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 inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.

We assessed the effect of gastrin on ductal secretion in normal and bile duct-ligated (BDL) rats. The effect of gastrin on ductal secretion was examined in the presence of proglumide, a specific antagonist for gastrin receptor (GR). We isolated pure cholangiocytes from normal and BDL rats and assessed gastrin effects on secretin receptor (SR) gene expression and intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. We examined the presence of GR mRNA in cholangiocytes by reverse transcription polymerase chain reaction (RT-PCR). In normal or BDL rats, gastrin produced no changes in spontaneous bile secretion. Simultaneous infusion of gastrin inhibited secretin-induced choleresis and bicarbonate output in BDL rats. In the presence of proglumide gastrin did not inhibit secretin-induced choleresis in BDL rats. Gastrin decreased in cholangiocytes from BDL rats 1) SR gene expression and 2) secretin-induced cAMP levels. With the use of RT-PCR, GR mRNA was detected in cholangiocytes. Similar to what is shown for secretin and somatostatin, we propose that the opposing effects of secretin and gastrin on cholangiocyte secretory activity regulate ductal secretion in rats.

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.

Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes.

Accumulation of bile acids (BA) and cholangiocyte proliferation occur in cholestasis, but BA effects on the proliferative and secretory capacity of cholangiocytes are undefined. Cholangiocyte proliferation coupled with increased expression of H3 histone and secretin receptor (SR) genes and secretin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) levels is limited to large cholangiocytes. We isolated pooled small and large cholangiocytes and studied the effect of taurocholic (TC) and taurolithocholic (TLC) acids on proliferation, by measurement of H3 histone gene expression, and secretion, by measurement of SR gene expression, cAMP levels, and Cl-/HCO3- exchanger activity. In pooled cholangiocytes, TC and TLC increased H3 histone (12-fold) and SR (3-fold) gene expression and both spontaneous (1.4-fold) and secretin-induced (4-fold) cAMP response. TC and TLC increased H3 histone (10-fold) and SR (2-fold) gene expression and secretin-induced cAMP response and Cl-/HCO3- exchanger activity (3-fold) only in large cholangiocytes. In large cholangiocytes, BA may have a signaling function in the modulation of ductal secretion.

Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion.

We have shown that agonist-regulated ductal secretion is limited to large cholangiocytes. To directly study cholangiocyte heterogeneity along the length of the normal biliary tree, we defined the genetic and functional expression of agonist-induced ductal secretion in intrahepatic bile duct units (IBDU) of different sizes. Small IBDU (< 15-microns diam) were separated from large IBDU (> or = 15-microns diam), and then ducts of different sizes were characterized by morphometric analysis, gene expression, secretin-induced adenosine 3',5'-cyclic monophosphate (cAMP) synthesis, and secretion by change in luminal size in response to agonists. IBDU diameters ranged from 11 to 65 microns. Secretin increased ductal secretion solely in large IBDU. Forskolin induced a modest increase in ductal secretion in small IBDU but markedly increased ductal secretion in large IBDU. Secretion increased Cl-/HCO3- exchanger activity and cAMP levels in large but not small IBDU. Secretin receptor and Cl-/HCO3 exchanger mRNAs were detected only in large IBDU. We propose that agonist-induced ductal secretion occurs in large (> or = 15-microns diam) but not small (< 15-microns diam) intrahepatic ducts.

Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion.

BACKGROUND & AIMS: After partial hepatectomy, liver regeneration occurs with the return of hepatocyte mass to normal, Limited data exist regarding the renewal of the biliary tree after partial hepatectomy. This study tested the hypothesis that, after partial hepatectomy, the biliary tree regenerates by proliferation of the remaining cholangiocytes, leading to an increase in secretin-induced ductal bile secretion. METHODS: After 70% partial hepatectomy, cholangiocyte proliferation was assessed in situ by morphometric analysis and In vitro by measurement of 3H-thymidine incorporation. Ductal secretion was estimated by measurement of secretin receptor gene expression and adenosine 3',5'-cyclic monophosphate (cAMP) levels in vitro and by the effect of secretin on ductal bile secretion in vivo. RESULTS: DNA synthesis was undetectable in control cholangiocytes, increased and peaked at day 3 after partial hepatectomy, and returned to normal by day 28. Morphometric analysis showed regrowth of the biliary tree beginning at day 1 with restoration by day 10. The expression of secretin receptor gene and secretin-induced cAMP levels and secretin-induced bicarbonate-rich choleresis increased during the period of bile duct renewal. CONCLUSIONS: After partial hepatectomy, the increase in secretin-induced ductal bile secretion observed during bile duct renewal results from proliferation of remaining cholangiocytes.

Characterization of a calcium binding domain in the VP1 protein of the avian polyomavirus, budgerigar fledgling disease virus.

Calcium ions appear to play a major role in maintaining the structural integrity and assembly of papovavirus virions and are likely involved in the process of viral uncoating. Recently it was reported that the purified recombinant VP1 protein of budgerigar fledgling disease virus (BFDV) was capable of assembling into capsid-like particles in the presence of calcium. It is now reported that the major capsid protein VP1 of BFDV binds calcium ions in an in vitro calcium binding assay. Two deletions were made in the VP1 protein to identify a calcium binding domain and to further characterize the role of calcium ions in the capsid assembly process. Recombinant VP1 lacking a putative calcium binding domain (Asp-237-Asp-248) failed to bind radioactive 45Ca2+ yet associated into capsomeres. These capsomeres were similar in shape to the wild-type VP1 but were unable to assemble into capsid-like particles. Likewise, recombinant VP1 lacking ten carboxyl terminal amino acids (Glu-334-Arg-343) also formed capsomeres that were unable to assemble into capsid-like particles. In contrast to the VP1 protein with the internal deletion, the protein with the truncated carboxyl terminus bound 45Ca2+ in the in vitro assay. These results have identified a calcium binding domain (Asp-237-Asp-248) for the BFDV VP1 protein and a crucial role for the VP1 carboxyl terminal amino acids (Glu-334-Arg-343) in capsid assembly.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly.

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Homotransplant renal artery stenosis.

Transplant renal artery stenosis occurred in 12 of 101 consecutive kidney transplants. Stenoses were all located in the renal artery distal to the anastomosis. Two separate forms of stenosis are recognized: angulation and segmental. All transplant patients with severe diastolic hypertension, refractory to medical management, and an audible abdominal bruit should undergo angiography. Surgical correction of the stenosis was accomplished in nine of 12 patients with cure of their hypertension.