isolated rat hepatocytes and isolated rat liver mitochondria

2004 ◽  
Vol 90 (2-3) ◽  
pp. 381-387 ◽  
Author(s):  
Pornpen Pramyothin ◽  
Withaya Janthasoot ◽  
Nushjira Pongnimitprasert ◽  
Siriwan Phrukudom ◽  
Nijsiri Ruangrungsi
Keyword(s):  
Rat Liver ◽  
Usnic Acid ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat Liver ◽  
Hepatotoxic Effect ◽  
Isolated Rat

Separation of the Intracellular Secretory Compartment of Rat Liver and Isolated Rat Hepatocytes in a Single Step Using Self-Generating Gradients of Iodixanol

1999 ◽  
Vol 276 (1) ◽  
pp. 88-96 ◽  
Author(s):  
Dietmar Plonne ◽  
Ian Cartwright ◽  
Werner Linß ◽  
Rolf Dargel ◽  
John M. Graham ◽  
...  
Keyword(s):  
Rat Liver ◽  
Rat Hepatocytes ◽  
Single Step ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

The mechanism of inhibition of ureogenesis by acidosis

1984 ◽  
Vol 4 (10) ◽  
pp. 819-825 ◽  
Author(s):  
J. P. Monson ◽  
R. M. Henderson ◽  
J. A. Smith ◽  
R. A. Iles ◽  
M. Faus-Dader ◽  
...  
Keyword(s):  
Ammonium Chloride ◽  
Rat Liver ◽  
Rat Hepatocytes ◽  
Ph Sensitive ◽  
Perfused Rat Liver ◽  
Carbamoyl Phosphate ◽  
Isolated Rat Hepatocytes ◽  
Mechanism Of Inhibition ◽  
Cytosol Ph ◽  
Isolated Rat

In perfused rat liver a decrease of cytosol pH, determined with pH-sensitive microelectrodes7 from 7.2 to 6.85 is associated with a 50% fall in ureogenesis from ammonium chloride. In isolated rat hepatocytes the fall in ureogenesis due to acidosis is associated with decrease in the mitochondrial and cytosolic concentration of citrulline. Limitation of carbamoyl phosphate synthesis and thus citrulline supply could be responsible for the inhibition of ureogenesis observed.

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.

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