Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Intracellular distribution of serum albumin and its possible precursors in rat liver

1. The fractionation of intracellular albumin labelled with radioactive l-leucine was studied in rat liver by means of isoelectric focusing. 2. Isoelectric fractionation was compared with ion-exchange chromatography for purification of radioactive intracellular albumin obtained by antibody precipitation. Similar results were obtained with both methods of separation. Purified albumin contains only a minor amount of the radioactivity. The remainder is associated with albumin-like protein(s). 3. The albumin-like protein has the properties of a precursor of plasma albumin. 4. The distribution and turnover of radioactive albumin in rough and smooth microsomal fractions and in a Golgi-rich fraction were studied. 5. It is concluded that newly synthesized albumin, as such, appears only momentarily if at all in any intracellular structure before its appearance in the plasma. 6. It is also concluded that the rate-limiting step in the secretion of plasma albumin is the conversion of precursor(s) into albumin. We can find no evidence to suggest that there is any significant transport of albumin, as such, during the course of secretion.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Immunochemical studies on biosynthesis of rat plasma angiotensinogen and its regulation by cortisol

Glucocorticosteroid hormones increase the level of rat plasma angiotensinogen by increasing its rate of synthesis. Two forms of plasma angiotensinogen have been purified differing with respect to molecular weight and affinity to concanavalin A. Immunochemical studies using antibodies raised against the separated forms of angiotensinogen revealed cross-reactivity with both antigens. Both antibodies were able to quantitatively precipitate the angiotensinogen activity present in rat serum samples. Cortisol increased the total amount of plasma renin substrate without changing the relative amounts of both angiotensinogen forms. mRNA coding for plasma angiotensinogen was determined by in vitro translation of poly(A)-containing RNA and immunochemical analysis of translation products. Angiotensinogen mRNA could be detected in total poly(A)-containing RNA isolated from rat liver, but not in mRNA isolated from brain, although angiotensinogen has been reported to be present in the latter organ. The level of hepatic mRNA coding for plasma angiotensinogen was high in rats treated with cortisol, but not detectable in animals depleted from endogenous glucocorticosteroids by bilateral adrenalectomy.     

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.     

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR-albumin complexes. Photoirradiation results of BR-albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Improved response of isolated liver cells to glucagon in the presence of rat serum.

The stimulation by glucagon of gluconeogenesis from 10 mM lactate and urea formation in intact isolated rat liver cells is enhanced from about 33 % to 61 % and 92 %, respectively, by the addition of rat serum (30 % v/v) to the incubation medium. This effect is exerted by (a) dialysable, heat-labile serum component(s), different from known stimulators of gluconeogenesis, such as fatty acids, lysine, ammonium chloride or calcium, which do not improve the response of hepatocytes to glucagon. The factor(s) could provide a useful tool for the study of glucagon action(s) on liver metabolism.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Cholecystokinin binding and degradation by isolated rat liver cells

The present studies were directed to examine and quantify binding and degradation of radiolabelled cholecystokinin (CCK) peptides by isolated rat liver cells. After incubation with liver cells (4.5 x 10(6) cells/ml) at 14 degrees C, minimal binding (less than 5%) of labelled CCK33 was detected. When labelled nonsulfated (nsCCK8) and sulfated CCK8 (sCCK8) were incubated, 16.2 +/- 1.8% (mean +/- S.E.) and 7.2 +/- 0.1% of 125I-nsCCK8 and 125I-sCCK8, respectively, were bound to the cell fraction. However, no inhibition of binding of either labelled nsCCK8 or sCCK8 was observed when incubated in the presence of excess unlabelled peptide (10 ng-10 micrograms). Preferential binding of labelled sCCK8, the biologically active form of the octapeptide, appeared to be to the nonparenchymal liver cell, rather than the hepatocyte, fraction; when corrected for cell size and protein content, binding of sCCK8 was approximately 15-times greater by the nonparenchymal cell population. When incubated with hepatocytes at 37 degrees C for 60 min, no degradation of labelled sCCK8 was detected by high pressure liquid chromatography. In contrast, progressive degradation of sCCK8 was observed when the peptide was incubated with the nonparenchymal cells. The results of these studies confirm previous observations that CCK33 is not bound by the liver. They further demonstrate that to some degree CCK8 is preferentially bound and degraded by hepatic nonparenchymal cells; however, this binding appears to be noncompetitive and, therefore, probably not receptor-mediated.     

Rapid uptake by liver sinusoidal cells of serum albumin modified with retention of its compact conformation

The clearance from the blood and the conformation of serum albumin modified by nitroguanidination and labeled with ^{1 2 5} I have been studied. Like formaldehyde-denatured albumin, but in contrast to native albumin, the nitroguanidinated derivative is rapidly cleared from the blood and taken up in lysosomes of liver sinusoidal cells. Although 94% of the free amino groups were blocked by nitroguanidination, we could not detect significant conformational changes using gel filtration, determination of reducible disulfide groups, and titration of tyrosine residues.It is concluded that extensive denaturation is no prerequisite for the uptake of albumin derivatives in liver sinusoidal cells. It is suggested that the nitroguanidinated protein, in contrast to native albumin, is bound on membrane receptors of sinusoidal cells. The nitroguanidino groups themselves might be bound on these receptors, but it seems equally possible that the blocking of positive charges of the albumin molecule or minor, local conformational changes of the protein are sufficient for the binding on the receptors.     

Synthesis of a complementary DNA to rat liver albumin mRNA.

NADPH reduces both liver microsomal cytochrome P-450 and cytochrome $\text{b}{}_5$. In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome $\text{b}{}_5$ which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome $\text{b}{}_5$ directly reduces cytochrome P-450 in rat liver microsomes.     

Degradation of serum amyloid A by isolated perfused rat liver

Degradation of serum amyloid A (SAA) was studied in the isolated perfused rat liver. Radioiodinated SAA was reconstituted with high density lipoproteins (HDL) and administered to rats. Plasma was taken 1 h later, and the HDL were isolated for use as tracer. HDL-bound 125I-SAA was cleared from the plasma of intact animals at a rate similar to SAA in native human HDL. Catabolism of SAA and HDL apoproteins was studied in parallel in the perfused liver. In a 3-h perfusion, 21% of SAA was degraded in contrast to 13% of apoC-III, 7% of apoA-I, and 6% of apoA-II. SAA1 (47% in 3 h) was degraded more rapidly than SAA5 (37%) although their in vivo clearance rates were similar. Degradation of SAA was inhibited when lipoproteins were added to the perfusate. At a protein concentration of 0.15 mg/ml, low density lipoproteins inhibited 47%, HDL 62%, and SAA-rich HDL 75%. Lipid-free normal HDL (0.3 mg/ml perfusate) did not appreciably affect SAA degradation; however, delipidated SAA-rich HDL (0.3 mg of protein/ml; 0.02 mg of SAA/ml) inhibited SAA degradation by 40%. Isolated perfused mouse liver proved more effective than rat liver in degrading SAA (5.3% versus 2.8%/g of liver/h). Degradation appeared to be mediated by cell-associated enzymes since perfusate, which had been recirculated through the liver for 3 h, accounted for less than 15% of the total degradation. Partial (38%) hepatectomy did not significantly reduce apoA-I clearance but reduced that of SAA by 16%, providing additional evidence for hepatic SAA catabolism. We conclude from these studies that SAA is catabolized independently of other HDL proteins, that association with lipoproteins retards SAA clearance, and that SAA catabolism is, in part, a specific process.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

The separation of intracellular serum albumin from rat liver

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-(14)C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10-25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70-90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.