Thuy-Anh Tran-Thi

Regulation of endothelin-1 action on the perfused rat liver

Endothelin‐1 (ET‐1) was found to be a very potent stimulus for contraction and glycogenolysis in the perfused rat liver. At 1 nM it caused a dramatic increase in portal pressure of 22.1 ± 2.7 cm water and enhanced the glucose output up to 3‐fold. Extracellular Ca2+ and protein kinase C were involved in the signal transduction of ET‐1. ET‐1 action does not seem to be mediated by endogenous eicosanoids. The effects of ET‐1 were significantly reduced in the presence of 1 μM Iloprost, a prostaglandin I2 analogue, or by 100 μM sin‐1, a nitric oxide donor. In cultured hepatocytes, glycogenolysis was also stimulated by ET‐1 although to an extent too small to explain the high glucose output found in the perfused liver.

Induction of rat α2‐macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell‐derived factor

Turpentine injection into rats elicits enhanced secretion of acute phase proteins inducing α2‐macroglobulin (α2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of α2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose‐dependent stimulation of α2M synthesis of up to 4‐fold after 10–12 h was observed. However, the presence of low concentrations (10−9 M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of α2M in hepatocytes requires the synergistic action of glucocorticoids and a non‐dialysable factor secreted by Kupffer cells.

Murine interleukin 1 stimulates α2‐macroglobulin synthesis in rat hepatocyte primary cultures

Interleukin 1 α2‐Macroglobulin Rat hepatocyte

Studies on synthesis and degradation of eicosanoids by rat hepatocytes in primary culture

The potential of hepatocytes in primary cultures to degrade the prostanoids produced by Kupffer cells and to synthesize eicosanoids, especially leukotriene B4, after treatment with D-galactosamine was studied. Hepatocytes in primary cultures showed a substantial capability to degrade all the prostanoids produced by stimulated Kupffer cells. The rate of degradation, approx. 2 pmol/min per 10(6) hepatocytes, was nearly the same for the prostaglandins D2, E2 and F2a. Lower rates were determined for thromboxane B2 (0.4 pmol/min per 10(6) cells) and for 6-ketoprostaglandin F1a (0.2 pmol/min per 10(6) cells). The degradation products of these prostanoids lacked biological activity, e.g., reactivity with specific antibodies and the ability to contract segments of rabbit femoral artery. In the presence of 30 microM arachidonic acid, hepatocytes produced only very small amounts of prostaglandins and thromboxane, ranging from less than or equal to 22 to 50 fmol/30 min per 10(6) cells. Neither untreated nor D-galactosamine-treated hepatocytes released significant amounts of leukotriene B4. Hepatocytes appear to be the site of degradation rather than synthesis of eicosanoids in the liver.

Induction of acute phase proteins by dexamethasone in rat hepatocyte primary cultures

The effect of dexamethasone on the synthesis of acute phase proteins has been studied in primary cultures of rat hepatocytes. In the absence of dexamethasone no detectable amounts of alpha 2-macroglobulin were synthesized by hepatocytes cultured for 1 day. alpha 2-Macroglobulin synthesis was induced by dexamethasone concentrations of 10(-8) M or higher with a maximum at a concentration of 10(-7) M. alpha 1-Acid glycoprotein was synthesized in the absence of dexamethasone; however, its synthesis was also greatly stimulated by dexamethasone concentrations of 10(-8)-10(-6) M. Synthesis of alpha 1-proteinase inhibitor was stimulated only 1.4-fold at a dexamethasone concentration of 10(-7) M. The kinetics of induction of alpha 2-macroglobulin and alpha 1-acid glycoprotein were studied at a dexamethasone concentration of 10(-7) M. After an initial lag phase of 3 h the synthesis of both proteins showed a steady increase during 2 days. Synthesis of albumin remained unchanged under these experimental conditions. Unlike alpha 2-macroglobulin and alpha 1-acid glycoprotein tyrosine aminotransferase activity increased already during the first 3 h of induction by dexamethasone with a maximum at 12 h followed by a slight decrease.

Biosynthesis and Secretion of α1‐Antitrypsin in Primary Cultures of Rat Hepatocytes

The biosynthesis and secretion of alpha 1-antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an alpha 1-antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of alpha 1-antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. Pulse-chase experiments showed that about 30 min after its synthesis the transformation of the 49000-Mr alpha 1-antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000-Mr protein was secreted by the hepatocytes. The 54000-Mr alpha 1-antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [3H]galactose and [3H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation of N-asparagine-linked oligosaccharide chains, an unglycosylated alpha 1-antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of alpha 1-antitrypsin by 60-70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both alpha 1-antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of alpha 1-antitrypsin.

Net prostaglandin release by perfused rat liver after stimulation with phorbol 12-myristate 13-acetate

Phorbol myristate acetate, which was shown previously to elicit eicosanoid synthesis in primary cultures of Kupffer cells, led to a net release of prostaglandins (PG) D2 and E2 from the perfused rat liver. While a substantial amount of PGD2 (the major prostaglandin of Kupffer cells) left the liver, very little PGE2 was found in the effluent. Considerable amounts of immunologically reactive PGD2 and E2 were secreted with the bile. PGE2 rather than PGD2 was able to stimulate glycogenolysis and to increase perfusion pressure. These effects were, however, strongly dependent on the direction of the flow. If the liver was perfused in a retrograde fashion, i.e., from the vena cava to the portal vein, phorbol myristate acetate or PGE2 exerted only minor effects. These observations suggest a topological heterogeneity of producer and responder cells, respectively, in the liver sinusoid.

Unglycosylated rat α1-proteinase inhibitor has a six-fold shorter plasma half-life than the mature glycoprotein

The plasma half-lives of glycosylated and unglycosylated alpha 1-proteinase inhibitor-radioactively labeled with [35S]methionine in rat hepatocyte primary cultures - were determined in the rat. Unglycosylated alpha 1-proteinase inhibitor was synthesized by hepatocytes in the presence of tunicamycin. Media from hepatocytes containing 35S-labeled glycosylated or unglycosylated alpha 1-proteinase inhibitor were injected into the tail veins of rats. At different times after injection alpha 1-proteinase inhibitor was isolated from plasma by affinity chromatography with anti-alpha 1-proteinase inhibitor Sepharose. Radioactivity measurements revealed a plasma half-life of 170 min for glycosylated alpha 1-proteinase inhibitor and of 30 min for the unglycosylated form of the inhibitor.

N-terminal amino acid sequences of precursor and mature forms of α-1-antitrypsin

α‐1‐Antitrypsin is found in hepatocytes as a high‐mannose glycoprotein (M r 49000), extracellularly as a complex‐type glycoprotein (M r 54000). Deglycosylation of both forms with peptide: N‐glycosidase led to proteins of identical app. M r (41000). The sequence of 26 N‐terminal amino acids of rat α1‐antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized α1‐antitrypsin showed a cluster of hydrophobic amino acids. A pre‐peptide of 24 amino acids is proposed. There is no evidence for the existence of a propeptide.

Synthesis of α2-macroglobulin in rat hepatocyte and in a cell-free system

The biosynthesis and secretion of α2‐macroglobulin was studied in rat hepatocytes primary cultures. After immunoprecipitation of α2‐macroglobulin from a cell homogenate and the hepatocyte medium, two forms of α2‐macroglobulin with app. M r of 176000 and 182000, respectively, were identified. A precursor—product relationship for the two α2‐macroglobulin forms was demonstrated by a pulse‐chase experiment. The cellular form of α2‐macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of α2‐macroglobulin remained unaffected. On the other hand, only the medium form of α2‐macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. M r of 162000.