Expression of matrix metalloproteinase-2 and -9 and of tissue inhibitor of matrix metalloproteinase-1 in liver regeneration from oval cells in rat.

Oval cells participate in liver regeneration when hepatocyte replication is impaired. These precursor cells proliferate in periportal regions and organize in ductules. They are surrounded by a basement membrane, the degradation of which by matrix metalloproteinases (MMP) might trigger their terminal differentiation into hepatocytes. We studied the expression of MMP-2 and MMP-9 and that of one of their tissue inhibitors (TIMP-1) in a model of hepatic regeneration from precursor cells. Regeneration was induced by treating rats with 2-acetylaminofluorene followed by partial hepatectomy. MMP-2 and MMP-9 hepatic expression paralleled oval cell number with a peak at day 9-14 after hepatectomy. They were mainly detected in oval cells. TIMP-1 mRNA and oncostatin M receptor mRNA, a major regulator of TIMP-1 synthesis, markedly increased from day 1 after surgery until day 9 and then declined; they were mainly detected in interlobular bile duct cells and oval cells until day 14. In agreement with the in vivo data, the WB-F344 liver precursor cell line expressed MMP-2 and MMP-9, as well as TIMP-1 and oncostatin M receptor. These data suggest that (a) early increased TIMP-1 synthesis by biliary and oval cells favors basement membrane deposition around proliferating ductular structures through MMP inhibition, (b) delayed increased MMP expression, concomitant to decreased TIMP-1 synthesis, leads to basement membrane degradation, preceding oval cell differentiation, (c) the oncostatin M pathway might play a major role in increased TIMP-1 synthesis.

Three alternative promoters of the rat gamma-glutamyl transferase gene are active in developing lung and are differentially regulated by oxygen after birth.

The rat gamma-glutamyl transferase mRNA transcripts I, II, and III are derived from three alternative promoters, P(I), P(II), and P(III). In the adult only mRNA III is expressed in the lung. We show that mRNA III gene expression is developmentally regulated in the fetal lung; it is first expressed in gestation. In contrast to the adult lung, the fetal lung expresses mRNA I, II, and III. The switch from the fetal to the adult pattern of gammaGT mRNA expression begins within the first 24 h of birth and is complete by 10 d of age. gammaGT mRNA II disappears within 24 h, mRNA I disappears by 10 d leaving mRNA III as the sole transcript. Alveolar epithelial type 2 cells (AT2) isolated from the adult lung express only mRNA III. When cultured in 21% O2 mRNA III is maintained, but when cultured in 3% O2 the fetal pattern of mRNA I, II and III expression is induced. When AT2 cells in hypoxia are exposed to carbon monoxide, mRNA II is suppressed suggesting that a heme-binding protein (responsive to oxygen) may suppress mRNA II expression and may be responsible for the decrease in lung mRNA II seen after birth. A reporter gene under the control of DNA sequences from the gammaGT P(III) promoter is activated in transient transfection studies in response to hyperoxia, while a deletion construct retaining an antioxidant responsive element is not. Oxygen appears to regulate each of the alternative promoters of the gammaGT gene, such that P(II) is rapidly repressed by a heme-dependent mechanism, P(I), is more gradually repressed by a nonheme mechanism and P(III) is activated by a putative oxygen response element. We hypothesize that similar oxygen-dependent mechanisms regulate other genes in the developing lung at birth.

Synthesis and immunocytochemical localization of alpha 2u globulin in the duct cells of the rat submaxillary gland.

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.

Immunocytochemical localization of gamma-glutamyltranspeptidase, GP-2 and amylase in the rat exocrine pancreas: the concept of zymogen granule membrane recycling after exocytosis.

We localized gamma-glutamyltranspeptidase (GGT) in the rat pancreas by immunocytochemistry using the protein A-gold technique. The enzyme was found in the apical and zymogen granule (ZG) membranes of the pancreatic acinar cell. With ZG at the onset of exocytosis, labeling was seen over membrane, whereas content was unreactive. In the acinar lumen, the enzyme was generally associated with small vesicles previously described as "pancreasomes." This observation corroborates a recent proposal that a membrane-shedding process is associated with exocytosis in the exocrine pancreas. It also implies that some elements of the ZG membrane are not recycled after exocytosis. The cellular distribution of GGT was compared with GP2, another glycoprotein component of the ZG membrane, and differences in localization indicate different fates for these two proteins. Indeed, GP2 shows a strong signal with the basolateral membrane, whereas in the case of GGT the signal is barely detectable. The reverse situation is observed on the apical plasma membrane, GGT producing a much stronger signal than GP2. The failure to detect GGT in lysosomal structures, combined with the fact that some endocytic-like vacuoles in the vicinity of the apical plasma membrane give a positive reaction, supports the view that some GGT molecules are recycled in the ZG membrane after exocytosis. Our observations clearly demonstrate that a fraction of the protein components of the ZG membrane are not recycled after exocytosis, raising new questions regarding the concept of membrane recycling associated with regulated secretion.

Disposable filters for microaggregate removal from extracorporeal circulation.

A comparative study of three commercially available blood filters for use in extracorporeal circuits was made in dogs. All filters were efficient in removing infused microclots from the circulation, but all caused mild thrombocytopenia and two produced minimal hemolysis. In dogs infused with microclots, only those animals without blood filters in the infusing circuit showed evidence of pulmonary microembolism at autopsy. It was concluded that while the filters have minimal disadvantages, their potential in reducing microembolism in extracorporeal circuits far outweighs these disadvantages.

Nature of deposits in a tubular membrane oxygenator after prolonged extracorporeal circulation: a scanning electron microscope study.

Although silicone fibers are among the most compatible with tissue and blood, numerous deposits are observed after their prolonged usage in a capillary membrane oxygenator, even when the blood has been properly heparinized. The scanning electron microscope (SEM) study shows that the morphology of these deposits varies greatly, depending upon the part of the unit from which the sample is taken. The area close to the inlet is the most severely affected. The outlet zone is affected to a lesser degree, and the areas in between are only slightly affected.

Gamma-glutamyl transpeptidase gene organization and expression: a comparative analysis in rat, mouse, pig and human species.

Gamma-glutamyl transpeptidase (GGT) is an enzyme located at the external surface of epithelial cells. It initiates extracellular glutathione (GSH) breakdown, provides cells with a local cysteine supply and contributes to maintain intracellular GSH level. GGT expression, highly sensitive to oxidative stress, is a part of the cell antioxidant defense mechanisms. We describe recent advances in GGT gene structure and expression knowledge and put emphasis on the complex transcriptional organization of that gene and its conservation among different species. GGT gene structure has been elucidated in rat and mouse where a single gene is transcribed from multiple promoters into several transcripts which finally yield a unique polypeptidic chain. Analysis of rat, mouse, human and pig cDNA and gene sequences reveals a large conservation of the transcriptional organization of that gene. This complex structure provides flexibility in GGT expression controlled at the promoter level, through multiple regulatory sites, and at RNA level by alternate 5' untranslated sequences which may create a diversity in the stability and translational efficiency of the different transcripts. In conclusion, transcription of the GGT gene from several promoters offers multiple DNA and RNA targets for various oxidative stimuli and contributes to a broad antioxidant cell defense through GGT induction and subsequent cysteine supply from extracellular glutathione.

[Effect of glycodihydrofusidate, a structural analog of bile salts, on the hepatic transport of bromosulfophthalein in rats]. / Influence du glycodihydrofusidate, un analogue structural des sels biliaires, sur le transport hépatique de la bromesulfonephtaléine chez le rat.

The interaction between bromosulfophthalein and glycodihydrofusidate in their transport by the liver were studied. In vitro, glycodihydrofusidate, a bile salt analogue, inhibited bromosulfophthalein uptake by isolated rat liver cells. This inhibition was similar to that previously described by adding sodium taurocholate to the medium; the inhibition was only partial and could no longer be detected at high bromosulfophthalein concentrations (20 microM). These results suggest that glycodihydrofusidate, like sodium taurocholate can compete with bromosulfophthalein for a common carrier in the liver cell membrane. In vivo, in the rat submitted to a saturating infusion of bromosulfophthalein, the addition of glycodihydrofusidate to the perfusate induced a 65 p. 100 decrease in the biliary excretion of bromosulfophthalein, a decrease in the water flow (47 p. 100) and a slight diminution in the bile salt output (14 p. 100). In experiments where glycodihydrofusidate-bromosulfophthalein interactions did not occur at the sinusoidal level, the biliary excretion of the dye was inhibited by glycodihydrofusidate. This suggests a common pathway for the two molecules. Our results are consistent with the existence of two different bromosulfophthalein carrier systems present at either pole of the hepatocyte. However only one is shared with bile salts and glycodihydrofusidate. This same hypothesis might account for many other experimental results as well.

Inhibition by sulfobromophthalein of mitochondrial translocation of anions and adenine nucleotides: effects upon liver adenosine triphosphate and possible correlation with inhibition of bile flow in the rat.

Unconjugated sulfobromophthalein (BSP) inhibits state III respiration of rat liver mitochondria. It competitively inhibits the translocation into mitochondria of citrate, malate, phosphate and adenosine diphosphate, as studied by the inhibitor stop method. A double-beam spectrophotometric study strongly suggests that glutamate translocation is similarly inhibited. After perfusion of 65 mumol/hr/100 g for 90 minutes, bile flow is inhibited by 82% and liver adenosine triphosphate (ATP) falls by 60%. The amount of mitochondrial BSP can be computed form the amount of [35S] BSP still bound to mitochondria that are prepared at the end of such experiments; the amount of BSP lost during the isolation procedure is estimated from parallel experiments following binding of BSP in vitro. Comparison of the kinetic constants of mitochondrial transport and of their inhibition by BSP on the one hand and of liver concentration of substrates and BSP on the other gives rise to the conclusion that a strong inhibition of transports, mainly of phosphate, occurs in vivo and is responsible for the concomitant decrease in bile flow.

[Blood microfiltration. Scanning electron microscopic study of the aggregates retained by a transfusion filter made of polyurethane foam]. / Microfiltration du sang. Etude par microscopie électronique à balayage des agrégats retenus par un filtre à transfusion en mousse de polyuréthane

Preliminary studies of the transfusion filter Bentley PF 127, a polyfilter type with a graded serie of diameters of microfenestration are reported. Dog blood has been used in all instances of the trial phase. Variations of the hematological factors as well as biochemical disparities have been examined and all deposits were assessed by means of scanning electron microscope. Amounts of deposits increased with the blood age. As far as banked dog blood develops less microaggregates during storage than human blood, the SEM pictures reported are a plea for banked blood microfiltration in any transfusion to human beings. The deposits which were trapped in the polyurethane foam, had previously passed through a screen filter with pore size slightly wider than the standard one (250 microns instead of 170 microns). Unfortunately the possibility of thrombus formation is serious as far as banked blood is rather fragile, and due to a slow flow rate, the time of blood contact with the filter is enough to allow thrombus development. However, the amounts of clots greatly increased with the age of the blood. The importance of filtration by adsorption was not very visible. The future of such a depth filter is questionable: should we prefer a transfusion screen filter with small pore size, the efficiently of which is determined by its pore size, and which traps the microaggregates by mechanical retention, or a depth filter which is supposed to retain the microaggregates regard less of the size but which could be very easily thrombus invaded and does not allow a suffisant blood flow rate for patients needing large amounts of blood in period of initial resuscitation? The debate is open but we should recognize that a screen filter with small pore size is widely used in the hospitals.

Effects of phenobarbital and rose bengal on the ATPases of plasma membranes of rat and rabbit liver.

The effects of drugs which change the bile-salt-independent fraction of bile flow on Na(+)K(a+) and Mg(2+) activated ATPases were studied in membrane fractions rich in bile canaliculi. The administration of phenobarbital caused no induction of these enzymes which could explain the increased bile flow observed in the rat. Rose bengal, in addition to its strong photoxidative inhibition of both ATPases, inhibits the Na(+)K(+) ATPase of rat and rabbit bile canaliculi in the absence of light. A closely related substance, uranine, inhibits neither bile flow nor Na(+)K(+)ATPase. Inhibition of this enzyme by rose bengal may therefore be responsible for the observed effects of this dye on bile flow independent of bile salts.

Multiple forms of gamma-glutamyl transpeptidase messenger ribonucleic acid are expressed in the adult rat testis and epididymis.

The expression of gamma-glutamyl transpeptidase (GGT) mRNA in tissues of the adult rat male reproductive tract was examined. Northern blot analysis of total RNA revealed that GGT mRNA expression occurs primarily in the initial segment and caput epididymidis. Multiple GGT mRNAs of varying sizes were detected in the testis and in different regions of the epididymis: testis, 2.4 and 2.8 kb; efferent ducts, 2.2 and 2.5 kb; initial segment, 2.2, 2.4, and 2.5 kb; caput, 2.2, 2.4, and 2.5 kb; corpus, 2.2 and 2.5 kb; cauda, 2.2 and 2.5 kb; ductus deferens, 2.2 and 2.4 kb. Ribonuclease (RNase) H removal of the poly(A) tail from testicular and epididymal GGT mRNA revealed that multiple GGT mRNAs were not generated by differences in the length of the poly(A) tail. Northern blot analysis of poly(A)+ mRNA with four GGT mRNA 5' untranslated region (UTR)-specific cRNA probes showed that the multiple GGT mRNAs expressed in the testis and epididymis were due to differences in the lengths and nucleotide compositions of the 5' UTR. We hypothesize that transcription of multiple GGT mRNAs from different promoters on the single-copy GGT gene is the molecular basis that underlies the region-specific expression of GGT mRNAs along the rat male reproductive tract.

The gamma-glutamyl transpeptidase gene is transcribed from a different promoter in rat hepatocytes and biliary cells.

Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adult rat liver by Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization analysis. We show that the GGT activity in the 18-day-old fetus results from the transcription of the gene from the promoter P3 in the hepatocytes. In contrast, the GGT promoter P4 activity appears to be specific of biliary cells in normal as well in cholestatic liver. Thus, sequences unique to the GGT transcripts initiated on these two alternate promoters provide unique molecular probes to discriminate between the biliary and the hepatocytic phenotypes in liver differentiation and cell lineage studies.

Identification of a second promoter which drives the expression of gamma-glutamyl transpeptidase in rat kidney and epididymis.

In rat, gamma-glutamyl transpeptidase (GGT) is encoded by multiple mRNAs (mRNAI, mRNAII, mRNAIII, and mRNAIV) that differ only in their 5' untranslated regions and are transcribed from a single-copy gene. Using oligonucleotides designed from the 5' untranslated sequences of the GGT mRNAII and mRNAIII, we amplified a 3.4-kb genomic sequence which contains the promoter region for mRNAII. The sequence flanking the two initiation start sites for mRNAII contains consensus motifs for several potential regulatory proteins and a TATA-like element at the expected position 26 bp upstream from the predominant start site. The sequence from positions -528 to +72 associated with the chloramphenicol acetyltransferase (CAT) reporter gene drives a promoter activity in LLC-PK1, a pig kidney cell line. Deletion analysis revealed that the region from nucleotides -528 to -322 mediates an activation of the promoter activity, whereas the sequence from -322 to -114 has a negative effect. Furthermore, the structural organization of the 5' end of the GGT gene reveals that the GGT mRNAIII is transcribed from a third promoter located upstream from the promoter II on the GGT gene. By Northern blot analysis, the promoter II was found to be expressed only in the kidney and in the epididymis. We also identified two new mRNA species which are expressed in the H5 hepatoma cells. Therefore, the GGT gene expression reveals a strong tissue- or cell-specific pattern which is based on the transcription of several mRNA species from multiple promoters.

A specific distal promoter controls gamma-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells.

In rat undifferentiated hepatoma cells, the gamma-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.

Species differences in the localisation of gamma-glutamyl transpeptidase immunopositive cells at the blood-brain interface.

The expression of gamma-glutamyl transpeptidase (GGT) has been associated with the emergence of a functional blood-brain barrier. We have undertaken a precise localisation of this enzyme in the cerebral cortical vessels of different species, by immunocytochemistry using a polyclonal antibody and light and electron microscopy. GGT immunoreaction product was present on the luminal surface of endothelial cells in 1-day-old to 3-month-old rats, whereas in the mouse, monkey and human cortex, this protein was present in astrocytic endfeet around the vessels. No labelling was observed in the other cellular components of the vessel walls, such as pericytes, fibroblasts, smooth muscle cells and perivascular cells. The localisation of GGT in astrocytes in mice, monkeys and humans suggests that these cells could play a role in the detoxication of lipophilic xenobiotic substances that cross the endothelial barrier. In these species, astrocytes can be viewed as a second line of defense against xenobiotics, beyond the capillary endothelium.

In vitro biosynthesis and membrane insertion of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit. The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments. We have investigated the first steps of GGT biosynthesis and processing in a cell-free system. mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis. In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme. Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000). This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H. Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain. This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.