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.

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.

[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.

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.

Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland.

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5' ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.

Expression of the rat gamma-glutamyl transpeptidase gene from a specific promoter in the small intestine and in hepatoma cells.

In the small intestine and in HTC hepatoma cells, the gamma-glutamyl transpeptidase (GGT) single-copy gene is transcribed into a 2.5 kb and a 2.2 kb mRNA. Cloning of the GGT cDNA sequences from HTC cells demonstrates that the 2.5 kb mRNA (mRNA(IV-1)) differs from the other rat GGT transcripts by a 371-base unique leader sequence which maps in the gene as 2 separate exons upstream of the 3 promoters which have been previously characterized. We established that the transcription of these two mRNAs is initiated on a new promoter (promoter IV) and occurs in the small intestine, in the epididymis, and in some hepatoma cells. The primary transcript initiated on GGT promoter IV is then alternatively spliced into the 2.5 kb mRNA(IV-1) or the 2.2 kb mRNA(IV-2) which is shorter in its 5'-untranslated sequence. The rat GGT gene exhibits a complex transcriptional organization leading to the transcription of five mRNAs from four independent promoters in a tissue-specific manner. The expression of the GGT promoter IV in the HTC hepatoma cells as well as in the small intestine could reveal that the HTC-transformed cells originate from liver precursor cells which still have the capacity to evolve toward different lineages. Thus, the GGT promoter IV will be valuable to isolate factors involved in the differentiation and carcinogenic processes.

Effect of fasting on organic anion uptake by isolated rat liver cells.

In order to determine if the delayed clearance of organic anions observed in vivo after fasting can be related to an alteration of cell membrane carriers, kinetics of sulfobromophthalein (BSP) uptake were determined in isolated rat liver cells obtained from 48-hr starved rats. Surprisingly, in fasted rats the existence of two carriers can be directly revealed by classical kinetic plots. The high-affinity component, inhibited by Na+-taurocholate, has a Km of 0.5 +/- 0.2 microM and a Vmax of 0.2 +/- 0.1 nmole per min per 10(6) cells; the low-affinity component, which is not sensitive to Na+-taurocholate, has a Km of 21.2 +/- 3.2 microM and a Vmax of 4.8 +/- 0.9 nmoles per min per 10(6) cells. Comparison with control cells shows that fasting does not modify the total capacity of the liver cell membrane carriers to take up BSP. However, alterations in the kinetic parameters of the two uptake components were observed: a 53% decrease in the affinity of the low-affinity component and a 50% reduction in the capacity of the high-affinity uptake. These alterations, together with the observed decrease in hepatic blood flow and/or the increase in BSP efflux from the hepatocytes, could be involved in the delayed clearance of BSP and other anionic compounds occurring in vivo after fasting.

Control of gamma-glutamyl transpeptidase expression by glucocorticoids in the rat pancreas. Correlation with granule formation.

Glucocorticoids are known to promote the formation of zymogen granules in acinar cells of the exocrine pancreas in vivo as well as in vitro. To gain insight into the mechanism of this regulation, we studied the effects of glucocorticoids on the synthesis of two components of the secretory granule membrane, the glycoprotein 2 (GP-2) and the gamma-glutamyl transpeptidase (GGT). It was demonstrated that following adrenalectomy, degranulation of pancreatic acinar cells is accompanied by a sharp decrease in GGT and GP-2 synthesis as measured by mRNA and protein accumulation. The decline of GGT synthesis was prevented by glucocorticoid replacement therapy, whereas GP-2 synthesis could be maintained with either glucocorticoid or estradiol treatment. These in vivo observations were corroborated and extended in an in vitro study using AR42J pancreatic cells. With this cell line, it was demonstrated that dexamethasone induces the formation of zymogen granules and the accumulation of a specific GGT transcript (mRNA III) by decreasing its degradation rate. At the same time, the GP-2 mRNA level was not modified by the hormonal treatment. These data demonstrate that glucocorticoids exert a positive control on the GGT expression in pancreatic cells at a post-transcriptional level. GGT, an enzyme of the glutathione metabolism, could play a significant role in protein packaging in secretory cells.