Inhibition by colchicine of fibrinogen translocation in hepatocytes.

Abstract

Colchicine inhibits the secretion into plasma of lipoproteins (12,18), albumin (11), and other proteins (11) produced by the liver. Electron microscopy has shown that, after administration of colchicine, the electron-dense, nonsecreted lipoproteins accumulate in cytoplasmic vesicles (12) and also in the Golgi apparatus (18) of hepatocytes; however, the accumulation site of the electron-transparent, nonsecreted proteins is unknown. In the present report, we demonstrate that fibrinogen, an electron-transparent protein synthetized by the liver cells (16), accumulates not only in the Golgi apparatus, but also within the endoplasmic reticulum when its secretion into plasma is inhibited by colchicine.Fibrinogen was demonstrated in the hepatocytes by means of antirat fibrinogen antibodies labeled with peroxidase, a method which can be used to detect antigens under light and electron microscopy (3). MATERIALS AND METHODSAdult male rats were injected with colchicine, 2.5 mg/kg, intraperitoneally, and were sacrificed 4, 6, 8, 16, 24, or 32 h after injection; rats, injected with 0.9% NaCI or with 2.5 mg/kg of lumicolchicine (a mixture of colchicine isomers prepared by irradiation of colchicine with ultraviolet light [20]) and sacrificed at the same time intervals, acted as controls; the experimental groups corresponding to each time interval consisted of five animals.Antirat fibrinogen antibodies were prepared as follows: a sheep was immunized by an intradermic injection of 7 mg of rat fibrinogen added with Freund's complete adjuvant, followed by an intramuscular injection of fibrinogen with Freund's adjuvant every month for 4 mo; rat fibrinogen was prepared according to the method of Poison et al. (14). Antibodies against antigens other than fibrinogen were eliminated by an immunoadsorbent technique (4) using an immunoadsorbent prepared from normal rat serum; purified antirat fibrinogen antibodies were finally obtained by the same technique, using an immunoadsorbent prepared from normal rat plasma. From those purified antibodies, Fab fragments were prepared by digestion with I% papain (15) and purified by filtration on a diethylaminoethyl-cellulose column (DE 52, Whatman Biochemicals Ltd., Maidstone, England) with sodium phosphate buffer 0.01 M pH 8.0; the Fab fragments were concentrated and labeled with horseradish peroxidase (RZ 3.0, Sigma Chemical Co., St. Louis, Mo.) according to Avrameas (2). For control reactions, Fab fragments were also prepared from normal sheep gamma globulins obtained by ammonium sulfate precipitation followed by filtration on a diethylaminoethyl-cellulose column (DE 52) with sodium phosphate buffer 0.017 M pH 6.9; those Fab fragments were also concentrated and labeled with horseradish peroxidase.Liver specimens were processed according to a technique previously described (6). Briefly, the specimens were at once fixed in 10% paraformaldehyde (prepared as described by Karnovsky [10]) buffered with phosphate buffer 0.1 M pH 7.4 for 6 h at 4~ After fixation, the liver pieces were washed for 24-48 h...

Control of the acute phase response. Demonstration of C-reactive protein synthesis and secretion by hepatocytes during acute inflammation in the rabbit.

“…This conclusion is consistent with our findings in serum, in which the rate of rise in CRP concentration was markedly decreased after colchicine administration. Such an effect of colchicine on secretion of other hepatocyte-preduced plasma proteins has been shown (14)(15)(16)(17). Electron microscopic studies indicated that the perinuclear staining found in large numbers of cells after colchicine administration appeared to represent CRP-filled RER.…”

“…In addition, since colchicine has been shown to inhibit secretion of a variety of plasma proteins by the cells in which they are synthesized (14)(15)(16)(17), the effect of administration of this drug upon cellular localization of CRP during acute inflammation was investigated.…”

Functional Hepatocellular Heterogeneity for the Production of Plasma Proteins

“…In the former period, between 1964 and 1980, most au thors working in this field described the fol lowing situation: in the normal state, a rela tively low percentage of hepatocytes (be tween 1 and 36% according to the protein) were actively engaged in synthesis, and plasma protein-synthesizing hepatocytes were randomly distributed in the hepatic lobule with sometimes a preferential locali zation around the portal triads or the centrilobular vein. For example, this was the case for albumin, some acute phase reactant pro teins such as C-reactive protein, a2-macroglobulin, ai-acid glycoprotein, haptoglobin, some coagulation proteins such as prothrom bin or fibrinogen and of ceruloplasmin and transferrin [1], However, even during this period, three observations did not fit well with these data: (1) when an increase in the hepatic synthesis of prothrombin [24], trans ferrin [25] or albumin [26] was induced, the percentage of hepatocytes engaged in the syn thesis of these three proteins increased strik ingly to shift from a small percentage to almost all hepatocytes; (2) inhibition of plasma protein secretion by microtubule-depolymerizing drugs, such as colchicine, which augment the amount of intracellular proteins, resulted in an increase in the num ber of fibrinogen-synthesizing hepatocytes [27], and (3) observation of serial liver sec tions clearly demonstrated that during an acute inflammatory reaction, the same hepa tocytes were able to synthesize at least four different acute phase reactant proteins at the same time [28], At the end of the 1970s, introduction of membrane-permeabilizing agents in immunocytochemistry completely changed the situation. As discussed pre As recalled in the introduction, hepato cytes, which synthesize the bulk of plasma proteins, are not specialized in the produc tion of a given plasma protein.…”

Synthesis and secretion of albumin in rats during treatment with a carcinogenic dose of N-2-acetylaminofluorene

“…The change in differentiation of smooth endoplasmic reticulum caused by N-2-AAF is a long-term effect, in contrast to the reversible short-term effect reported for colchicine and fibrinogen. Colchicine causes a decrease in secretion of albumin accompanied by an accumulation in the Golgi apparatus (Dorling et al, 1975;Redman et al, 1975), while fibrinogen causes a decrease in secretion of albumin which is accompanied by an accumulation initially in the rough microsomes, but subsequently in the Golgi apparatus (Feldmann et al, 1975).…”