Biomedical subjects

G Alpini

Publications and source records attributed to G Alpini.

51 records · Page 3Linked to original sources

PubMed1994

Butyrate synchronization of hepatocytes: modulation of cycling and cell cycle regulated gene expression.

To develop a model for studies of liver growth control, we characterized cell cycle synchronization of liver-derived cells with sodium butyrate. Exposure of cultured HTC (rat hepatoma) cells to 5 mM butyrate arrested cell growth in a reversible manner. Flow cytometric analysis revealed that butyrate-treated HTC cells were restricted in G0/G1, as well as S/G2M phases. After release from butyrate arrest, HTC cells underwent synchronous cycles of DNA synthesis and transited through S phase. Inhibition of cell growth by butyrate was associated with a complex pattern of cell cycle regulated gene expression, including a decoupling of c-fos and c-jun gene expression. Transcription of c-fos, as well as c-jun increased with butyrate arrest, whereas steady rate mRNA levels of c-jun only were increased, suggesting additional regulation of c-fos. In addition, butyrate-arrested cells exhibited a transcriptionally determined accumulation of H3 histone, C-Ha-ras and ornithine decarboxylase mRNAs, suggesting that cell cycle-related check points following the onset of S phase were modulated. An increase in c-myc mRNA levels in butyrate-arrested cells was post-transcriptionally regulated. After release from butyrate-arrest, the abundance of immediate early, as well as S phase regulated, gene expression changed coordinately with S phase cell transitions. Thus, exposure of HTC cells to butyrate modulates cell cycle regulated gene expression, inhibits cycling, and results in accumulation of cells in specific compartments. Synchronization of liver cells with butyrate should, therefore, provide a useful model for defining cell cycle-related events in response to various mitogenic stimuli.

Animals↗

PubMed1994

Recent advances in the isolation of liver cells.

The development of new and refined separation techniques--including FACS, FFE, CFE and isopyknic gradients--has had a profound impact on the ability of investigators to isolate specific cell types from the liver. Although some of these techniques, such as FFE, may be of limited preparative value, they are nonetheless important analytical tools that detect subtle differences among cell subpopulations. The isolation of highly purified preparations of liver cells in large yields requires the use of more conventional purification methods such as CFE and isopyknic centrifugation. Immunological approaches represent a key development for the isolation of specific liver cell types, especially when they are used in combination with other techniques. Excellent, reliable and relatively simple techniques now exist to isolate highly purified preparations of hepatocytes, cholangiocytes, KCs, SCs, FSC, myofibroblasts and pit cells. Additional work is necessary to refine techniques for the isolation of dendritic cells and lymphocytes.

Animals↗

PubMed1993

Models for hepatic progenitor cell activation.

Activation of liver progenitor cells was studied in rat liver induced to regenerate after carbon tetrachloride (CCl4) or D-galactosamine (GalN) injury. A change in the concentration of histone-3 mRNA was used as a marker for cell proliferation and the fetal form of alpha-fetoprotein (AFP) mRNA as a marker for fetal hepatoblasts. gamma-Glutamyltranspeptidase (GGT) and glutathione-S-transferase P were used as markers for activation of putative liver progenitor cells. After CCl4 administration, the proliferative response was high but confined primarily to parenchymal cells. No changes in the relative expression of albumin, glutathione-S-transferase P or insulin-like growth factor-II were observed. On the other hand, the level of AFP mRNA was increased modestly and predominantly in the nonparenchymal cell (NPC) fraction. After GalN administration, proliferation of NPC began within 24 hr, primarily in the portal area around the bile ducts. Activated cells were bile "duct-like" in appearance, had scant cytoplasm, and a pale, oval-shaped nucleus. On Day 2, they formed rows and clusters, expanding from the portal zone and invading the parenchyma, as well as proliferating in regions of focal necrosis. On Days 3 and 5, NPC expressing histone-3 mRNA expanded further, forming pseudoducts and islet-like structures (NPC structures) throughout the hepatic lobule. Proliferating NPC were positive for GGT. Some GGT-positive cells on Days 3 and 5 were also positive for fetal AFP mRNA. Expression of fetal AFP mRNA lagged behind that of GGT by 24 hr, was highest on Day 5, and then declined. Expression of albumin mRNA and glucose 6-phosphatase decreased during the first 48 hr after GalN administration and then resumed. These findings indicate that after GalN injury, the liver responds with activation of putative progenitor cells that proliferate and then differentiate through the hepatocyte lineage, whereas the regenerative response after CCl4 administration is primarily through proliferation of preexisting hepatocytes.

Albumins↗

PubMed1992

Widespread differentiation stage-specific expression of the gene encoding phosphoprotein p19 (metablastin) in mammalian cells.

p19 is a highly conserved 19 kD cytosolic protein that undergoes phosphorylation in response to diverse extracellular factors in mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. To gain insights regarding its function, we analyzed the expression of p19 mRNA in a variety of cell types during induction of differentiation. Murine erythroleukemia cells showed a moderate increase followed by a marked decrease in the abundance of p19 mRNA during induction of differentiation. In murine C2 myoblasts and primary fetal rat osteoblasts, p19 mRNA was abundant in replicating cells and decreased to undetectable levels during differentiation. In resting human peripheral blood lymphocytes, p19 mRNA was virtually undetectable but was strongly induced during blast transformation of both B and T cells. In rat liver, p19 mRNA was abundant on embryonic day 17 and decreased during early postnatal development. Upon fractionation of adult rat liver cells by centrifugal elutriation, p19 mRNA was not detected in hepatocytes while a low level was observed in a fraction enriched in non-parenchymal epithelial cells. CCl4-induced liver regeneration resulted in induction of p19 mRNA in hepatocytes. Primary cultures of embryonic and neonatal rat brain were analyzed by indirect immunofluorescence using co-staining with stage-specific markers. p19 expression was restricted to immature neurons and oligodendrocyte precursors. In contrast to the other cell types examined, the neuronal and glial precursors that express p19 were shown, using BrdU labeling, to be postmitotic both in primary culture and in vivo. The data demonstrate widespread, stage-specific expression of p19 and suggest that the protein exerts a general, lineage-independent function during induction of differentiation of mammalian cells. In view of the available evidence on the stimulation of serine phosphorylation of p19 by several growth factors, our working hypothesis is that phosphorylation of p19 may be involved in the mechanism by which growth factors control cell differentiation.

Animals↗

PubMed1992

Secretin stimulates bile ductular secretory activity through the cAMP system.

Although convincing evidence has been obtained to support a ductular origin of secretin choleresis, the precise mechanism of the choleretic effect of the hormone is poorly understood. The present studies were carried out to 1) further clarify the anatomic site at which secretin stimulates bile flow and 2) establish the signal transduction system underlying this effect. To this end, parenchymal and nonparenchymal liver cells, the latter enriched in bile duct cells, were isolated from rats with ductular cell hyperplasia, and the effect of secretin on intracellular formation of both adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (IPs) was compared with that observed with glucagon and [Tyr10,13,Phe22,Trp25]secretin (SG-secretin). In the pancreas, secretin stimulates both messenger systems, while SG-secretin activates only the cAMP cascade. In isolated hepatocytes, both secretin and SG-secretin failed to increase formation of cAMP and IPs, which were instead activated by glucagon. In isolated bile duct cells, secretin induced formation of both cAMP and IPs, while SG-secretin stimulated solely the cAMP system, as in the pancreas. Glucagon did not stimulate either messenger system in this cell preparation. In vivo, both secretin and SG-secretin stimulated a bicarbonate-rich fluid in rats with bile ductular cell hyperplasia and in normal guinea pigs, which was demonstrated to originate at the distal biliary epithelium. These findings support the existing view that glucagon stimulates canalicular bile flow, while secretin increases secretory activity at the bile ductules and/or ducts. More importantly, they indicate that stimulation of ductular secretory activity by secretin is mediated by the cAMP system and does not involve the IP signal transduction pathway.

Animals↗

PubMed1992

Distribution of albumin and alpha-fetoprotein mRNAs in normal, hyperplastic, and preneoplastic rat liver.

The nature of bile duct-like (oval) cells proliferating during chemical hepatocarcinogenesis has been controversial. To investigate this issue further, the authors compared the hepatic distribution of albumin (ALB) and alpha-fetoprotein (AFP) mRNAs in rats in which oval cell proliferation was induced by feeding a choline-devoid diet containing 0.1% ethionine (CDE, a hepatocarcinogenic diet) with that in normal rats and in rats in which biliary epithelial cell hyperplasia was induced by either bile duct ligation or feeding alpha-naphthylisothiocyanate (ANIT). Northern blot analysis in parenchymal and nonparenchymal liver cells isolated from these animals demonstrated that ALB mRNA was present in the hepatocytes of both control and experimental animals, whereas this transcript was detected in nonparenchymal epithelial cells only in CDE-fed rats. Alpha-fetoprotein mRNA was not seen in either parenchymal or nonparenchymal cells isolated from normal or hyperplastic livers induced by bile duct ligation or ANIT feeding. In CDE-fed rats, however, both parenchymal and nonparenchymal cell populations displayed AFP message. In situ hybridization directly demonstrated nonparenchymal cell expression of both ALB and AFP transcripts in CDE-fed rats. Most surprisingly, ALB and AFP mRNAs were also detected by in situ hybridization in occasional nonparenchymal cells located in portal tracts near the limiting plate in normal liver, as well as under conditions associated with bile duct hyperplasia. Immunohistochemical studies of intermediate filament proteins, cytokeratin 19 (a marker of glandular epithelia), vimentin (a marker of mesenchymal lineage), and desmin (a marker of muscle cell differentiation) demonstrated that oval cells, as well as normal and hyperplastic bile duct cells, were positive for cytokeratin 19 and negative for both vimentin and desmin. Cytokeratin-positive oval cells formed duct profiles and were connected to preexisting ductules and ducts. These results are construed to suggest that oval cells proliferating during CDE hepatocarcinogenesis are derived from epithelial cells within the biliary tree. The presence of cells with similar morphologic appearance, periportal location, and AFP and ALB expression in normal liver suggests that these cells may be the progenitors of oval cells induced by some carcinogenic regimens.

Animals↗

PubMed1992

Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells.

The origin of bile duct-like cells (oval cells) proliferating during chemical hepatocarcinogenesis is highly controversial. To illuminate this issue, we induced oval cell proliferation by feeding rats a choline-devoid diet containing 0.1% ethionine (CDE), a hepatocarcinogenic diet, for up to 60 days. At various times we studied 1) oval cell morphology by light and electron microscopy, 2) the immunohistochemical expression of albumin and intermediate filament proteins by the various hepatic cells, 3) hepatic incorporation of [3H]thymidine by histoautoradiography, 4) the fractional area occupied by duct-like structures in liver cross sections, 5) the biliary tree volume in vivo to establish the possible continuity of the proliferated structures to the existing biliary lumina, and 6) spontaneous bile flow rate and the choleretic responsiveness to the hormone secretin, which stimulates ductular secretory activity. The results demonstrated the following: 1) oval cells resemble bile duct cells with respect to their histologic and ultrastructural appearance and their formation of duct-like structures; 2) as normal and hyperplastic bile duct cells induced by bile duct ligation, oval cells are positive for cytokeratins 7 and 19 (markers of glandular epithelia) and 8 and 18 (markers of simple epithelia) and are negative for vimentin and desmin, markers of mesenchymal and muscular differentiation, respectively; 3) in general, oval cells are negative for albumin, which is expressive of hepatocyte lineage, even though a few are positive for this protein, particularly those morphologically resembling small hepatocytes; 4) after initiation of the CDE diet, DNA synthesis begins in biliary epithelial cells; and 5) the degree of oval cell proliferation parallels the increase in biliary tree volume, spontaneous bile flow rate, and responsiveness to secretin choleresis, as in bile duct cell hyperplasia induced by biliary obstruction. Although the involvement of a periductular progenitor compartment cannot entirely be eliminated, these findings are construed to indicate that oval cells proliferating during CDE hepatocarcinogenesis are biliary epithelial cells. In our view, oval cells represent the two-dimensional expression of spatially expanded cholangioles and intrahepatic bile ductules and/or ducts.

Animals↗

PubMed1991

Distribution of glucose-6-phosphatase activity in normal, hyperplastic, and preneoplastic rat liver.

The significance of glucose-6-phosphatase (G6P) expression by bile duct-like cells proliferating during hepatocarcinogenesis in the histogenesis of hepatocellular carcinoma is not clear. To this end, we measured the histochemical and biochemical activity of G6P in normal rat liver, and in rat livers in which bile duct-like proliferation was induced by either hyperplastic (bile duct ligation for 14 days or feeding alpha-naphthylisothiocyanate for 28 days) or neoplastic (feeding a choline-devoid diet containing 0.1% ethionine for 60 days) regimens. In normal, hyperplastic, and preneoplastic livers, G6P histochemical activity was confined to the hepatocytes; proliferated bile duct-like cells, like normal bile ducts, did not display visible G6P staining. When the enzyme activity was determined biochemically, however, hydrolysis of glucose-6-phosphate was observed in both parenchymal and nonparenchymal liver cells isolated from all experimental animals. In elutriated nonparenchymal fractions, G6P activity was directly proportional to the number of cells positive for gamma-glutamyl transpeptidase and cytokeratin no. 19 (markers of bile duct cells) and inversely proportional to the number of cells positive for vimentin (marker of mesenchymal cells). These results indicate that, while by light microscopy hepatic G6P histochemical activity is detectable only in the hepatocytes, the biochemical activity is also expressed in proliferating bile duct-like cells. However, the nonparenchymal activity is observed during both neoplastic and hyperplastic liver growth, thus indicating that the presence of this enzyme in bile duct-like cells proliferating during hepatocarcinogenesis should not necessarily be construed as supporting their stem cell nature nor their neoplastic commitment.

1-Naphthylisothiocyanate↗

PubMed1990

von Willebrand factor antigen is not an accurate marker of rat and guinea pig liver endothelial cells.

To determine whether von Willebrand Factor (vWF) is a valid marker of liver endothelial cells, we determined vWF immunoreactivity in rat and guinea pig liver sections and in smears of elutriated nonparenchymal cells isolated from these two species. In frozen sections, positive staining for vWF was seen only in the endothelium lining large hepatic vessels in both species, and no immunoactivity was detected in the sinusoids. On the other hand, immunohistochemical staining for vimentin (a marker of mesenchymal cells) showed positive reaction throughout the vascular and sinusoidal endothelial cells in both the rat and guinea pig liver. In fractions of elutriated rat and guinea pig nonparenchymal liver cells, which included almost exclusively liver endothelial cells, only 25-40% of the cells displayed a positive reaction for vWF. However, when these same fractions were stained for vimentin, 70-95% of the cells exhibited immunoreactivity. Most of the vWF-negative cells were not red and white blood cells, biliary epithelial and Kupffer cells, and hepatocytes, and had ultrastructural features of sinusoidal endothelial cells. We conclude that in both the rat and guinea pig, liver sinusoidal endothelial cells do not exhibit vWF immunoreactivity. Thus, in routine immunohistochemical assays, vWF is not an accurate marker of rat and guinea pig liver endothelial cells. Vimentin is more appropriate for this purpose, provided that other mesenchymal cells are separated or independently identified.

Animals↗

PubMed1989

Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes.

In the present study we have isolated and purified fractions of nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% alpha-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36-40 ml/min. In this fraction 1.8-3.8 x 10(6) cells per liver were recovered and up to 55% of them were positive for gamma-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 x 10(6) per liver, and as many as 60%-85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or alpha- naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.

Animals↗

PubMed1989

Bile secretory function of intrahepatic biliary epithelium in the rat.

To shed light on ductular fluid secretion, hepatic histology and ultrastructure, cell proliferation and phenotypes, and several aspects of biliary physiology were studied in rats with ductular cell hyperplasia induced by either biliary obstruction (0-14 days) or 1-naphthylisothiocyanate (ANIT) feeding (0-28 days). In both groups of experimental animals, bile duct hyperplasia and spontaneous bile flow and secretin-induced choleresis increased with time of treatment in a linear fashion. Measurements of [14C]mannitol biliary entry and of biliary tree volume showed that the increase in both spontaneous and secretin-stimulated bile flow originated at the proliferated biliary structures. Ultrastructural examination, [3H]thymidine incorporation, and histochemical and immunohistochemical staining for various markers demonstrated that in both hyperplastic reactions the proliferated cells were the progeny of preexisting biliary epithelial cells and retained their characteristics. These results indicate that the increased bile secretory activity associated with either biliary obstruction or ANIT intoxication reflects a quantitative change due to the proliferation of biliary epithelial cells. Thus both models of bile ductular cell hyperplasia lend themselves to assessment of the transport function of intrahepatic biliary epithelium and its contribution to normal bile formation. In the present studies, we have estimated that net ductular secretion in the normal rat accounts for 10-13% of spontaneously secreted hepatic bile.

1-Naphthylisothiocyanate↗

PubMed1988

Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.

To establish the role of the biliary epithelium in bile formation, we studied several aspects of biliary physiology in control rats and in rats with ductular cell hyperplasia induced by a 14-d extrahepatic biliary obstruction. Under steady-state conditions, spontaneous bile flow was far greater in obstructed rats (266.6 +/- 51.9 microliters/min per kg) than in controls (85.6 +/- 10.6 microliters/min per kg), while excretion of 3-hydroxy bile acids was the same in the two groups. Infusion of 10 clinical units (CU)/kg per h secretin produced a minimal choleretic effect in controls (+3.8 +/- 1.9 microliters/min per kg) but a massive increase in bile flow in the obstructed animals (+127.8 +/- 34.9 microliters/min per kg). Secretin choleresis was associated with an increase in bicarbonate biliary concentration and with a decline in [14C]mannitol bile-to-plasma ratio, although solute biliary clearance significantly increased. Conversely, administration of taurocholate (5 mumol/min per kg) produced the same biliary effects in control rats and in rats with proliferated biliary ductules. In the obstructed animals, the biliary tree volume measured during taurocholate choleresis (67.4 +/- 15.8 microliters/g liver) was significantly greater than that determined during the increase in bile flow induced by secretin (39.5 +/- 10.4 microliters/g liver). These studies indicate that, in the rat, the proliferated bile ductules/ducts spontaneously secrete bile and are the site of secretin choleresis. Furthermore, because the proliferated cells expressed phenotypic traits of bile ductular cells, our results suggest that whereas under normal conditions the biliary ductules/ducts in the rat seem to contribute little to bile formation, secretion of water and electrolytes is a property of biliary epithelial cells.

Amylases↗

PubMed1988

Bile acid secretion and pool size during phenobarbital induced hypercholeresis.

An increase in bile flow after phenobarbital administration occurs in the rat and other species; however, the mechanism(s) of the choleretic effect is incompletely understood and the role of the increase in liver weight is controversial. We therefore measured bile flow, bile acid secretion and pool size in male Sprague-Dawley rats pretreated with phenobarbital (75 mg/kg/day) for 6 days; liver weight, liver cell volume and DNA content were also evaluated. Phenobarbital treatment increased liver weight and mean hepatocyte volume by 39 and 26%, respectively, while total DNA content did not change, thus indicating that the hepatomegaly results principally from hypertrophy rather than hyperplasia. Bile flow was significantly higher in treated rats when expressed per unit of body weight (64.6 +/- 2.4 (S.E.) vs 53.3 +/- 1.6 microliter/min/kg; P less than 0.05) but was unchanged when expressed per gram of liver (1.40 +/- 0.04 vs 1.37 +/- 0.06 microliter/min/g; P greater than 0.5). The initial bile acid secretion rate and pool size were both significantly reduced in the phenobarbital group compared to controls (1224.2 +/- 110.4 vs 1656.6 +/- 163.2 nmol/kg/min and 562.8 +/- 41.5 vs 814.3 +/- 78.3 mumol/kg; both P less than 0.05), whereas the basal synthetic rate was unchanged. These findings suggest that the enlarged, phenobarbital-treated hepatocyte produces more bile than the normal cell, despite the decreased secretion of bile acids. Therefore, the drug-induced choleresis involves a selective increase in the bile acid-independent fraction of bile flow.

Animals↗

PubMed1987

Phenobarbital specifically increases the hepatocellular uptake of sulfobromophthalein-glutathione.

The transport of sulfobromophthalein glutathione was studied in perfused livers isolated from phenobarbital treated and control rats. Phenobarbital increased the cell size and the uptake of sulfobromophthalein glutathione. The effect on uptake is specific since in phenobarbital treated livers each unit of hepatocyte surface area takes up more sulfobromophthalein glutathione than controls. The cellular hypertrophy does not involve all cell functions; total and specific content of cytosolic fatty acid binding protein for example, were unchanged by phenobarbital. The increase in Vmax and influx rate constant for sulfobromophthalein glutathione uptake suggest that phenobarbital increases the amount of membrane carriers or their rate of cycling.

Animals↗

PubMed1986

Water and nonelectrolyte permeability of isolated rat hepatocytes.

We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3H2O, [14C]urea, [14C]erythritol, [14C]mannitol, [3H]sucrose, and [3H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3H2O, 98.6 +/- 18.4; [14C]urea, 18.2 +/- 5.3; [14C]erythritol, 4.8 +/- 1.6; [14C]mannitol, 3.1 +/- 1.4; [3H]sucrose, 0; [3H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [14C]erythritol and [14C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [3H]sucrose and [3H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway.

Animals↗