Properties of the adenylate cyclase system of isolated hepatocytes following freezing-thawing

The effect of various regimen of freezing and thawing on the functional state of one of the most important receptor-regulatory cell systems, i. e. adenylate cyclase complex (ATP-pyrophosphate lyase cyclating E C 4.6.1.1.), has been studied on isolated rat hepatocytes. Basal and fluoride-stimulating activity and enzyme susceptibility to the regulatory effect of isoproterenol, adrenaline and noradrenaline were shown. Regardless of the regimen used, freezing and thawing decrease the stimulating effect of adrenergetic agents on adenylate cyclase system of isolated hepatocytes. The reason of it is probably the damage of a receptor site. The functional properties of catalytic enzyme subunit practically do not change. The results obtained show the necessity of correction of metabolic cell responses mediated by adenylate cyclase system during their freezing and thawing.     

Respiratory and detoxication activity of two fractions of hepatocytes isolated by a non-enzymatic method]

Slow centrifugation of isotonic sucrose medium containing the suspension of hepatocytes, obtained after disaggregation of liver, which has been preliminarily perfused by EDTA solution, results in the formation of two fractions of cells of different colour. Fraction 1 contains intact and metabolically active hepatocytes. Cells of fraction 2 have a damaged membrane, but they are capable of realizing biotransformation of xenobiotics in the presence of exogenous NADPH as well as maintaining efficiency of oxidative phosphorylation of mitochondria under the transfer to the medium simulating cytoplasm.     

Role of albumin in the biotransformation of biphenyl by isolated rat hepatocytes]

The role of albumin in biotransformation of biphenyl, a lipophilic xenobiotic, by isolated rat hepatocytes has been studied. It is shown that in the absence of albumin biphenyl is quickly and almost completely bound by cells. The rate of formation of 4-hydroxybiphenyl, the reaction product, depends on the substrate concentration (in the range 8.5-70 microM) and conforms with the Mikhaelis-Menten equation. An increase in the biphenyl concentration in the incubating medium to 140 microM induces no changes in the rate of its biotransformation. Serum albumin, while binding biphenyl, also reduces its effective concentration in a cell, which prevents the cytochrome-P-450-dependent monooxygenase system of hepatocytes from the inhibiting effect of high concentrations of the xenobiotic.     

Classification of the isolated hepatocytes

At this present, enzyme perfusion method is a routine technique to isolate hepatocytes from rat liver for the physiological and pathological experiments. This study described a way of the classification of freshly isolated hepatocytes. First of all, the hepatocytes were fractionated with parenchymal and non-parenchymal cells by low speed centrifugation. And then these cells were subfractionated with a newly developed Percoll linear density gradient method. The fractionated parenchymal cells were divided with cells of periportal and centrilobular areas, respectively. Furthermore, their characteristics were confirmed functionally and morphologically. Non-parenchymal cells (NPC) include Kupffer cells, endothelial cells and fat storing cells (FSC, Ito cells). These isolated NPC are fractionated with a method as mentioned above or centrifugal alutriation method. In this paper, fractionation and classification of Kupffer cells and FSC were discussed with the measurement of fluorescent intensity of vitamin A and the morphological observation of cytoskeleton in culture. Especially, transport of vitamin A into FSC were detected autoradiographically.     

Evaluation of mitochondrial function in isolated rat hepatocytes and mitochondria during oxidative stress

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.     

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes]

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.     

Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogensis from ammonia in isolated rat hepatocytes.

1. Citrate isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions, thereof, obtained by the digitonin and silicone oil fractionation technique. 2. Caculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV. 3; Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, is demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reserved. 4. It is concluded that during urogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 muM, below the Km (isocitrate) of the NAD-linked enzyme but in the range of that for the NADP-linked enzyme. 5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate. 6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.     

A factor influencing the process of chromatin condensation in hepatocytes of regenerating rat liver]

It was shown with the model supramolecular chromatin systems that the activity of the factor of chromatin condensation changes in the process of liver regeneration. No activity of the factor of chromatin condensation was found 20 hrs after operation. Within 32-33 hrs (in the period of increased mitotic frequency) its activity approaches that prior to the operation. The nature of the activity of the factor of chromatin condensation is discussed with respect to the functional state of chromatin.     

Status of the detoxication function of the liver after vagal denervation

In 14 and 25 days of bilateral subdiaphragmatic+ vagotomy perimeter of the endoplasmic reticulum (EPR) of hepatocytes, content of the enzyme (cytochrome R-450) built into the membrane in microsomal fractions, speed of hydroxylation of substrates and oxygen absorption decreased and the section area of EPR and duration of the hexonal sleep increased. The results demonstrate certain disturbances of the detoxication function of the denervated liver.     

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.     

Energy metabolism of isolated hepatocytes at various levels of oxidative phosphorylation uncoupling]

The energy metabolism changes in isolated hepatocytes at different levels of proton conductivity of cellular membranes were studied. The low doses of the uncoupler which increased hepatocyte respiration rate but did not markedly affect the mitochondrial potential caused: the reduction in total adenine nucleotide contents (ATP + ADP + AMP), the oxidation of mitochondrial NADH, the increase in the rates of glycogenolysis and net flux via phosphofructokinase without any changes in the rates of glucose, lactate and pyruvate accumulation. High doses of the uncoupler which eliminated completely oxidative phosphorylation decreased Atkinson's energy charge down to 0.5, reduced cytoplasmic NADH, induced a further increase in the glycogenolysis rate, increased the rates of glucose and lactate accumulation, heightened glucose-6-phosphate content and lowered contents of 3-phosphoglycerate and 2-oxoglutarate.     

Some characteristics of the process of forming autophagocytic vacuoles in the hepatocytes of regenerating rat liver]

This work deals with the previously described process of cellular autophagocytosis in regenerating rat liver. The experimental data obtained indicate that the early appearance of acid phosphatase positive cytoplasmic vacuoles (with diameter more than 2 mu) occur only in a certain portion of the parenchymal cell population (nearly 30% of the total number of hepatocytes) and mainly in the central area of the hepatic lobule. It may be concluded that the observed reaction of the cell vacuolar apparatus concerns hepatocytes that undergo the entry into the mitotic cycle much later than does the main part (ca 60%) of cell population.     

CAPS in search of a lost function

Ca2+-dependent activator protein for secretion (CAPS) is an evolutionarily conserved secretory protein that was previously thought to mediate Ca2+-triggered fusion of dense-core vesicles. In an elegant study of CAPS1-deficient mice, Speidel et al. (this issue of Neuron) now show that CAPS function may have been misunderstood. CAPS appears to act upstream of fusion in the biogenesis or maintenance of mature secretory vesicles, raising the possibility of a completely new type of function for an essential component of the secretory machinery.     

Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis

The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis induced by a combination of CCl₄ and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different. Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was 50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes.     

Dexamethasone restores hormonal inducibility of ornithine decarboxylase in primary cultures of rat hepatocytes

Induction of ornithine decarboxylase by various hormones was studied in quiescent primary cultures of adult rat hepatocytes maintained in a chemically defined medium. The following results were obtained: Enzyme activity rose transiently during the first day of cultivation in hormone-untreated cells. During this phase, insulin increased ornithine decarboxylase activity. Inducibility by insulin was maintained for more than 40 h only after pretreatment with 0.1 microM dexamethasone. Enzyme activity could be induced by 1 nM insulin and peaked after 7 h. Inducibility by glucagon and growth hormone required pretreatment with the glucocorticoid hormone. Ornithine decarboxylase activity was maximal 5 h after glucagon addition. Concentrations down to 0.1 nM were effective. Pretreatment with dexamethasone was most effective, when the hormone was present during the first 20 h of cultivation. The effect of the glucocorticoid during the pretreatment phase was diminished by colchicine and to a lesser extent by cytochalasine B. We suggest that part of the permissive effect of dexamethasone could be mediated by changes in the cytoskeleton and the function of hormone receptors. The fact that induction of ornithine decarboxylase was exerted by several hormones despite the absence of cell proliferation and DNA synthesis may indicate that polyamine biosynthesis has an important role in the quiescent hepatocyte.     

Binding of calcium ions to the isolated asialo-glycoprotein receptor. Implications for receptor function in suspended hepatocytes

The binding of calcium ions by the isolated asialoglycoprotein receptor of hepatocytes and the inter-relationship between the calcium ion concentration and receptor function have been studied. The isolated receptor binds calcium ions only in the presence of asialoglycoprotein. The asialo-glycoprotein receptor complex binds 4 calcium ions; the binding exhibits marked positive cooperativity, and the association constant at half-saturation of the binding sites was of the order of 10*5) M-1 as determined from a Hill plot. The isolated receptor was almost saturated at a calcium ion concentration of 0.1 mM. The binding capacity of isolated hepatocytes for asialo-glycoproteins increased, however, even when the calcium concentration was increased above this level. This may be explained by the exposure of increasing numbers of functional receptors on the surface of the cell with increasing membrane potential, and this explanation is supported by analogous observations in the presence of 5 mM La3+.