Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

Oxygen and substrate dependence of hepatic cellular respiration: sinusoidal oxygen gradient and effects of ethanol in isolated perfused liver and hepatocytes

The oxygen dependence of hepatic cellular respiration was studied by employing simultaneous organ spectrophotometry of cytochromes and hemoglobin, the latter used as an intrasinusoidal optical oxygen probe. The Km of cytochrome aa3 for oxygen was found to be 6.8 microM in the isolated perfused liver and 0.3 microM in suspensions of isolated hepatocytes. The results indicate that the sinusoid-to-cell pO2 gradient is about 5 torr. Optical determination of the average effective pO2 indicates that the axial sinusoidal O2 profile does not conform to zero-order O2 uptake in the liver. Because of extensive NAD+ reduction, ethanol increases the thermodynamic driving force of oxidative phosphorylation, and it also increased the oxygen consumption in both the perfused liver and the hepatocyte suspension, but had no effect on the grade of steady-state cytochrome aa3 reduction, the cellular energy state [ATP]/[ADP].[Pi], or the Km of cytochrome aa3 for oxygen. The results indicate that hepatic energy metabolism is oxygen independent at very low O2 concentrations, but that the sinusoidal axial O2 concentration is anomalous, probably due to the spatial arrangement of the metabolizing systems.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Cholic acid uptake and isolated rat hepatocytes.

Cholic acid uptake was studied in isolated rat hepatocytes using a centrifugal filtration technique to allow rapid sampling. Hepatocytes were found to adsorb as well as to transport cholic acid. The adsorption was characterized by a capacity of 24 nmol X mg cell protein-1 and an association constant of 0.59 X 103 M-1. Cholic acid uptake was linear with respect to concentration at or below 10 degree C, suggesting a unsaturable uptake process which was considered to represent simple diffusion and is quantitated by a diffusion coefficient of 1.76 pmol cholic acid X min-1 X mg protein-1 X muM-1. Above 10 degrees C the uptake curve was biphasic. After subtracting the unsaturable component from uptake rates at higher temperatures, a curve showing saturable kinetics resulted. The apparent Km and V values at 37 degrees C were calculated to be 31muM and 0.8 nmol X min-1 X mg protein-1 respectively. This saturable uptake process was temperature-dependent with an activation energy of 13 kcal X mol-1 (5.44 X 104 J X mol-1) and was inhibited by oligomycin and KCN. Countertransport was demonstrated with cholic, taurocholic and chenodeoxycholic acids. The results suggest that cholic acid is transported by an energy-dependent carrier-mediated process in addition to simple diffusion by hepatocytes, and that the postulated carrier has affinity for other bile acids.     

Non-enzymatic lipid peroxidation of microsomes and mitochondria isolated from liver and heart of pigeon and rat

Studies were carried out to determine the level of ascorbate-Fe2+ dependent lipid peroxidation of mitochondria and microsomes isolated from liver and heart of rat and pigeon. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria and microsomes from rat liver than in the same organelles obtained from pigeon. In both mitochondria and microsomes from liver of both species a significant decrease of arachidonic acid was observed during peroxidation. The rate C18:2 n6/C20:4 n6 was 4.5 times higher in pigeon than in rat liver. This observation can explain the differences noted when light emission and unsaturation index of both species were analysed. A significant decrease of C18:2 n6 and C20:4 n6 in pigeon liver mitochondria was observed when compared with native organelles whereas in pigeon liver microsomes only C20:4 n6 diminished. In rat liver mitochondria only arachidonic acid C20:4 n6 showed a significant decrease whereas in rat liver microsomes C20:4 n6 and C22:6 n3 decreased significantly. However changes were not observed in the fatty acid profile of mitochondria and microsomes isolated from pigeon heart. In the heart under our peroxidation conditions the fatty acid profile does not appear to be responsible for the different susceptibility to the lipid peroxidation process. The lack of a relationship between fatty acid unsaturation and sensitivity to peroxidation observed in heart suggest that other factor/s may be involved in the protection to lipid peroxidation in microsomes and mitochondria isolated from heart.     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria.

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions. The release process under optimized conditions (approx. 50 mumol/1 FMN, 1 mmol/l succinate, 0.35 mmol/1 Fe(III) (as ferritin iron), 37 degrees C and pH 7.40) amounts to 0.9--1.2 nmol iron/mg protein per min. The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

45Ca2+ uptake and phospholipid methylation in isolated rat liver microsomes

The effects of glucagon, epinephrine and insulin on hepatic phospholipid methylation were studied. Glucagon, either injected into rats or added to perfused livers, stimulated methylation in subsequently isolated microsomes. Epinephrine also increased phospholipid methylation. Insulin by itself did not influence the rate of the reaction, but, when administered prior to glucagon, it blocked the effect of the latter. The possibility that the observed stimulation of phospholipid methylation might be causally linked to the reported stimulation by glucagon of 45Ca2+ uptake in subsequently isolated liver microsomes was examined. Both the substrate and the competitive inhibitor of the methylation reaction, S-adenosylmethionine and S-adenosylhomocysteine, had profound effect on the rate of phospholipid methylation, without having comparable effects on Ca2+ uptake. S-adenosylmethionine in increasing concentration stimulated methylation four-fold, while no significant changes in 45Ca2+ uptake were seen. S-adenosylhomocysteine did not inhibit 45Ca2+ uptake even at levels causing more than 95% decrease in methylation. In conclusion, while both phospholipid methylation and 45Ca2+ uptake seem to be hormonally controlled, the correlation between these two processes was not sufficient to support the notion that the changes in 45Ca2+ uptake are caused by the changes in phospholipid methylation.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

Some properties of the thiamine uptake system in isolated rat hepatocytes

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.     

Latency of inosine-5'-diphosphatase in microsomes isolated from rat liver.

The latency of inosine-5'-diphosphatase has been studied in microsomes isolated from rat liver. The appearance of latent activity was the result of an increase in the Vmax of the enzyme. This was observed when assays were carried out in the presence of sodium deoxycholate, after microsomes were treated wtih phospholipase C, or at pH 10.3 and after microsomes were subjected to nitrogen cavitation. The apparent Km of inosine-5'-diphosphatase for IDP was unchanged when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with sodium deoxycholate or subjected to nitrogen cavitation, approximately 75% of the inosine-5'-diphosphatase activity was released from the membrane, and the apparent Km of the enzyme for IDP increased 4- and 2-fold, respectively. Microsomal cisternae were loaded with lead phosphate by incubation with glucose-6-P and Pb2+, and the release of this lead phosphate following the addition of EDTA to the medium was determined to estimate the permeability of the microsomal membrane. When microsomes were treated with sodium deoxycholate, phospholipase C, or at alkaline pH, the microsomal membrane became almost completely permeable to EDTA under conditions where there was little or no increase in the activity of inosine-5'-diphosphatase. Microsomes were treated at pH 10.3 and then adjusted slowly to pH 7.5. The activity of inosine-5'-diphosphatase decreased to the same activity observed in untreated preparations. The results seem of exclude the possibility that latent inosine-5'-diphosphatase activity is the result of an increased permeability of the membrane to IDP. They are, however, consistent with the presence of a noncompetitive inhibitor of the enzyme in the microsomal membrane.     

Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

Cellular and lysosomal uptake of methylamine in isolated rat hepatocytes.

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.