Jo Lynne Phinizy

Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4damages only large ducts. CCl4 or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4 administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4 converts to its radicals) only in large cholangiocytes. CCl4induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-α

BACKGROUND/AIMS We determined the role of gastrin in the regulation of cholangiocarcinoma growth. METHODS We evaluated for the functional presence of cholecystokinin (CCK)-B/gastrin receptors in the cholangiocarcinoma cell lines, Mz-ChA-1, HuH-28 and TFK-1. We determined the effect of gastrin on the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. We evaluated the effect of gastrin on growth and apoptosis of Mz-ChA-1 in the absence or presence of inhibitors for CCK-A (L-364, 718) and CCK-B/gastrin (L-365, 260) receptors, the intracellular Ca2+ chelator (BAPTA/AM), and the protein kinase C (PKC)-alpha inhibitor, H7. We evaluated if gastrin effects on Mz-ChA-1 growth and apoptosis are associated with membrane translocation of PKC-alpha. RESULTS Gastrin inhibited DNA synthesis of Mz-ChA-1, HuH-28 and TFK-1 cells in a dose- and time-dependent fashion. The antiproliferative effect of gastrin on Mz-ChA-1 cells was inhibited by L-365, 260, H7 and BAPTA/AM but not L-364, 718. Gastrin induced membrane translocation of PKC-alpha. The inhibition of growth of Mz-ChA-1 cells by gastrin was associated with increased apoptosis through a PKC-dependent mechanism. CONCLUSIONS Gastrin inhibits the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. Gastrin inhibits growth and induces apoptosis in Mz-ChA-1 cells through the Ca2+-dependent PKC-alpha. The data suggest a therapeutic role for gastrin in the modulation of cholangiocarcinoma growth.

α-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca2+- and PKC-dependent stimulation of cAMP

Acetylcholine potentiates secretin‐stimulated ductal secretion by Ca2+‐calcineurin–mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin‐stimulated ductal secretion via activation of protein kinase C (PKC)‐γ. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of α‐1A/1C, ‐1β and β‐1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the α‐1 and β‐1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP3 and Ca2+ levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC‐α inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. α‐1 and β‐1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin‐stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin‐stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Gö6976. Phenylephrine increased IP3 and Ca2+ levels and activated PKC‐α and PKC‐β‐II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca2+/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:1116–1127))

Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.

We studied the expression of endothelin-1 (ET-1) receptors (ETA and ETB) and the effects of ET-1 on cholangiocyte secretion. The effects of ET-1 on cholangiocyte secretion were assessed in normal and bile duct-ligated (BDL) rats by measuring 1) basal and secretin-induced choleresis in vivo, 2) secretin receptor gene expression and cAMP levels in small and large cholangiocytes, and 3) luminal expansion in response to secretin in intrahepatic bile duct units (IBDU). ETA and ETB receptors were expressed by small and large cholangiocytes. ET-1 had no effect on basal bile flow or bicarbonate secretion in normal or BDL rats but decreased secretin-induced bicarbonate-rich choleresis in BDL rats. ET-1 decreased secretin receptor gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced luminal expansion in IBDU from normal or BDL rats. The inhibitory effects of ET-1 on secretin-induced cAMP synthesis and luminal duct expansion were blocked by specific inhibitors of the ETA (BQ-610) receptor. ET-1 inhibits secretin-induced ductal secretion by decreasing secretin receptor and cAMP synthesis, two important determinants of ductal secretion.

Gastrin reverses established cholangiocyte proliferation and enhanced secretin-stimulated ductal secretion of BDL rats by activation of apoptosis through increased expression of Ca2+-dependent PKC isoforms

Abstract: We posed these questions: (i) Does administration of gastrin to 1‐week bile duct ligation (BDL) rats inhibits established cholangiocyte proliferation and ductal secretion? (ii) Is gastrin inhibition of cholangiocyte proliferation and secretion of BDL rats associated with enhanced apoptosis? (iii) Are gastrins effects on cholangiocyte function associated with increased expression of protein kinase C (PKC) isoforms; and (iv) Is gastrin stimulation of cholangiocyte apoptosis regulated by the Ca2+‐dependent PKC pathway?

Gamma-interferon inhibits secretin-induced choleresis and cholangiocyte proliferation in a murine model of cirrhosis

BACKGROUND/AIMS Cholangiocyte proliferation is associated with increased secretin receptor gene expression and secretin-induced choleresis. Since gamma-interferon has antiproliferative effects, we tested the hypothesis that gamma-interferon inhibits ductal proliferation and secretin-stimulated choleresis associated with cirrhosis. METHODS Mice were treated with 0.1 ml of 25% carbon tetrachloride intraperitoneally twice weekly and 5% alcohol in drinking water for 12 weeks to induce cirrhosis and subsequently gamma-interferon 10(5) intramuscularly was administered daily for 10 weeks. We measured the effects of carbon tetrachloride and gamma-interferon on liver collagen content by morphometric analysis and hydroxyproline content. We measured the effects of gamma-interferon on ductal mass by morphometry and on ductal secretion by assessment of secretin receptor gene expression and secretin-induced choleresis. RESULTS Compared to controls, there was an increase in liver hydroxyproline content of carbon tetrachloride-treated mice with histologic evidence of cirrhosis. Gamma-interferon treatment significantly decreased collagen liver content with loss of histologic features of cirrhosis. Morphometry revealed an increased number of bile ducts in cirrhotic mice as compared to controls or cirrhotics who received gamma-interferon. Secretin receptor mRNA levels were higher in cirrhotic mice compared to controls but this increase was inhibited by gamma-interferon. Secretin stimulated ductal secretion in cirrhotic mice but not control or cirrhotic mice who received gamma-interferon. CONCLUSIONS We have established a murine model for cirrhosis and have shown, consistent with our hypothesis, that gamma-interferon decreases collagen content, ductal mass and secretin-induced choleresis incirrhotic mice.

Taurohyodeoxycholate- and tauroursodeoxycholate-induced hypercholeresis is augmented in bile duct ligated rats

BACKGROUND/AIMS Taurohyodeoxycholate (THDCA) and tauroursodeoxycholate (TUDCA) induce more bile flow per molecule excreted compared to endogenous bile acids. The aim of this study is to determine if the hypercholeretic effect of tauroursodeoxycholate or taurohyodeoxycholate in normal and bile duct ligated (BDL) rats is due to increased ductal secretion. METHODS Normal or BDL rats were infused with tauroursodeoxycholate or taurohyodeoxycholate and bile flow, bicarbonate, bile salt, cholesterol, and phospholipid secretion were measured. Cholangiocytes were stimulated with taurohyodeoxycholate or tauroursodeoxycholate, and secretin-stimulated secretion was measured. RESULTS Taurohyodeoxycholate and tauroursodeoxycholate increased bile flow more in BDL than normal rats. Tauroursodeoxycholate increased bicarbonate secretion more in BDL compared to normal rats. Taurohyodeoxycholate when infused with taurocholate increased bile flow (but not phospholipid excretion) to a greater degree in BDL compared to normal rats. Taurohyodeoxycholate and tauroursodeoxycholate decreased secretin-stimulated cholangiocyte secretion. CONCLUSIONS Consistent with a ductal origin for bile acid-induced hypercholeresis, taurohyodeoxycholate and tauroursodeoxycholate produced a greater hypercholeresis in BDL than normal rats. Tauroursodeoxycholate- (but not taurohyodeoxycholate-) stimulated hypercholeresis is associated with increased HCO(3)(-) secretion. Tauroursodeoxycholate increases biliary HCO(3)(-) secretion by a mechanism unrelated to secretin-stimulated cholangiocyte secretion. Taurohyodeoxycholate-induced hypercholeresis in BDL rats is unrelated to enhanced phospholipid excretion.

Effects of Age, Sex, Cataract, and Cataract Surgery on Serum γ-Crystallin Concentration

Purpose: To determine if measurement of lens protein in serum is a feasible means to gain information on the physiologic status of the lens in human subjects. Methods: The γ-crystallin concentration was measured by a sandwich radioimmunoassay in the sera of 280 subjects aged 25–94 years. Medical records were reviewed for diagnoses of cataract and aphakia. Results: There was no effect of age or sex on the serum γ-crystallin concentration. There were 57 subjects with cataract and 27 with aphakia. γ-Crystallin was higher in all cataract groups and lower in aphakia. The mean γ-crystallin concentrations for selected subject groups were as follows: clear lens 301 pg/ml; pure nuclear cataract 344 pg/ml; pure cortical cataract 439 pg/ml and aphakia 255 pg/ml. Conclusions: This is the first published report to show that lens protein is measurable in serum and to demonstrate the feasibility of using serum assays of lens proteins to gain information on the physiological status of the lens. Our results confirm the hypothesis that molecular and cellular events leading to cataract cause increased leakiness of lens cell membranes with release of lens proteins appearing in the blood. It is conceivable that measurement of lens proteins in serum might find future use in the evaluation of cataract risk, potentially cataractogenic and anticataractogenic agents, retained lens fragments after phacoemulsification, secondary cataract, phacolytic glaucoma, anaphylactic endophthalmitis, eye injuries, and other eye diseases.

Regulation of cholanglocyte apical bile acid transporter (ABAT) activity by biliary bile acids: Different potential compensatory changes for intrahepatic and extrahepatic cholestasis

Regulation of Cholangiocyte Apical Bile Acid Transporter (ABAT) Activity by Biliary Bile Acids: Different Potential Compensatory Changes for Intrahepafic and Extrahepatic Cholestasis Gianfranco Alpini, The Texas AM Shannon Glaser, Jo Lynne Phinizy, Scott & White Memorial Hosp, Temple, TX; Noriatsu Kanno, The Texas AM Heather Francis, Scott & White Memorial Hosp, Temple, TX; Mikel Ludvik, The Texas AM Gene Lesage, Scott & White Memorial Hosp, Temple, TX

2. FUNCTIONAL HETEROGENEITY OF INTRAHEPATIC CHOLANGIOCYTES

Publisher Summary This chapter discusses the recent findings that demonstrate that cholangiocytes are heterogeneous with regard to morphology, secretory activity to gastrointestinal hormones/peptides and bile salts and proliferative/apoptotic responses to liver injury/toxins. On the basis of these findings, a scheme is proposed for mapping the morphological, secretory, proliferative and apoptotic events in the intrahepatic biliary tree. This model proposes that bile ducts are morphologically heterogeneous with small ducts lined by small cholangiocytes, and large ducts lined by large cholangiocytes. Large bile ducts, express secretin and somatostatin receptors, cystic fibrosis transmembrane regulator (CFTR), and Cl − /HCO − , and respond to these two hormones with changes in ductal secretion. Pathologically, small and large ducts respond differentially to specific injury/toxins. Following bile duct ligation (BDL), only large cholangiocytes proliferate, whereas following carbon tetrachloride (CCl 4 ) administration, damage and loss of large duct function leads to de novo proliferation and secretion of small cholangiocytes (resistant to CCl 4 ) to compensate for the loss of large duct function. In partial hepatectomy, both small and large cholangiocytes respond with increases in proliferation and secretion. The presence of cholangiocyte heterogeneity may well endow the biliary system with physiological advantages involving bile secretion, cholehepatic shunting, and countercurrent bile and blood flow. Human cholangiopathies differentially target the small and large ducts leading to cholangiocyte proliferation/loss.