Comparison of the effects of a preliminary hepatic washing and of saponin on the intracellular penetration of peroxidase-labeled anti-rat albumin antibodies in hepatocytes

The effect of a preliminary hepatic washing with saline before liver fixation either by perfusion or immersion was compared to the effect of saponin, a membrane-permeabilizing agent, in order to ascertain which procedure is best to obtain a homogeneous distribution of albumin-containing hepatocytes in the hepatic lobule. Albumin was located in the hepatocytes by peroxidase-labeled antibodies using light and electron microscopy. The efficacy of the two procedures on the intracellular penetration of labeled antibodies in liver sections was judged by preparing transverse ultrathin sections. Both procedures yielded similar results. Liver fixation by perfusion with saponin and without a preliminary washing, however, distributes albumin-containing hepatocytes more homogeneously in the hepatic lobule and enables labeled antibodies to penetrate more satisfactorily. In contrast, when the liver is fixed by immersion, the preliminary washing is the only way to obtain an even distribution of albumin-containing hepatocytes, as saponin is not effective under these conditions. In conclusion, the localization of albumin in the hepatocytes must be adapted according to the technique used to fix the liver.     

Comparison of the effects of a preliminary hepatic washing and of saponin on the intracellular penetration of peroxidase-labeled anti-rat albumin antibodies in hepatocytes

Summary The effects of a preliminary hepatic washing with saline before liver fixation either by perfusion or immersion was compared to the effect of saponin, a membrane-permeabilizing agent, in order to ascertain which procedure is best to obtain a homogeneous distribution of albumincontaining hepatocytes in the hepatic lobule. Albumin was located in the hepatocytes by peroxidase-labeled antibodies using light and electron microscopy. The efficacy of the two procedures on the intracellular penetration of labeled antibodies in liver sections was judged by preparing transverse ultrathin sections. Both procedures yielded similar results. Liver fixation by perfusion with saponin and without a preliminary washing, however, distributes albumin-containing hepatocytes more homogeneously in the hepatic lobule and enables labeled antibodies to penetrate more satisfactorily. In contrast, when the liver is fixed by immersion, the preliminary washing is the only way to obtain an even distribution of albumin-containing hepatocytes, as saponin is not effective under these conditions. In conclusion, the localization of albumin in the hepatocytes must be adapted according to the technique used to fix the liver.     

Transport and intracellular localization of cortisol-asialotranscortin complexes in rat liver

The influence of desialylation of human transcortin on transport of the transcortin-cortisol complex into the liver cells and its intracellular distribution was investigated in perfused rat liver. Under experimental conditions used the half-time of cortisol in perfusion medium was decreased more than 200 times in presence of asialotranscortin compared to that of native transcortin. Experiments with [3H]cortisol and [131I]asialotranscortin demonstrated a simultaneous uptake of cortisol and asialotranscortin by the hepatocytes. Distribution of [3H]cortisol and [131I]asialotranscortin in subcellular fractions showed the following pathway of cortisol-asialotranscortin complex: cell membrane, lysosomes, cytoplasm. The complex dissociates in lysosomes.     

The acute effect of ethanol on albumin, fibrinogen and transferrin synthesis in the rat

Decrease of absolute synthesis of albumin and fractional synthesis of transferrin was observed within 3h of orally administering ethanol (4ml/kg) to rats maintained on a 40%-protein diet. In contrast, absolute synthesis of fibrinogen was unaffected. With this ethanol intake, the changes in protein synthesis occurred without significant ultrastructural change in the liver. When the ethanol intake was greater (8ml/kg) ultrastructural disruption was observed. However, both the decrease of plasma protein synthesis and the ultrastructural alterations could be prevented by the simultaneous administration of a mixture of amino acids with the ethanol. The latter findings, not reported hitherto, suggest that ethanol may interfere with hepatic plasma protein synthesis and ultrastructure more through a disturbance of amino acid metabolism than through direct physical damage to the hepatocyte. An Appendix outlines the deconvolutional method used to correct for losses of labelled protein in the period during which measurements were made. The principle may also be applied to labelled plasma urea. The details of the calculations are given in a supplementary paper that has been deposited as Supplementary Publication 50007 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin

A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.     

The occurrence and intracellular localization of sucrase activity in rat liver

The presence of an enzyme in rat liver which hydroluzes sucrose is demonstrated in this report. The hydrolysis of sucrose was studied in vivo after injecting [¹⁴C]sucrose into rats, and a method was developed for the extraction and analysis of the radioactive sugars stored within subcellular particles. The results show that, besides sucrose, glucose and fructose are also found in the lysosomal fraction of the liver homogenate. In vitro studies reveal the presence of a sucrase, although the activity of the enzyme is very low. Intracellular distribution studies indicate that sucrase is present in the lysosomes as well as in the microsomes, the microsomal enzyme having a pH optimum different from that of the lysosomal enzyme.     

Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes

In order to investigate the regulation of synthesis of some of the plasma proteins, especially fibrinogen, at the cellular level, we have chosen to work with suspensions of hepatocytes isolated by the perfusion of rat liver with crude bacterial collagenase. By adding soybean trypsin inhibitor to the collagenase and by avoiding mechanical damage, we have prepared cell suspensions that synthesize and secrete fibrinogen and albumin and that survive for longer than twenty hours. The fibrinogen secreted is clottable and shows the same pattern in acrylamide gel electrophoresis as fibrinogen purified from rat plasma. After a three hour lag, the rate of synthesis of fibrinogen, as measured by a solid-phase radioimmunoassay, continually accelerates, so that rates several fold greater than the $\text{in vivo}$ rate have been observed after twenty hours incubation. Cycloheximide (0.1 mM) completely abolished the appearance of fibrinogen in the medium; whereas colchicine (0.3 mM) reduced the rate by 85%. Insulin and cortisol succinate enhanced fibrinogen synthesis and secretion. The albumin secretion profile differs in several respects from that of fibrinogen, reflecting differences in intracellular pool levels and probably distinct regulatory mechanisms.     

Opposite effect of albumin on the erythrocyte aggregation induced by immunoglobulin G and fibrinogen

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG1.8/A and v = a'F1.5 (A + b'), respectively (v, the velocity of erythrocyte aggregation; A, G and F, the concentrations of albumin, IgG and fibrinogen, respectively; a, a' and b', constant). The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.     

Digitonin perfusion of rat liver. A new approach in the study of intra-acinar and intracellular compartmentation in the liver.

Perfusing a rat liver with digitonin in the concentration range 2-20 mg/ml results in complete decolorization of the organ within 45-250 s. Decolorization progresses with time in the direction of flow, and it is therefore possible, by collecting the eluate, to obtain material from specific intracellular compartments of hepatocytes in different zones in the microcirculatory unit of the liver. The results demonstrate that cytoplasmic marker enzymes from periportal or perivenous hepatocytes can be collected with as little contamination from the other compartment as is obtained in micro-dissection studies. Furthermore, a fraction enriched in mitochondrial marker enzymes can be achieved with only 10-20% contamination by cytoplasmic material.     

Molecular properties and intracellular localization of rat liver nuclear scaffold protein P130

We examined the molecular basis of rat P130, a nuclear scaffold protein, and its functions. P130 comprising 845 amino acid residues possesses several functional domains and yields an electrophoretically distinctive isoform, P123, by altering its phosphorylation status in association with translocation across the nuclear membrane and from the digitonin-extractable fraction of the nucleus to the nuclear scaffold. The functional domains, NLS, NES, and zinc-finger bearing DNA-binding domains, ZF1 and ZF2, aid these translocations. P130 binds RNA through two RNA-binding domains (RB1 and RB2) similar to those of hnRNPs I and L. Microsome- and polysome-localized P130 and P123 were found in rat liver and Ac2F hepatoma cells. This localization required prior entry of P130 to the nucleus, but did not require RB1 and RB2. Thus, P130 initially purified from rat liver nuclear scaffold has the potential to play a variety of roles in biological events not only in the nuclear scaffold but also in various subcellular compartments. P130 (AB205483) is identical to matrin 3 (M63485 and BC062231), although the primary structure of rat matrin 3 has been revised, since it was first published.