Production Of Plasma Proteins Research Articles

Inhibition by the mushroom toxins α-amanitin and phalloidin of hepatopoietin-induced 3H-thymidine incorporation into rat liver DNA and of plasma protein production in hepatocyte cultures

F. M. Fouad, W. D. Marshall, P. G. Farrell, M. Goldberg and G. Ruhenstroth-Bauer, Inhibition by the mushroom toxins α-amanitin and phalloidin of hepatopoietin-induced 3H-thymidine incorporation into rat liver DNA and of plasma protein production in hepatocyte cultures. Toxicon 25, 1265 – 1271, 1987.—In primary cultures of rat hepatocytes the induction of 3H-thymidine uptake into DNA of liver cells by the liver cell proliferation factor hepatopoietin demonstrates that this factor is active not only in vivo but also in vitro. Addition of the mushroom toxins α-amanitin or phalloidin to liver cell culture decreased the uptake of 3H-thymidine into hepatocytes (in the absence or presence of hepatopoietin) as well as the attachment of the hepatocyte cultures. Mushroom toxins also inhibited the production of plasma proteins in hepatocyte cultures. The inhibition, observed at toxin concentrations from 10 −5 to 10 −7 M, was dose-dependent. At low concentrations of phalloidin the inhibition appears to be selective for certain proteins.

Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken.

Identity has been established between chicken hemopexin and alpha 1-globulin "M," a plasma known for the hormone responsiveness of its synthesis in monolayer cultures of embryonic chicken hepatocytes (Grieninger, G., Plant, P. W., Liang, T. J., Kalb, R. G., Amrani, D., Mosesson, M. W., Hertzberg, K. M., and Pindyk, J. (1983) Ann. N. Y. Acad. Sci. 408, 469-489). Identification was based on immunological cross-reactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5- and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.

Plasma-protein production by rat hepatoma cells in culture, their variants and revertants

A series of subclones of the H4II line of the Reuber H35 rat hepatoma produce substantial amounts of three plasma proteins, transferrin, α 1-antitrypsin and fibrinogen. Immunocytochemical staining demonstrated that each of these proteins is synthesized by essentially every cell of these cell populations. Cells of dedifferentiated variant clones either cease to produce the proteins, or exhibit a substantial reduction that is accompanied by variability in the synthetic activity of individual cells of the population, As previously observed with regard to angiotensinogen production, the variant clones clearly divide into two categories: those that show only a reduction in synthesis are able to give rise to revertants, whereas the negative clones fail to do so. Revertant cells exhibit a dramatic restoration of the synthesis of plasma proteins, which in some cases, exceeds by severalfold the rates seen in the differentiated clones of origin. In addition, the revertant cells synthesize α-fetoprotein, a function that is not expressed by H4II cells or its daughter subclones. Immunocytochemical staining revealed that, with regard to several plasma proteins including albumin, fibrinogen and α-fetoprotein, the cell populations of revertant clones are very heterogeneous, for only a fraction of the cells synthesizes each protein. Hybrid cells resulting from several types of crosses, exhibited extinction of the plasma proteins, the exception being transferrin, whose production was maintained, but at a reduced level and in only a fraction of the cells. Taken together, our results show that the expression of albumin and transferrin can be dissociated from one to another, and from that of fibrinogen, α 1-antitrypsin and angiotensinogen.

Insulin-like growth factors (IGF I and IGF II) mimic the effect of insulin on plasma protein synthesis and glycogen deposition in cultured hepatocytes

Monolayer cultures of chick embryo hepatocytes were used to compare the effects of insulin-like growth factors, IGF I and IGF II, with insulin on two hepatic functions: plasma protein production and glycogen deposition. Just as with exposure of the cells to insulin, addition of either IGF I or IGF II to the otherwise hormone-free medium elicited a dramatic change in the production of secretory proteins as well as the development of glycogen deposits equivalent to in vivo (fed) levels. No major differences between insulin, IGF I or IGF II were observed in terms of the degree of stimulation or potency of the hormones.

Direct assignment of orosomucoid to human chromosome 9 and alpha 2HS-glycoprotein to chromosome 3 using human fetal liver x rat hepatoma hybrids.

The production of plasma proteins has been monitored in somatic cell hybrids between a rat hepatoma cell line (7777) and human fetal liver cells. Production of 14 plasma proteins was assayed in concentrated serum-free culture supernatants by electroimmunoassay. Alpha 2HS-glycoprotein (AHSG) was produced by 10 of 19 hybrids; concordancy for presence or absence of protein production was 100% for human chromosome 3. Orosomucoid (ORM) was produced in 8 of 19 hybrids, with a concordancy for presence or absence of protein of 94.7% with human chromosome 9. The chromosome location for genes for these two proteins, previously assigned by linkage studies, is confirmed by direct assignment. These studies have also suggested possible chromosomal assignments for loci for alpha 1-antichymotrypsin and C1 esterase inhibitor. Other genes for proteins which could not be assigned to specific chromosomes using these hybrids were: complement C3, ceruloplasmin, hemopexin, inter-alpha-trypsin inhibitor, prealbumin, retinol-binding protein, transferrin and apolipoproteins CII, B, and sinking-pre-beta [Lp(a)].

Maintenance of differentiated rat hepatocytes in primary culture.

Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (ii) analyzed for the production of albumin and other plasma proteins; and (iii) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11-19 micrograms/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically defined medium with Me2SO enables maintenance of differentiated hepatocytes in culture for extended periods of time.

Morphological Aspects of Plasma Protein Synthesis and Secretion by the Hepatic Cells

Publisher Summary This chapter presents a review of morphological aspects of plasma protein synthesis and secretion by the hepatic cells. The aim of the review is to examine the following problems: (1) identification of the cells responsible for plasma protein synthesis—the problems of their specialization and location in the hepatic lobule, (2) production of several plasma proteins at a given time by the protein-synthesizing cells and possible interference by some plasma proteins in the production of others, (3) behavior of synthesizing cells in experimental and pathological conditions, and (4) arguments for the production of plasma proteins by cells outside the liver. Since the introduction of immunohistological methods, attempts to locate the PP-containing hepatocytes in the normal hepatic lobule have produced varied results. For instance, when IF and IPO were first used, random distribution of albumin-containing hepatocytes was observed by light microscopy in the hepatic lobule. The recent introduction of membrane-permeabilizing agents has remarkably improved the results obtained with immunohistochemical methods.

Human keratinocytes and monocytes release factors which regulate the synthesis of major acute phase plasma proteins in hepatic cells from man, rat, and mouse.

Human keratinocytes and activated monocytes produces factors which can stimulate the proliferation of thymocytes. The same activity has also been implicated in regulating the expression of plasma proteins in liver cells during the acute phase reaction. To assess whether factors produced by such cells can directly influence liver cells to change the production of acute phase plasma proteins, we studied in tissue culture the response pattern of hepatic cells from three species: human hepatoma cells ( HepG2 cells), and primary cultures of rat and mouse hepatocytes. Conditioned media from the squamous carcinoma COLO-16 cells, normal epidermal cells, and activated peripheral monocytes were able to stimulate the synthesis of specific acute phase plasma proteins: alpha 1-antichymotrypsin in HepG -2 cells, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, alpha 1-acute phase protein, and alpha 2-macroglobulin in rat hepatocytes, and alpha 1-acid glycoprotein, haptoglobin, and hemopexin in mouse hepatocytes. Only in rat cells, dexamethasone was found to have further enhancing effect. The increased production of plasma proteins could be explained by an elevated level of functional mRNA. Comparing thymocyte-stimulating activities with the effects on plasma protein production, we found some difference both between the conditioned media of epidermal cells and monocytes, and between the responses of the three hepatic cell systems. Furthermore, gel chromatography of conditioned media resulted in partial separation of activities regulating liver cells and thymocytes. Since there is no strict correlation between thymocyte- and hepatocyte-stimulating activities, the presence of different sets of specific factors is assumed.

Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium

Three acute-phase proteins, haptoglobin, α 2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and α 2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, α 1-acute-phase protein, α 1-acid glycoprotein and α 1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acutephase proteins by liver cells.

In vitro production of pregnancy-associated plasma protein A by human decidua and trophoblast

Immunohistochemical techniques and direct measurements of pregnancy-associated plasma protein A (PAPP-A) have demonstrated the presence of PAPP-A in trophoblast and decidua. The purpose of the present study was to investigate the possibility that these tissues are capable of producing PAPP-A in vitro. Trophoblast and decidua were obtained from term deliveries and from legal surgical terminations of pregnancy (7 to 12 weeks). In addition to trophoblast and decidua, myometrium was also obtained during two hysterectomies in the first trimester of pregnancy. Tissues were incubated in medium 199 at 37 degrees C under an oxygen/carbon dioxide atmosphere. Media containing either pregnancy-associated serum or non-pregnancy-associated serum were changed after 8 hours of incubation in medium 199 alone. In addition to PAPP-A, human placental lactogen (hPL) and prolactin (Prl) were measured in homogenates and media by radioimmunoassays in order to confirm the viability of the cultured tissues. Addition of pregnancy-associated serum to the media induced a significant release of PAPP-A from trophoblast and decidua when compared to that in control cultures. Non-pregnancy-associated serum had no effect. Myometrium did not release any measurable PAPP-A into the medium even in the presence of pregnancy-associated serum. Cycloheximide added to pregnancy-associated serum significantly inhibited the release of PAPP-A from trophoblast and decidua. These last tissues, irrespective of the culture condition, released significantly more PAPP-A as well as hPL and Prl than was initially present in the tissue. These data demonstrate that PAPP-A is released in vitro by trophoblast and decidua (but not by myometrium) and that this release can be magnified by a factor present only in pregnancy-associated serum. The release of PAPP-A, hPL, and Prl is considered as a de novo production since concentration of these proteins are higher in media and tissues after incubation compared to concentrations initially present in the tissue before culture and since cycloheximide significantly inhibits the release of PAPP-A, Prl, and hPL from the cultured tissues.

Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis.

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes.

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.

Rapid and selective shutoff of plasma protein production in herpes simplex virus type 2-infected hepatoma cells

The effect of herpes simplex virus type 2 (HSV-2) infection of hepatoma McA-RH7777 cells on the production of alpha-fetoprotein (AFP) and several other secreted plasma proteins was examined. Cells infected with HSV-2 were labeled with [35S]methionine for various times postinfection (p.i.). Culture media and intracellular extracts were immunoprecipitated with plasma protein antibodies and examined by polyacrylamide gel electrophoresis. The relative levels of AFP and several other plasma proteins decreased substantially in infected cells compared with mock-treated controls; however, the level of at least one secreted plasma protein was unchanged after infection and the level of another increased after infection. Hybridization analyses with AFP cDNA showed that AFP RNA decreased to 43 and 30% of controls at 3.5 and 6.5 hr p.i., respectively. These observations were supported by the results of cell-free translation of isolated poly(A)-containing RNA. HSV-2 proteins were not detectable at times p.i. when AFP synthesis, secretion, and mRNA levels were significantly diminished. No decrease in AFP levels was observed in cells infected with ultraviolet-irradiated virus. We conclude that HSV-2 infection of McA-RH7777 cells leads to a rapid decrease in synthesis and secretion of specific plasma proteins which is apparent, at least for AFP, at the level of mRNA. This shutoff is not common to all McA-RH7777 cell proteins and, although seen early after infection, most likely requires the expression of a virus gene(s).

Induced underglycosylation of PLC/PRF/5 human hepatoma cells. Brief report.

The production of HBsAg and human plasma proteins by PLC/PRF/5 human hepatoma cell line was studied in the presence of glycosylation inhibitors. The data presented here suggest that HBsAg release in culture medium by these cells is not reduced by an inhibition of glycosylation and that Tunicamycin may play a role in a mobilization of the intracellular pool of antigen.

Decreased albumin and increased fibrinogen secretion by single hepatocytes from rats with acute inflammatory reaction.

During the acute inflammatory reaction (AIR), the plasma concentration of several plasma proteins, including fibrinogen, is increased as a result of enhanced production by the liver. Whether this increased production influences the production of the other plasma proteins at the single hepatocyte level is not known. Accordingly, we simultaneously measured albumin and fibrinogen secretion by single hepatocytes isolated from normal rats and from rats sacrificed 24 hr after a turpentine–induced AIR, using an hemolytic plaque test. In normal rats, 74, 82, and 98% of the hepatocytes were detected as secreting albumin, after 1, 2, and 4 hr of incubation, respectively. Simultaneously, 0, 32, and 62% of the hepatocytes were detected as secreting fibrinogen at the same incubation times. For secretion of both proteins, a significant correlation was established between the diameter of a plaque–forming hepatocyte and its hemolytic plaque. In rats with AIR, the formation of hemolytic plaques detecting albumin secretion was slowed down compared to normal rats, since only 0, 36, and 78% of the hepatocytes were forming hemolytic plaques after 1, 2, and 4 hr of incubation, respectively. Simultaneously, the formation of hemolytic plaques detecting fibrinogen secretion was increased compared to normal rats, since at the same incubation times, 52, 78, and 93% of the hepatocytes formed hemolytic plaques. A significant correlation between the diameter of a plaque–forming hepatocyte and its hemolytic plaque was still maintained for both protein secretion. These results demonstrate that during the AIR enhanced fibrinogen production is the result of an increased secretion rate per cell, and decreased albumin secretion is the result of a lowered secretion rate per cell, generalized for all hepatocytes. These data strongly suggest that there is a reciprocal modulation of fibrinogen and albumin secretion at the single hepatocyte level during the AIR.

Serum stimulation of plasma protein synthesis in culture is selective and rapidly reversible

Primary hepatocyte monolayers, derived from chick embryos, can be cultured from the onset in a completely chemically defined medium, free of added hormones. The liver cells synthesize and secrete a wide spectrum of plasma proteins for several days in this serum-free environment. Addition of fetal bovine serum elicits a 3–5-fold increase in the production of certain plasma proteins: fibrinogen, albumin, and the α 1- globulin M. This effect of serum is selective; transferrin and plasminogen syntheses are enhanced less than 1.5-fold. Significant stimulation is observed with 0.1% fetal bovine serum, and half-maximal values for individual plasma proteins are obtained with concentrations ranging between 0.4 and 1%. The stimulatory activity of serum shows no developmental or species specificity. Plasma is as active as serum derived from the same blood sample. The hepatocytes respond rapidly to serum, significant changes in albumin synthesis occurring less than 1 h after serum addition or removal. The effect of short exposure is fully reversible. These results establish the capacity of low concentrations of serum to stimulate plasma protein synthesis and underscore the importance of studying the effects of hormones and other factors under serum-free conditions. The findings suggest that, in addition to the classical hormones, ubiquitous but as yet uncharacterized serum components play a role in controlling this major hepatic function.

Influence of autohemotransfusion on the quantitative and qualitative composition of plasma proteins in patients with thyrotoxic goiter

Changes in the concentration of plasma proteins were studied in 45 patients with thyrotoxic goiter, who were injected with autologous blood during the operation. Exfusion of 250 ml of blood in patients before surgery and its reinfusion during surgery did not cause protein metabolism disorders. On the contrary, after hemexfusion, the production of plasma proteins increased. As a result of this, as well as reinfusion of autologous blood, the concentration of albumin increased; the content of G-globulins returned to normal.

Nouvelle méthode de préparation de l'hémopexine de rat

Three acute-phase proteins, haptoglobin, α2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and α2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, α1-acute-phase protein, α1-acid glycoprotein and α1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acutephase proteins by liver cells.

Serum vitamin A, retinol-binding protein, and prealbumin concentrations in protein-calorie malnutrition: I. A functional defect in hepatic retinol release

The components of the plasma vitamin A transport system have been examined in 33 Egyptian children with protein-calorie malnutrition and in 15 control children. Twenty-one children with classical kwashiorkor had significantly decreased concentrations of serum retinol-binding protein (RBP), pre-albumin (PA), and vitamin A as well as of carotenoids, albumin, and total protein. After treatment with calories and proteins but without supplemental vitamin A, 11 patients with kwashiorkor were clinically judged to be cured; these patients demonstrated highly significant increases in the concentrations of vitamin A, RBP, and PA by the end of the 2nd week with further progressive increases occurring by the 4th week. The concentrations of vitamin A, RBP, and PA were comparable to or above those of the control children at discharge. Six children with kwashiorkor who were considered improved showed lesser increases in the concentrations of the three components of the retinol transport system. Six children with marasmus had pretreatment concentrations of vitamin A and RBP not significantly different from controls. Over the wide concentration range observed in kwashiorkor during treatment, the serum vitamin A, RBP, and PA concentrations were highly significantly correlated with each other. These findings suggest that the low serum vitamin A levels in kwashiorkor largely reflect a functional impairment in the hepatic release of vitamin A rather than vitamin A deficiency per se. Hepatic release of vitamin A is apparently impaired because of defective hepatic production of plasma proteins, including the plasma transport proteins for retinol because of a limiting supply of substrate for protein synthesis. When substrate is provided by dietary calories and protein, the hepatic production of plasma proteins increases, plasma RBP and PA concentrations rise, and hence, plasma vitamin A concentration increases.

Studies on plasma protein synthesis with a new liver perfusion apparatus.

SummaryAn apparatus has been devised to perfuse livers under conditions which simulate those existing in the body. It consists of two identical perfusion sets so the metabolism of two livers can be compared under the same conditions. The perfused liver continues to produce plasma protein for 10 hours. After this time there is no vacuolation or necrotic change in any part of the liver; only slight atrophic changes in hepatic cells are present. A replacement of the blood plasma of the perfusate by Ringer-Locke solution caused an increase in plasma protein production and a decrease in the synthesis of liver tissue protein. When PVP was added to Ringer-Locke solution the production of plasma protein decreased. In both perfused liver and in vivo globulin appears to have a higher rate of synthesis than albumin. Gamma-globulin shows a faster turnover rate than albumin and alpha-globulin shows a still faster rate.