Nicola Tavoloni

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.

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.

Bile acid structure and bile formation in the guinea pig

The effects of intravenous infusions (1-4 mumol/min/kg) of 14 bile acids, cholic, deoxycholic, ursodeoxycholic, chenodeoxycholic, dehydrocholic, and their glycine and taurine conjugates, on bile flow and composition and on the biliary permeation of inert carbohydrates have been studied in the guinea pig bile fistula. Hydroxy bile acids were eliminated in bile without major transformation, except for conjugation (over 90%) when unconjugated bile acids were infused. During infusion of dehydrocholate and taurodehydrocholate, 77-100% of the administered dose was recovered in bile as 3-hydroxy bile acids, thus indicating that reduction of the keto group in position 3 was virtually complete. All bile acids produced choleresis at the doses employed: the strongest choleretic was deoxycholate (81.78 microliters/mumol), the weakest was taurodehydrocholate (10.2 microliters/mumol). Choleretic activity was directly and linearly related to bile acid hydrophobicity, as inferred by HPLC, both for similarly conjugated bile acids, and for bile acids having the same number, position, or configuration of the hydroxyl groups. In all instances, the rank ordering was: deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate. During choleresis produced by any of the bile acids tested, bicarbonate concentration in bile slightly declined, but the calculated concentration in bile-acid-stimulated bile (45-57 mmol/l) was always higher than that measured in plasma (23-26 mmol/l). Biliary concentrations of cholesterol (20-68 mumol/l) and phospholipid (14-63 mumol/l) were very low during spontaneous secretion, and declined even further following bile acid choleresis. None of the infused bile acids consistently modified biliary excretion of cholesterol and phospholipid. Consistent with a previous observation from this laboratory, all hydroxy bile acids reversibly diminished [14C]erythritol and [14C]mannitol biliary entry during choleresis, while they increased or failed to modify that of [3H]sucrose and [3H]inulin. The rank ordering for the inhibitory effect on [14C]erythritol and [14C]mannitol permeation was: 3 alpha,7 alpha,12 alpha-trihydroxy greater than 3 alpha,7 alpha-dihydroxy greater than 3 alpha,7 beta-dihydroxy greater than 3 alpha,12 alpha-dihydroxy bile acids.(ABSTRACT TRUNCATED AT 400 WORDS)

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

SummaryThe 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.

Does Adriamycin Undergo an Enterohepatic Circulation

Abstract To determine whether adriamycin (ADR) and/or its metabolites undergo an enterohepatic circulation following an iv administration, anesthetized rats with biliary and urinary fistulae were intraduodenally perfused with bile flowing from bile duct-cannulated rats which had been injected iv with 20 mg/kg [14C]ADR. During a 6-hr duodenal perfusion period, 27.3% of the ADR injected to bile donor rats was recovered as total radioactivity in the intestinal perfusate of bile recipient rats and approximately 1.6 and 0.18% were excreted in their bile and urine, respectively. The intestinal tissue of bile recipient rats contained concentrations of the total drug equivalents ranging from 1.98 μg/g in the initial portions of the duodenum to 12.21 μg/g in the distal parts of the ileum. Minimal levels were observed in the heart, lung, kidney, and liver (0.25–0.86 μg/g tissue). In order to estimate the amount of the total [14C]ADR equivalents perfused into the small intestine of the bile recipient rats, [14C]ADR 20 (mg/kg) was injected iv to a separate group of anesthetized rats in which bile was continuously collected for a 6-hr period. In these experiments, total radioactivity excreted through the biliary route accounted for 30.6% of the injected dose. These combined results indicate that approximately 10% of the total [14C]ADR equivalents eliminated in bile over a 6-hr period are reabsorbed from the lumen of the small intestine of the anesthetized rat, an amount which roughly represents 3% of the injected ADR.

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.