scholarly journals Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1977 ◽  
Vol 168 (3) ◽  
pp. 401-408 ◽  
Author(s):  
Björn Åkesson
Keyword(s):  
Molecular Species ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Phospholipid Metabolism ◽  
Control Points ◽  
Phospholipid Synthesis ◽  
Intact Cells

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [14C]ethanolamine incorporation into phospholipids, whereas the incorporation of [3H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [3H]glycerol and hepatocytes, the appearance of 3H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [3H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of 3H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-14C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [14C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.

Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

1990 ◽  
Vol 10 (6) ◽  
pp. 573-577 ◽  
Author(s):  
H. K. Metcalfe ◽  
R. D. Cohen ◽  
J. P. Monson
Keyword(s):  
Plasma Membrane ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Similar Magnitude ◽  
Potential Mechanism ◽  
Lactate Transport ◽  
Hormonal Modulation ◽  
Cultured Rat Hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.

Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels

1992 ◽  
Vol 8 (3) ◽  
pp. 235-242 ◽  
Author(s):  
D. J. Mann ◽  
A. J. Strain ◽  
E. Bailey
Keyword(s):  
Malic Enzyme ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Specific Expression ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Species Being ◽  
Rat Tail

ABSTRACT The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.

scholarly journals Purification and characterization of autophagosomes from rat hepatocytes

1998 ◽  
Vol 335 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Per Eivind STRØMHAUG ◽  
Trond Olav BERG ◽  
Monica FENGSRUD ◽  
Per O. SEGLEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Protein Markers ◽  
Intact Cells ◽  
Cytoskeletal Elements ◽  
Intermediate Density ◽  
Glucose Regulated Protein

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

scholarly journals Compartmentation of glucose 6-phosphate in hepatocytes

1987 ◽  
Vol 248 (3) ◽  
pp. 927-931 ◽  
Author(s):  
N Kalant ◽  
M Parniak ◽  
M Lemieux
Keyword(s):  
Primary Cultures ◽  
Specific Radioactivity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Nuclei ◽  
Glucose Phosphorylation

Rat hepatocytes were incubated with 14C-labelled hexoses, and the specific radioactivities of glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate were determined. (1) When suspensions of freshly isolated hepatocytes were incubated with [14C]glucose, the specific radioactivities of glucose 1-phosphate and fructose 6-phosphate were severalfold higher than that of glucose 6-phosphate. The ratios of the specific radioactivities decreased with time of incubation. These relationships were also found when incubations were carried out with primary cultures of rat hepatocytes or with crude homogenates of hepatocytes, but not with isolated nuclei. (2) When cells were incubated with [14C]fructose, the ratios of the specific radioactivities were higher than with [14C]glucose, and also decreased with time. (3) Paired incubations were carried out with a mixture of galactose and fructose, with one or other sugar being labelled with 14C. The specific radioactivity of glucose released into the medium was greater than that of glucose 6-phosphate when fructose was labelled, but not when galactose was labelled. Furthermore, glucose 6-phosphate and glucose in the medium differed with regard to the distribution of 14C between C-1 and C-6. These results are interpreted as evidence that glucose 6-phosphate in hepatocytes does not exist as a homogeneous pool, but that subcompartments exist which are associated with glucose phosphorylation, gluconeogenesis and glycogenolysis.

Alterations of bile acid and bumetanide uptake during culturing of rat hepatocytes

1990 ◽  
Vol 258 (4) ◽  
pp. C700-C712 ◽  
Author(s):  
W. Follmann ◽  
E. Petzinger ◽  
R. K. Kinne
Keyword(s):  
Bile Acid ◽  
Cultured Cells ◽  
Driving Forces ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Maximum Velocity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Transport Activity ◽  
Isolated Rat Hepatocytes

Uptake by the multispecific bile acid transport system of [3H]taurocholate, [14C]cholate, and [3H]-bumetanide into primary cultures of rat hepatocytes was compared with their uptake into freshly isolated rat hepatocytes. The uptake maximum velocity (Vmax) of all compounds declined in primary culture, whereas the Michaelis constant (Km) values remained stable. Loss of uptake was not due to the reduction of driving forces as evaluated from the level of ATP and the activity of Na(+)-K(+)-ATPase. No alpha-fetoprotein was detectable in culture supernatants. Neither growth factors (glycylhistidyl-lysine, epidermal growth factor), peroxisome and cell proliferators (nafenopin, dimethyl sulfoxide), nor bile acids prevented the loss of transport in hepatocyte culture. However, addition of dibutyryl adenosine 3'5'-cyclic monophosphate protracted the transport activity significantly. When cultured rat hepatocytes with reduced transport were detached by trypsin, cells rounded up and showed the same uptake capacity for bumetanide, cholate, and taurocholate as seen in freshly isolated hepatocytes. "Cryptic" transport activity in the lower basolateral membrane facing the support was found using an incubation chamber for cultured hepatocytes, which allowed us to distinguish simultaneously between uptake via the upper and lower basolateral membrane of the cultured cells.

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

1987 ◽  
Vol 253 (5) ◽  
pp. G613-G621 ◽  
Author(s):  
R. W. Van Dyke ◽  
B. F. Scharschmidt
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Solute Transport ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Sodium Content ◽  
Intracellular Sodium ◽  
Intact Cells ◽  
Sodium Dependent ◽  
Dependent Transport

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

scholarly journals Enhancement of glycogen concentrations in primary cultures of rat hepatocytes exposed to glucose and fructose

1988 ◽  
Vol 251 (3) ◽  
pp. 795-802 ◽  
Author(s):  
M A Parniak ◽  
N Kalant
Keyword(s):  
Glycogen Synthesis ◽  
Relative Proportion ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Phosphorylase Activity ◽  
Isolated Rat Hepatocytes ◽  
Glucose Carbon ◽  
Fasted Rats ◽  
Gluconeogenic Substrate

Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.

scholarly journals Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures

1982 ◽  
Vol 208 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Ronald J. Gerson ◽  
Zahir A. Shaikh
Keyword(s):  
Time Course ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Fresh Medium ◽  
Control Value ◽  
Dispositional Differences ◽  
Insight Into ◽  
Hepatocyte Monolayer

The administration of inorganic Cd and Hg in vivo has been shown to result in markedly different metal concentrations in rat liver. Primary cultures of rat hepatocytes were utilized to gain insight into the dispositional differences between these chemically similar metals. Hepatocyte monolayer cultures were exposed to several concentrations of Cd or Hg (3, 10 and 30μm) in serum-containing medium for 30min. The cells were then washed and incubated in fresh medium for the remainder of the experiment. Hepatocytes exposed to Cd accumulated significantly more metal than hepatocytes exposed to equimolar concentrations of Hg. In cells exposed to 3μm-Cd there was an initial loss of Cd from the hepatocytes when placed in fresh medium, followed by a gradual re-uptake of metal, concomitant with increased binding to metallothionein. In hepatocytes exposed to 3 and 10μm-Cd, 87 and 77% of the intracellular Cd was bound to metallothionein within 24h. Loss of Hg from hepatocytes pulsed with 30μm-Hg was also observed upon the addition of fresh medium and continued for the duration of the experiment. No time-dependent increase in Hg binding to metallothionein was observed. A maximum of about 10% of the intracellular Hg was found associated with metallothionein in hepatocytes exposed to 30μm-Hg. Studies utilizing [35S]cysteine incorporation indicated significant increases in the amount of metallothionein synthesized in hepatocytes exposed to 3 and 10μm-Cd (300% of control value) and 30μm-Hg (150% of control value) 24h after metal pulsing. Time-course studies revealed a 6–12h lag in metallothionein synthesis, followed by a significant elevation in [35S]cysteine incorporation into metallothionein between 12 and 24h. These studies suggest that (a) isolated hepatocytes differentiate between Cd and Hg and preferentially accumulate the former, and (b) Cd strongly stimulates the induction of, and preferentially binds to, metallothionein, whereas Hg induces weakly, and does not preferentially bind to, metallothionein.

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