FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression.

We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase activity, which requires Mn2+ and is insensitive to inhibition by okadaic acid. FIN13 is localized in the nuclei of transiently transfected cells. Cotransfection of FIN13-expressing plasmids with a plasmid that expresses the neomycin resistance gene inhibits the growth of drug-resistant colonies in NIH 3T3, HeLa and Rat-1 cells. In transiently transfected cells, FIN13 inhibits DNA synthesis and results in the accumulation of cells in G1 and early S phases. Similarly, the induction of expression of FIN13 under the control of a tetracycline-regulated promoter in NIH 3T3 cells leads to growth inhibition, with accumulation of cells in G1 and early S phases. Thus, overexpression and/or unregulated expression of FIN13 inhibits cell cycle progression, indicating that the physiological role of this phosphatase may be that of regulating the orderly progression of cells through the mitotic cycle by dephosphorylating specific substrates which are important for cell proliferation.

Variable transduction efficiency of murine leukemia retroviral vector on mammalian cells: role of cellular glycosylation.

To elucidate the cellular tropism of Moloney murine leukemia virus (MuLV), we have studied the transduction efficiency of a recombinant MuLV vector carrying the beta-galactosidase reporter gene on a variety of rodent cells. Under optimal conditions for in vitro cell transduction, primary cultures of adult rat fibroblasts derived from various organs were very poorly transduced by the ecotropic MuLV vector (0.02-0.12%) when compared to immortalized cells such as the F2408 (42%) and 3Y1 (defined as 100%) lines. Primary cultures of fibroblasts from neonatal (3.7%) or embryonic rat tissues (4.6%) and primary cultures of rat mammary epithelial cells (3-4%) were somewhat more susceptible. Immortalization of rodent fibroblasts with Polyomavirus Large T. SV40 Large T, and E6-E7 genes of human papilloma virus resulted in a modest or minimal increase in transduction efficiency, and introduction of the transforming genes v-Src, v-Ras, and v-Raf was in most instances associated with a decrease in MuLV vector entry. Variability of transduction efficiency was not related to differences in cellular growth rate and treatment of MuLV vectors in vitro with deoxyribonucleoside triphosphates and treatment of cells in culture with protease inhibitors failed to modify cellular entry of the MuLV vector. On the other hand, inhibition of cellular glycosylation with swansonine, 1-deoxymannojirimycin and, primarily, tunicamycin enhanced entry of the ecotropic vector by up to 220-fold, particularly into cells which were otherwise highly resistant. These findings demonstrate major differences in transduction efficiency of the ecotropic MuLV vector on rodent cells and indicate that cellular glycosylation plays a critical role in determining MuLV cellular tropism.

A simple procedure to determine the biological titer of recombinant retroviral vectors.

Retroviral vectors are widely used to deliver genetic material to live cells both in experimental and clinical settings. The ability of these vectors to transduce target cells is an important aspect of their clinical applicability and one of the factors determining their transduction efficiency is vector functional titer. Current methods for titrating retroviral vectors involve measuring the number of target cells in culture transduced by a given volume of vector solution. In this report, we describe a new procedure which allows one to estimate the actual number of infectious particles capable of transducing a permissive cell type. Vector biological titer is calculated from the fractional decline in transduction efficiency observed when a given volume of vector solution is sequentially added to multiple dishes containing permissive cells. Values determined this way are greater than those obtained from a single transduction experiment, with the difference being inversely proportional to the degree of cell permissiveness for vector entry. The present procedure is simple, reliable and expeditious. It will be useful to standardize vector biological titers determined in different laboratories, and help implement strategies for efficient gene delivery protocols.

Cellular aging is a critical determinant of primary cell resistance to v-src transformation.

Primary cell cultures are in general resistant to the transforming effect of a single oncogene, a finding considered consistent with the multistage theory of carcinogenesis. In the present studies, we examined whether cellular age, differentiation stage, and/or tissue origin of primary cells plays a role in determining their response to v-src transformation. To study the role of cellular age, rat mammary fibroblasts were isolated from a 50-day-old female rat and infected with a recombinant retrovirus carrying a v-src gene after 2, 7, 14, 21, and 28 days of continuous growth. To determine whether cellular differentiation is important, fibroblasts were isolated from embryos at 12 and 16 days of gestation, from newborns, and from a 30-day-old rat and similarly infected. Finally, the role of primary-cell histogenesis was assessed by infecting primary cultures of fibroblasts isolated from the mammary gland, dermis, and lungs of a mature rat. When compared to 3Y1 cells, all preparations of primary cultures exhibited considerable resistance to v-src transformation. However, whereas primary cells isolated from different tissues responded similarly to the transforming effect of the oncogene, major differences were observed when cells were transduced at different stages of their in vitro life span. v-src was capable of inducing formation of foci and growth in soft agar in early-passage cells but failed to do so in primary cultures infected after 14 days of continuous passaging. Similarly, both the number of foci and the number of colonies in soft agar decreased with tissue donor age. The differential response of young and senescing cells could not be explained by mutations in v-src provirus, by differences in functional v-src expression, or by growth stimulation or suppression via paracrine mechanisms. Furthermore, v-src cooperated with an immortalizing gene, like simian virus 40 large T, polyomavirus large T, E6 and E7 of human papillomavirus, or an activated p53 mutant, to induce anchorage-independent growth of primary cultures but failed to do so with cytoplasmic transforming genes, like v-abl, v-ras, or v-raf, which did not confer indefinite division potential. These studies indicate that cellular aging is a critical determinant of primary-cell resistance to v-src transformation. It is suggested that v-src requires a nuclear auxiliary function for transformation which is present in early-passage cells, particularly when these cells are derived from embryonic tissue, but is lost as cells approach replicative senescence. This auxiliary function is provided by nuclear oncogenes but not cytoplasmic transforming genes.

Suppression of v-Src transformation in primary rat embryo fibroblasts.

To understand the mechanism for resistance of primary cultures of rat embryo fibroblasts (REFs) to oncogene-induced transformation, we studied the transforming ability of a recombinant retrovirus, ZSV, containing v-src and neo genes in REFs and in the rat cell line F2408. The susceptibility of REFs to p60v-src transformation was markedly reduced when compared with that of F2408 cells, despite high levels of expression of functional p60v-src tyrosine kinase in the two systems. In hybrid cells obtained by somatic cell fusion between F2408 cells transformed by v-src and uninfected REFs, the transformed phenotype was suppressed despite persistent expression of p60v-src tyrosine kinase. On the other hand, hybrid cells between v-src transformed F2408 cells and uninfected F2408 cells retained the transformed phenotypes. These results indicate that primary cells possess an intracellular function(s) that cause suppression of the transformed phenotype induced by the v-src gene. In ZSV-infected REFs, tyrosine phosphorylation of cellular proteins, including p125 focal adhesion kinase, p70 paxillin and p130 was similar to that in the ZSV-infected F2408 cells, indicating that tyrosine phosphorylation of these proteins is not sufficient for the expression of transformed phenotype. On the other hand, cellular fibronectin and one of integrin receptors were downregulated in the ZSV-transformed F2408 cells but not in ZSV-infected REFs, suggesting that fibronectin and/or its receptor might play a role in suppressing v-src transformation in primary rat cells.

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Liver carcinogenesis associated with feeding of ethionine in a choline-free diet: evidence against a role of oval cells in the emergence of hepatocellular carcinoma.

In an attempt to clarify the role of oval cells in the emergence of hepatocellular carcinoma, we fed rats a choline-free diet containing 0%, 0.05% or 0.1% ethionine. The incidence and nature of premalignant and malignant hepatic lesions were then related to the degree of oval cell proliferation. Intake of choline-free diet alone for up to 12 mo was associated with minimal oval cell proliferation; cholangiofibrosis, hepatocellular nodules and hepatocellular carcinoma were observed in 55%, 23% and 14% of the animals, respectively. When rats were given the choline-free diet with 0.05% ethionine, proliferation of oval cells was more pronounced; after a 6- to 12 mo feeding period, cholangiofibrosis (57%) was again observed. However, hepatocellular nodules (91%) and hepatocellular carcinoma (74%) were the most common lesions seen with this feeding regimen. Finally, rats fed the choline-free diet with 0.1% ethionine had massive oval cell proliferation and progressive loss of parenchymal liver tissue. Most of these animals died before they had consumed the choline-free diet with 0.1% ethionine for 12 mo. Rats in this group (96%) exhibited large and numerous cholangiofibrotic lesions, but hepatocellular nodules and carcinoma were not detected. In all animals of each experimental group, hyperplastic bile duct cells in areas of cholangiofibrosis and oval cells were positive for cytokeratin 19, an intermediate filament protein present only in bile duct cells in normal liver. Hepatocellular nodules and hepatocellular carcinoma were invariably negative for cytokeratin 19. We interpret these findings to suggest that oval cells are not involved in the histogenesis of hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon induces biliary protein excretion in guinea pigs.

This study was done to determine glucagon's effect on protein biliary excretion in anesthetized, bile duct-cannulated guinea pigs. Glucagon (1.4 nmol.min-1.kg-1) induced choleresis and increased protein biliary concentration from 0.12 +/- 0.04 to 0.20 +/- 0.6 mg/ml and protein output from 22.8 +/- 3.8 to 54.5 +/- 16.1 micrograms.kg-1.min-1. Protein biliary excretion increased during the first 10 min of glucagon infusion and progressively declined thereafter. Biochemical analysis of biliary protein revealed that the increase could be accounted for primarily by an increase in the lysosomal enzymes acid phosphatase and beta-glucuronidase. Biliary excretion of the canalicular membrane enzymes 5'-nucleotidase and alkaline phosphatase only modestly increased, whereas that of [14C]sucrose, a marker of paracellular fluid transport, was unaffected. On the other hand, glucagon enhanced biliary entry of horseradish peroxidase in a fashion similar to that observed with total endogenous protein. These effects were mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) system, since infusion of dibutyryl-cAMP at 0.5 mumol.kg-1.min-1 increased bile flow and biliary protein excretion in a time-dependent manner, as observed with glucagon. Glucagon's failure to sustain enhanced protein biliary output was not due to declining hepatic concentrations of cAMP or to depletion of hepatocellular lysosomal enzymes. These studies provide evidence that glucagon stimulates biliary excretion of protein in guinea pigs that can be accounted for by biliary discharge of enzyme originating from the canalicular membrane and, primarily, from the lysosomal compartment. Although the precise mechanism(s) underlying these effects remains to be elucidated, it is suggested that the increase in canalicular membrane enzyme excretion is due to glucagon's effect on exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopy identification of early proliferating cells in rat liver tissue during hyperplasia induced by lead nitrate.

Recent studies have suggested that hepatic stem cells may be involved in at least some forms of liver epithelial growth. To obtain further information on this controversial hypothesis, we treated rats with lead nitrate to induce liver growth and identified the cells undergoing early DNA synthesis by bromodeoxyuridine immunohistochemistry, using both light and electron microscopic detection methods. Eight hours after an intravenous injection of lead nitrate 100 mumol/kg, DNA synthesis was detected in a few scattered hepatocytes and in nonparenchymal cells in portal connective tissue. At the light microscopic level, identification of nonparenchymal cells was limited to bile duct epithelial cells. Other cell types were also labeled, but their identity could not be established. At the ultrastructural level, however, four types of nonparenchymal cells were identified as containing bromodeoxyuridine immunogold particles. These four types included bile duct epithelial cells, fibroblasts, macrophages and nondescript periductular cells. These periductular cells displayed certain ultrastructural features of bile duct cells but did not line a lumen or display microvilli on their apical membrane, nor did they reside within the bile duct basement membrane. Because proliferation of nonparenchymal cells in portal areas preceded that of hepatocytes, it is suggested that the former reaction reflects a direct mitogenic effect of lead nitrate and not an adaptive growth response secondary to parenchymal enlargement. However, whether DNA synthesis in periductular cells or bile duct cells reflects activation of hepatic stem cells cannot be established from the present morphological observations. If so, such a progenitor compartment must be dormant because it does not seem to play a functional role in this and other forms of adult liver epithelial growth.

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.

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.

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.

Phenobarbital does not increase early labeling of bilirubin from 4-[14C]-delta-aminolevulinic acid in man and rat.

delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five patients with Gilbert's syndrome and four healthy control subjects on two occasions: before and on days 10 through 14 of a course of phenobarbital (2.5 mg/kg/day). The resulting curves of [3H]-bilirubin and [14C]-bilirubin in plasma were analyzed by computer to determine a number of parameters of physiological interest. As expected, phenobarbital produced a highly significant fall in the plasma concentration of unconjugated bilirubin as a result of a significant increase in hepatic bilirubin clearance in all subjects; plasma bilirubin turnover was unaltered. Surprisingly, the drug produced no change in the incorporation of [14C]-delta-aminolevulinic acid into [14C]-early labeled bilirubin. To explain this unexpected finding, the effects of phenobarbital (75 mg/kg/day for 6 days) on incorporation of [14C]-delta-aminolevulinic acid and 2-[14C]-glycine into [14C]-early labeled bilirubin and on the activity of the enzyme delta-aminolevulinic acid synthase were studied in nonfasted, adult, male Sprague-Dawley rats. At the dose and duration of treatment used, phenobarbital administration increased total hepatic delta-aminolevulinic acid synthase activity and produced a significant increase of 70% in the incorporation of [14C]-glycine into early labeled bilirubin. By contrast, no increase in the incorporation of [14C]-delta-aminolevulinic acid into early labeled bilirubin was observed. These data suggest that delta-aminolevulinic acid is an inappropriate precursor for studies of the rate of heme biosynthesis, presumably because it bypasses delta-aminolevulinic acid synthase, the physiological rate-limiting enzyme in the heme biosynthetic pathway.

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.

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.

mechanism of glucagon choleresis in guinea pigs.

The present studies were carried out to clarify the mechanism of glucagon choleresis in guinea pigs. At the infusion rate of 1.4 nmol.min-1.kg-1, glucagon increased bile flow from 206.6 +/- 14.3 to 302.6 +/- 35.0 microliters.min-1.kg-1 and bicarbonate biliary concentration from 63.7 +/- 4.2 to 75.5 +/- 5.9 meq/l. Measurements of bile acid excretion in bile, the biliary tree volume, and of the hormone choleretic effect in guinea pigs with proliferated bile ductules/ducts induced by alpha-naphthylisothiocyanate feeding indicated that glucagon, unlike secretin, stimulated canalicular bile flow. Inhibition of prostaglandin synthesis by indomethacin administration (5 mg.kg-1.h-1) did not modify the choleretic effect of glucagon, and infusion of a glucagon analogue (TH-glucagon, 1.4 nmol.min-1.kg-1), which did not increase hepatic formation of adenosine 3'5'-cyclic monophosphate (cAMP), failed to stimulate bile flow. Like the parent hormone, however, TH-glucagon augmented plasma glucose levels and stimulated formation of inositol phosphates. Colchicine pretreatment (0.5 mg/kg ip) almost entirely prevented the choleretic effect of glucagon but did not modify spontaneous and bile acid-induced bile flow and the stimulatory effect of the hormone on glucose release and on hepatic formation of cAMP and inositol phosphates. Finally, glucagon produced a large increase in the biliary entry of horseradish peroxidase, even though this effect was transient and was not coupled to the increase in bile flow. These results indicate that glucagon choleresis in the guinea pig is not secondary to prostaglandin release, is canalicular in origin, involves bicarbonate secretion, is mediated by cAMP, and requires an intact microtubular system.

Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat.

Proliferation of bile duct-like structures is a hepatic cellular reaction observed in most forms of human liver disease and in a variety of experimental conditions associated with liver injury. Yet the origin, means of initiation, and significance of this hyperplasia are unknown. To clarify these issues we induced bile duct proliferation in rats by ligating the common bile duct and studied (a) hepatic incorporation of [3H]thymidine by histoautoradiography, (b) hepatic morphometry, (c) biliary tree volume using [3H]taurocholate as a marker of biliary transit time, (d) immunohistochemical expression of cytokeratin no. 19, (e) the effect of indomethacin, and (f) the role of increased biliary pressure, in the absence of physiological and biochemical evidence of cholestasis, on [3H]thymidine incorporation by the bile-duct cells. The results have demonstrated that (a) the proliferating bile duct-like cells are products of the extant biliary epithelium and retain its characteristics; (b) bile duct cells divide irrespective of the size of the duct in which they are located and form a system with a lumen continuous with the preexisting one; (c) bile duct proliferation results mainly in elongation, not in circumferential enlargement or sprouting of side branches; (d) portal macrophage infiltration does not play a role in the hyperplastic reaction, and (e) increased biliary pressure is the initiating factor in bile duct cell division. Our results provide evidence that under the present conditions, ductular metaplasia of hepatocytes does not occur and there is no functioning stem cell for biliary epithelial growth segregated in any particular duct size or within the portal connective tissue.

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.

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.

Bile secretory apparatus in the newborn dog: relationship between structural and functional immaturities.

In the dog, bile secretion is not fully mature at birth and develops during postnatal life. To try to establish morphologic correlates to the physiologic deficiencies, we examined the ultrastructure of hepatic parenchyma and biliary epithelium in a newborn puppy and in 3 puppies of 1, 3, and 7 days of age. At birth, the hepatocytes contain much glycogen and fat droplets, a small smooth endoplasmic reticulum and Golgi apparatus, rare autophagic vacuoles, and numerous lysosomes. The sinusoidal microvilli are short, and submembrane vesicles are few and small. The bile canaliculus is not dilated, but few and short microvilli and no pericanalicular vesicles are seen. The biliary epithelial cells are normal in size, but the luminal surface of the bile ductule has no microvilli and numerous blebs. These morphologic features change with maturation and, by the first week of life, the fine structure of the hepatocytes, bile ductular cells, and biliary passages resemble that observed in the adult liver. These findings provide morphologic support for the concept that, in the dog, the bile secretory apparatus is immature at birth and develops during postnatal life.