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Upregulation of secretin receptors on cholangiocytes after bile duct ligation
Regulatory Peptides 97 (2001) 1–6 www.elsevier.com / locate / regpep
Upregulation of secretin receptors on cholangiocytes after bile duct ligation P.S. Tietz, E.M. Hadac, L.J. Miller, N.F. LaRusso* Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Internal Medicine, and Department of Biochemistry and Molecular Biology, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA Received 5 January 2000; received in revised form 21 February 2000; accepted 1 March 2000
Abstract Secretin not only increases ductular bile secretion in vivo in rats after bile duct ligation (BDL) [1], but also increases cAMP levels and stimulates exocytosis in isolated cholangiocytes [2]. Although we have previously reported that secretin receptor mRNA was upregulated in cholangiocytes after BDL [3], the cholangiocyte secretin receptor has not been functionally characterized or quantified after BDL. In this work, we used a novel, photolabile and biologically active analogue of secretin to quantify and characterize secretin receptors on cholangiocytes isolated from normal and BDL rats. The cholangiocyte secretin receptor bound radioligand with high affinity and in a rapid, reversible, and temperature-dependent manner. While receptors on cholangiocytes from normal and BDL rats were functionally and biochemically identical, receptor density on cholangiocytes was increased 5-fold following BDL. The combination of increased cell number with increased functional secretin receptors per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion, explains the increased hormone-responsive choleresis observed after BDL and may reflect an adaptive response of the organism to cholestasis. 2001 Elsevier Science B.V. All rights reserved. Keywords: Pancreatic; Secretin; Receptor; Photoaffinity labeling
1. Introduction The liver is composed of a variety of resident cells, including hepatocytes and bile duct cells, or cholangiocytes [4]. These two cell types represent the only epithelial cells in the liver, are involved in both absorptive and secretory activities, and represent highly polarized units containing morphologically and functionally distinct plasma membrane domains [5]. Recent studies by us Abbreviations: BDL, bile duct ligation; BSA, bovine serum albumin; GGT, g-glutamyl transpeptidase; HPLC, high-performance liquid chromatography; IC 50 , 50% inhibitory concentration; Ki , receptor binding dissociation constant; KRH, Krebs–Ringers–Hepes solution with protease inhibitors; NRC, normal rat cholangiocyte; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; STI, soybean trypsin inhibitor *Corresponding author. Tel.: 1 1-507-284-1006; fax: 1 1-507-2840762. E-mail address: [email protected] (N.F. LaRusso).
provided evidence that cholangiocytes contribute to bile secretion in response to hormones, including secretin, by an exocytic process [2]. In addition, in vivo studies have shown that secretin stimulates bile flow in rats in whom cholangiocyte hyperplasia has been induced by bile duct ligation (BDL) [1]. To date, studies on the secretin receptor have been done primarily in pancreas and stomach with relatively little characterization of this receptor on cholangiocytes due in part to the lack of suitable experimental models. For example, Farouk et al. described specific secretin binding to rat intrahepatic biliary epithelium using in vitro tissue section autoradiography and competition-binding to crude biliary plasma membranes [6]. In addition, previous work from our laboratory has demonstrated the presence of secretin receptor mRNA in cholangiocytes [3] In this work, we have utilized three new technologies developed by us to functionally and biochemically characterize the secretin receptor on cholangiocytes, including:
0167-0115 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00109-9
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P.S. Tietz et al. / Regulatory Peptides 97 (2001) 1 – 6
(i) the preparation of isolated cholangiocytes [7]; (ii) the preparation of enriched plasma membranes from these cells [5]; and (iii) the development of a photolabile, high specific radioactivity probe for the secretin receptor [8]. Our results provide the first clear evidence of increased functional secretin receptor density on cholangiocytes after BDL, and provide a mechanistic explanation for the secretin-induced hypercholeresis seen in this animal model of cholangiocyte hyperplasia.
2. Methods
2.1. Materials Synthetic rat secretin, rat VIP, and somatostatin were from Peninsula Laboratories (Belmont, CA); Hepes, type B trypsin and bovine serum albumin (BSA, fraction V) were from Calbiochem (La Jolla, CA); Type II collagenase was from Worthington (Freehold, NJ); deoxyribonuclease I, sucrose and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (St. Louis, MO); soybean trypsin inhibitor (STI) was from Cooper Biochemical (Malvern, PA). All other reagents were of analytical grade.
2.2. Utilization of a rat secretin analogue The secretin analogue, ((Tyr 10 , pNO 2 –Phe 22 ) rat secretin-27), used in these studies was synthesized as we reported [8]. This was designed to provide a high-specific radioactivity, high-affinity ligand incorporating a photolabile residue within a pharmacophoric region, permitting ‘intrinsic’ photoaffinity labeling of the secretin receptor. This probe was fully biologically active at the pancreatic acinar cell secretin receptor, bound in a specific and high affinity manner, and effectively affinity labeled that molecule [8].
2.3. Radioiodination of secretin probe The secretin analogue was radioiodinated with [ 125 I]Na using the solid-phase oxidant N-chlorobenzenesulfonamide (Iodo-Beads, Pierce, Rockford, IL) as described previously [8]. The product was purified by reversed-phase HPLC to yield a specific radioactivity of 2000 Ci / mmol.
2.4. Animal model of selective cholangiocyte hyperplasia In all experiments, we used male Fisher 344 rats weighing between 250 and 300 g (Harlan Sprague–Dawley, Indianapolis, IN), which were fed Purina laboratory chow (Ralston Purina, St. Louis, MO) and permitted ad libidum access to food and water. To induce selective cholangiocyte hyperplasia, we performed BDL 3 weeks prior to cell isolation as previously described [9].
2.5. Animal model for in vivo studies In the experiments performed here, we employed an in vivo model previously reported by us [1] which allows comparative analysis of the effects of various infusates on the regulation of ductal bile flow. Two weeks prior to the in vivo experiments, sham rats were anesthetized and the bile duct manipulated through an abdominal incision. In BDL rats, a polyethylene PE-50 cannula was inserted into the common bile duct and heat sealed. After 2 weeks in sham-operated rats, we inserted a cannula in the bile duct and allowed the bile to flow freely. In BDL rats, a cannula inserted and heat-sealed closed 2 weeks prior to the experiment was externalized via a small abdominal incision and the sealed end clipped off to allow bile collection. Bile was collected every 10 min. After a 60-min equilibration period, secretin or secretin analogue (10 27 M) was administered over 30 min (0.1 ml every 3 min, total volume of 1.0 ml injected) via a femoral vein cannula. We continued bile collections every 10 min for 60 min after the infusion period. After the final collection point, the animal was exsanguinated.
2.6. Isolation of cholangiocytes for receptor binding studies Parenchymal and nonparenchymal liver cells were separated from livers of normal or BDL rats by collagenase perfusion, enzymatic digestion and mechanical disruption as previously described [7]. A second, higher activity digestion and several passes through a 22-gauge needle yielded a single-cell, cholangiocyte-enriched population. Cells were counted and purity assessed morphologically by staining for g-glutamyl transpeptidase (gGT) (a cholangiocyte-specific marker) as previously described [10].
2.7. Preparation of plasma membranes for photoaffinity labeling studies Cholangiocyte plasma membranes were prepared from BDL rats using isopycnic centrifugation on linear sucrose gradients by the method developed and reported by us [5]. All solutions for membrane isolation were prepared in the presence of 0.01% STI and 0.1 mM PMSF. Densities of all sucrose solutions were adjusted by refractometry at room temperature. We assayed the purified cholangiocyte plasma membrane fraction for alkaline phosphodiesterase I (APDI), a known plasma membrane marker [11].
2.8. Receptor binding studies Receptor binding characterization was performed using a 125 I-labeled secretin analogue (2000 Ci / mmol, specific activity), native porcine secretin for displacement, and 15 million cholangiocytes per tube. Non-specific binding was determined in the presence of 1 mM secretin. Time- and
P.S. Tietz et al. / Regulatory Peptides 97 (2001) 1 – 6
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temperature-dependency of binding were performed at 48C and room temperature. Bound and free radioligand were separated by centrifugation (15 000 3 g for 5 min) and washing, as we have reported [8]. All binding was performed at steady state, attained after 60 min at room temperature.
2.9. Photoaffinity labeling Photoaffinity labeling studies were performed as previously described [8]. Briefly, radiolabeled probe (75–100 pmol / l) was initially bound to purified cholangiocyte plasma membranes (100 mg). The labeled membranes were photolyzed for 30 min at 48C with a Rayonet ˚ lamps (Southern New photochemical reactor using 3000 A England Ultraviolet, Hamden, CT). Membrane proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gel, with the receptor identified by autoradiography. The molecular weight of the affinitylabeled protein was calculated by interpolation on a plot of log of molecular weights versus mobility of standard proteins. A 50% inhibitory concentration (IC 50 ) value for covalent labeling was determined using densitometric scanning of the autoradiographs.
2.10. Statistical analysis Data were analyzed using LIGAND and Prism software (GraphPad Software, San Diego, CA) analysis programs and Clinfo data management analysis system. All results are expressed as mean6S.E. unless otherwise indicated.
3. Results
3.1. Chemical characterization of the secretin analogue The synthetic secretin analogue was purified by HPLC with its structure verified by amino acid analysis as previously reported [8].
3.2. Effects of secretin and secretin analogue on bile flow, i.e., demonstration of biological activity In vivo, intravenous infusion of secretin analogue (10 27 M) caused an increase (144% maximal increase above baseline; P , 0.05) in bile flow in 2-week BDL, but not in sham-operated rats (Fig. 1). As previously reported by us [1] using this same experimental model, native secretin (10 27 M) had similar secretagogue activity, resulting in a 164% maximal increase above baseline (P , 0.05 in 2week BDL, and not in sham-operated rats) (Fig. 1). Bile flow returned to basal state approximately 30 min following infusion of either secretin or secretin analogue in BDL rats.
Fig. 1. Effect of secretin or secretin analogue (10 27 M) on bile flow in both sham-operated and bile duct-ligated rats. In separate experiments, secretin or secretin analogue was infused in both sham-operated and bile duct-ligated rats. Hormone was injected via a femoral vein cannula after a 60-min equilibration period. Infusion of secretin or the secretin analogue was over a 30-min period from 60 to 90 min. Bile was collected every 10 min and expressed as microliters per minute per kilogram body weight. Data are mean6S.E. for n $ 3 animals.
3.3. Functional characterization of rat cholangiocyte secretin receptor binding The cholangiocyte isolation method described yielded a partially purified, single cell suspension of 32.761.7 million cells per gram liver for BDL rats and 16.863.6 million cells per gram liver from normal rats. Cholangiocyte purity, as assessed by gGT positivity, was 63.662.4% for BDL and 35.265.0% for normal. In previous studies by us [7], we have shown that endothelial cells (which do not express secretin receptors) represent the major other cell type in this preparation. The time- and temperature-dependence of radioligand binding to functionally active cholangiocytes are shown in Fig. 2. Binding was rapid, reaching its plateau level within 5 min at both 22 and 48C. At 228C, this was 1.5–2 times greater specific binding than was achieved at 48C, perhaps reflecting internalization of bound ligand. Binding of the secretin analogue was inhibited by competing native secretin in a concentration-dependent manner on cholangiocytes from both normal and BDL rats (Fig. 3). Specific binding ranged between 50 and 60%. There was no significant difference in the affinity of the ligand (Ki ) for the receptor on cholangiocytes from normal versus BDL rats; 2168 and 27615 nM, respectively (P 5 0.4) (determined using ligand analysis, with two-site model not statistically better than a one-site model). Binding specificity was affirmed by a lack of displacement by somatostatin and with VIP displaying a much lower binding affinity than secretin, with 1 mM VIP competing for only 11% of saturable binding (Fig. 4). Receptor density per cell was significantly greater on cholangiocytes from BDL than those from normal rats; representing a
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Fig. 2. Time- and temperature-dependence of binding of 125 I-labeled secretin analogue to intact cholangiocytes isolated from BDL rats. Data are expressed as a percentage of control at 48C and room temperature (228C). Data are mean6S.E. for n > 3 animals.
Fig. 4. Affinity labeling of cholangiocyte plasma membranes isolated from BDL rats with 125 I-labeled secretin analogue and increasing concentrations of secretin and VIP up to 1 mM. Samples were reduced prior to electrophoresis on a 10% SDS–polyacrylamide gel and autoradiograms analyzed by quantitative densitometric scanning (Molecular Analyst). Data (n 5 2) are expressed as densitometric units, percent of maximum.
5-fold increase from 8,800 (6760) receptor molecules per cell in normal cholangiocytes to 34,400 (62,800) following BDL (P , 0.05).
3.4. Biochemical characterization of the cholangiocyte secretin receptor Photoaffinity labeling identified specific labeling at an Mr 5 57,000–62,000 band. The specificity of labeling was confirmed by the complete displacement by secretin, and poor displacement by VIP. Quantitative densitometric scanning of affinity-labeled receptor showed an IC 50 value of 1 and 600 nM for secretin and VIP, respectively. Fig. 3. Competition-binding curves for binding of 125 I-labeled secretin analogue to cholangiocytes isolated from normal and BDL rats. Assays were incubated for 60 min at room temperature; 100% binding represents the counts bound in the absence of competing peptide. Data are expressed as radioactivity bound (percent of control). Data are mean6S.E. for cells from n $ 3 animals for the secretin curves.
4. Discussion The major findings reported here relate to observed changes in the secretin receptor protein on cholangiocytes
P.S. Tietz et al. / Regulatory Peptides 97 (2001) 1 – 6
following BDL in the rat. Studies in vivo showed that both native secretin and a photolabile secretin analogue used to probe the structure of the secretin receptor caused an increase in bile flow in BDL, but not in sham-operated, rats. Receptor-binding studies in isolated cholangiocytes from both normal and BDL rats using this biologically active analogue of secretin showed time- and temperaturedependence, similar binding affinities, and similar biochemical characteristics. Receptor density calculations showed a significant increase in the number of secretin receptors on cholangiocytes after BDL. We previously reported that secretin induces a hypercholeresis in rats after BDL, an observation confirmed here with both secretin and secretin analogue [9]. We further suggested that this hypercholeresis might be attributable to either alterations in cholangiocyte function, an increased number of cells (since cholangiocytes are the only endogenous liver cells that proliferate after BDL), or both [3]. To address the first possibility, we employed the biologically active secretin analogue described here and isolated cholangiocytes. Compared to cholangiocytes from normal rats, BDL did not alter the affinity of the receptor for secretin. In contrast, the receptor density per cell was significantly greater on cholangiocytes from BDL than those from normal rats. These data provide the first evidence that the hypercholeresis seen after BDL in response to secretin reflects an increased number of secretin receptors per cell. To our knowledge, the cholangiocyte is the only epithelial cell in which the number of secretin receptors per cell is known to be increased after a proliferative stimulus. The possible physiological relevance of this observation is discussed below. For reasons that are yet unclear, the selective proliferation of cholangiocytes that occurs following BDL is accompanied by a selective upregulation of certain genes, including those for the secretin receptor [3]. In our previous studies, we found that the transcript for the secretin receptor increased 7- to 9-fold in cholangiocytes from BDL rats compared with controls [3]. The addition of our current data provides the first direct evidence that there is also an increased number of receptor molecules per cell following BDL. These data also show that the messenger RNA for the secretin receptor is, in fact, translated into a functional protein. The mechanism by which BDL increases the expression of secretin receptor message and protein remains unclear. Our observations relating to differences between normal and BDL cholangiocytes in response to secretin agree with and extend the findings of Farouk and co-workers [6,12]. Employing 125 I-labeled secretin receptor autoradiography on frozen sections of normal and BDL rat liver, they found that saturable secretin binding as measured by quantitative densitometry was increased after BDL. Scatchard analysis of the binding showed a one-site receptor model with a binding affinity constant of 5.361.1 nM. In agreement with our findings, they observed little displacement by
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other secretin family peptides such as VIP. Subsequent studies by Farouk et al. using 3-week BDL rats showed that 125 I-labeled secretin bound saturably to crude plasma membranes prepared from cholangiocytes and was competed off in a concentration-dependent manner (Ki 5 1.360.1 nM, Bmax 5 273623 fmol / mg protein). However, this group did not address the issue of receptor density. Not unexpectedly, based on our findings and those of others [3,6,12], the secretin receptor characterized on cholangiocytes closely resembles the secretin receptor, expressed in the pancreas [8]. We know that several conditions that lead to an increased number of cholangiocytes (e.g., ANIT feeding, partial hepatectomy, and BDL) result in an increase in both spontaneous bile flow and secretin-induced hypercholeresis [13–16]. In addition, we have now shown that BDL increases steady-state levels of secretin receptor mRNA, increases the rate of secretin receptor transcription, and increases the number of secretin receptor molecules per cholangiocyte. We propose that the increase in the rate of secretin transcription and the subsequent increase in the number of secretin receptor sites per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion is amplified by an increase in secretin receptor expression. These findings raise not only intriguing questions about the regulatory signals for selective gene expression that are apparently triggered by BDL but also about the relationship between the selective proliferation of individual cholangiocytes and the increase in secretin receptor gene transcription and mRNA translation. With the diversity of experimental models to study cholangiocyte biology available to us, we are now in a position to further explore these questions.
Acknowledgements This work was supported by Grants DK24031 (Dr. LaRusso) and DK46577 (Dr. Miller) from the National Institutes of Health and by the Mayo Foundation.
References [1] Tietz PS, Alpini GD, Pham LD, LaRusso NF. Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. Am J Physiol 1995;269:G110–8. [2] Kato A, Gores GJ, LaRusso NF. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J Biol Chem 1992;267:15523–9. [3] Alpini G, Ulrich CD, Phillips JO, Pham LD, Miller LJ, LaRusso NF. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol 1994;266:G922–8. [4] Jones AL. Anatomy of the liver. In: Hepatology, Philadelphia, PA: W.B. Saunders, 1982, p. Zakim M., Boyer T.D.
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[5] Tietz PS, Holman RT, Miller LJ, LaRusso NF. Isolation and characterization of rat cholangiocyte vesicles enriched in apical or basolateral plasma membrane domains. Biochemistry 1995;34(47):15436–43. [6] Farouk M, Vigna SR, McVey DC, Meyers WC. Localization and characterization of secretin binding sites expressed by rat bile duct epithelium. Gastroenterology 1992;102(3):963–8. [7] Ishii M, Vroman B, LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 1989;97:1236–47. [8] Ulrich CDI, Pinon DI, Hadac EM, Holicky EL, Chang-Miller A, Gates LJ, Miller LJ. Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors. Gastroenterology 1993;105(5):1534–43. [9] Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 1988;81:569–78. [10] Rutenberg AM, Kim H, Fishbein JW, Hanker JS, Wasserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of g-glutamyl transpeptidase activity. J Histochem Cytochem 1969;17:517–26.
[11] Beaufay HY, Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. J Cell Biol 1974;61:188–200. [12] Farouk M, Vigna S, Haebig J, Gettys TW, McVey DC, Chari R, Pruthi RS, Meyers WC. Secretin receptors in a new preparation of plasma membranes from intrahepatic biliary epithelium. J Surg Res 1993;54(1):1–6. [13] Alpini G, Lenzi R, Zhai WR, Slott PA, Liu MH, Sarkozi L, Tavoloni N. Bile secretory function of intrahepatic biliary epithelium in the rat. Am J Physiol 1989;257:G124–33. [14] Kossor D, Goldstein R, Ngo W, DeNicola DB, Leonard TB, Dulik DM, Meunier PC. Biliary epithelial cell proliferation following a-naphthylisothiocyanate (ANIT) treatment: relationship to bile duct obstruction. Fundam Appl Toxicol 1995;26:51–62. [15] LeSage G, Glaser S, Gubba S, Robertson WE, Phinizy JL, Lasater J, Rodgers R. Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion. Gastroenterology 1996;111:1633–44. [16] Slott PA, Liu MH, Tavoloni N. Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat. Gastroenterology 1990;99:466–77.
Upregulation of secretin receptors on cholangiocytes after bile duct ligation P.S. Tietz, E.M. Hadac, L.J. Miller, N.F. LaRusso* Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Internal Medicine, and Department of Biochemistry and Molecular Biology, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA Received 5 January 2000; received in revised form 21 February 2000; accepted 1 March 2000
Abstract Secretin not only increases ductular bile secretion in vivo in rats after bile duct ligation (BDL) [1], but also increases cAMP levels and stimulates exocytosis in isolated cholangiocytes [2]. Although we have previously reported that secretin receptor mRNA was upregulated in cholangiocytes after BDL [3], the cholangiocyte secretin receptor has not been functionally characterized or quantified after BDL. In this work, we used a novel, photolabile and biologically active analogue of secretin to quantify and characterize secretin receptors on cholangiocytes isolated from normal and BDL rats. The cholangiocyte secretin receptor bound radioligand with high affinity and in a rapid, reversible, and temperature-dependent manner. While receptors on cholangiocytes from normal and BDL rats were functionally and biochemically identical, receptor density on cholangiocytes was increased 5-fold following BDL. The combination of increased cell number with increased functional secretin receptors per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion, explains the increased hormone-responsive choleresis observed after BDL and may reflect an adaptive response of the organism to cholestasis. 2001 Elsevier Science B.V. All rights reserved. Keywords: Pancreatic; Secretin; Receptor; Photoaffinity labeling
1. Introduction The liver is composed of a variety of resident cells, including hepatocytes and bile duct cells, or cholangiocytes [4]. These two cell types represent the only epithelial cells in the liver, are involved in both absorptive and secretory activities, and represent highly polarized units containing morphologically and functionally distinct plasma membrane domains [5]. Recent studies by us Abbreviations: BDL, bile duct ligation; BSA, bovine serum albumin; GGT, g-glutamyl transpeptidase; HPLC, high-performance liquid chromatography; IC 50 , 50% inhibitory concentration; Ki , receptor binding dissociation constant; KRH, Krebs–Ringers–Hepes solution with protease inhibitors; NRC, normal rat cholangiocyte; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; STI, soybean trypsin inhibitor *Corresponding author. Tel.: 1 1-507-284-1006; fax: 1 1-507-2840762. E-mail address: [email protected] (N.F. LaRusso).
provided evidence that cholangiocytes contribute to bile secretion in response to hormones, including secretin, by an exocytic process [2]. In addition, in vivo studies have shown that secretin stimulates bile flow in rats in whom cholangiocyte hyperplasia has been induced by bile duct ligation (BDL) [1]. To date, studies on the secretin receptor have been done primarily in pancreas and stomach with relatively little characterization of this receptor on cholangiocytes due in part to the lack of suitable experimental models. For example, Farouk et al. described specific secretin binding to rat intrahepatic biliary epithelium using in vitro tissue section autoradiography and competition-binding to crude biliary plasma membranes [6]. In addition, previous work from our laboratory has demonstrated the presence of secretin receptor mRNA in cholangiocytes [3] In this work, we have utilized three new technologies developed by us to functionally and biochemically characterize the secretin receptor on cholangiocytes, including:
0167-0115 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00109-9
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(i) the preparation of isolated cholangiocytes [7]; (ii) the preparation of enriched plasma membranes from these cells [5]; and (iii) the development of a photolabile, high specific radioactivity probe for the secretin receptor [8]. Our results provide the first clear evidence of increased functional secretin receptor density on cholangiocytes after BDL, and provide a mechanistic explanation for the secretin-induced hypercholeresis seen in this animal model of cholangiocyte hyperplasia.
2. Methods
2.1. Materials Synthetic rat secretin, rat VIP, and somatostatin were from Peninsula Laboratories (Belmont, CA); Hepes, type B trypsin and bovine serum albumin (BSA, fraction V) were from Calbiochem (La Jolla, CA); Type II collagenase was from Worthington (Freehold, NJ); deoxyribonuclease I, sucrose and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (St. Louis, MO); soybean trypsin inhibitor (STI) was from Cooper Biochemical (Malvern, PA). All other reagents were of analytical grade.
2.2. Utilization of a rat secretin analogue The secretin analogue, ((Tyr 10 , pNO 2 –Phe 22 ) rat secretin-27), used in these studies was synthesized as we reported [8]. This was designed to provide a high-specific radioactivity, high-affinity ligand incorporating a photolabile residue within a pharmacophoric region, permitting ‘intrinsic’ photoaffinity labeling of the secretin receptor. This probe was fully biologically active at the pancreatic acinar cell secretin receptor, bound in a specific and high affinity manner, and effectively affinity labeled that molecule [8].
2.3. Radioiodination of secretin probe The secretin analogue was radioiodinated with [ 125 I]Na using the solid-phase oxidant N-chlorobenzenesulfonamide (Iodo-Beads, Pierce, Rockford, IL) as described previously [8]. The product was purified by reversed-phase HPLC to yield a specific radioactivity of 2000 Ci / mmol.
2.4. Animal model of selective cholangiocyte hyperplasia In all experiments, we used male Fisher 344 rats weighing between 250 and 300 g (Harlan Sprague–Dawley, Indianapolis, IN), which were fed Purina laboratory chow (Ralston Purina, St. Louis, MO) and permitted ad libidum access to food and water. To induce selective cholangiocyte hyperplasia, we performed BDL 3 weeks prior to cell isolation as previously described [9].
2.5. Animal model for in vivo studies In the experiments performed here, we employed an in vivo model previously reported by us [1] which allows comparative analysis of the effects of various infusates on the regulation of ductal bile flow. Two weeks prior to the in vivo experiments, sham rats were anesthetized and the bile duct manipulated through an abdominal incision. In BDL rats, a polyethylene PE-50 cannula was inserted into the common bile duct and heat sealed. After 2 weeks in sham-operated rats, we inserted a cannula in the bile duct and allowed the bile to flow freely. In BDL rats, a cannula inserted and heat-sealed closed 2 weeks prior to the experiment was externalized via a small abdominal incision and the sealed end clipped off to allow bile collection. Bile was collected every 10 min. After a 60-min equilibration period, secretin or secretin analogue (10 27 M) was administered over 30 min (0.1 ml every 3 min, total volume of 1.0 ml injected) via a femoral vein cannula. We continued bile collections every 10 min for 60 min after the infusion period. After the final collection point, the animal was exsanguinated.
2.6. Isolation of cholangiocytes for receptor binding studies Parenchymal and nonparenchymal liver cells were separated from livers of normal or BDL rats by collagenase perfusion, enzymatic digestion and mechanical disruption as previously described [7]. A second, higher activity digestion and several passes through a 22-gauge needle yielded a single-cell, cholangiocyte-enriched population. Cells were counted and purity assessed morphologically by staining for g-glutamyl transpeptidase (gGT) (a cholangiocyte-specific marker) as previously described [10].
2.7. Preparation of plasma membranes for photoaffinity labeling studies Cholangiocyte plasma membranes were prepared from BDL rats using isopycnic centrifugation on linear sucrose gradients by the method developed and reported by us [5]. All solutions for membrane isolation were prepared in the presence of 0.01% STI and 0.1 mM PMSF. Densities of all sucrose solutions were adjusted by refractometry at room temperature. We assayed the purified cholangiocyte plasma membrane fraction for alkaline phosphodiesterase I (APDI), a known plasma membrane marker [11].
2.8. Receptor binding studies Receptor binding characterization was performed using a 125 I-labeled secretin analogue (2000 Ci / mmol, specific activity), native porcine secretin for displacement, and 15 million cholangiocytes per tube. Non-specific binding was determined in the presence of 1 mM secretin. Time- and
P.S. Tietz et al. / Regulatory Peptides 97 (2001) 1 – 6
3
temperature-dependency of binding were performed at 48C and room temperature. Bound and free radioligand were separated by centrifugation (15 000 3 g for 5 min) and washing, as we have reported [8]. All binding was performed at steady state, attained after 60 min at room temperature.
2.9. Photoaffinity labeling Photoaffinity labeling studies were performed as previously described [8]. Briefly, radiolabeled probe (75–100 pmol / l) was initially bound to purified cholangiocyte plasma membranes (100 mg). The labeled membranes were photolyzed for 30 min at 48C with a Rayonet ˚ lamps (Southern New photochemical reactor using 3000 A England Ultraviolet, Hamden, CT). Membrane proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gel, with the receptor identified by autoradiography. The molecular weight of the affinitylabeled protein was calculated by interpolation on a plot of log of molecular weights versus mobility of standard proteins. A 50% inhibitory concentration (IC 50 ) value for covalent labeling was determined using densitometric scanning of the autoradiographs.
2.10. Statistical analysis Data were analyzed using LIGAND and Prism software (GraphPad Software, San Diego, CA) analysis programs and Clinfo data management analysis system. All results are expressed as mean6S.E. unless otherwise indicated.
3. Results
3.1. Chemical characterization of the secretin analogue The synthetic secretin analogue was purified by HPLC with its structure verified by amino acid analysis as previously reported [8].
3.2. Effects of secretin and secretin analogue on bile flow, i.e., demonstration of biological activity In vivo, intravenous infusion of secretin analogue (10 27 M) caused an increase (144% maximal increase above baseline; P , 0.05) in bile flow in 2-week BDL, but not in sham-operated rats (Fig. 1). As previously reported by us [1] using this same experimental model, native secretin (10 27 M) had similar secretagogue activity, resulting in a 164% maximal increase above baseline (P , 0.05 in 2week BDL, and not in sham-operated rats) (Fig. 1). Bile flow returned to basal state approximately 30 min following infusion of either secretin or secretin analogue in BDL rats.
Fig. 1. Effect of secretin or secretin analogue (10 27 M) on bile flow in both sham-operated and bile duct-ligated rats. In separate experiments, secretin or secretin analogue was infused in both sham-operated and bile duct-ligated rats. Hormone was injected via a femoral vein cannula after a 60-min equilibration period. Infusion of secretin or the secretin analogue was over a 30-min period from 60 to 90 min. Bile was collected every 10 min and expressed as microliters per minute per kilogram body weight. Data are mean6S.E. for n $ 3 animals.
3.3. Functional characterization of rat cholangiocyte secretin receptor binding The cholangiocyte isolation method described yielded a partially purified, single cell suspension of 32.761.7 million cells per gram liver for BDL rats and 16.863.6 million cells per gram liver from normal rats. Cholangiocyte purity, as assessed by gGT positivity, was 63.662.4% for BDL and 35.265.0% for normal. In previous studies by us [7], we have shown that endothelial cells (which do not express secretin receptors) represent the major other cell type in this preparation. The time- and temperature-dependence of radioligand binding to functionally active cholangiocytes are shown in Fig. 2. Binding was rapid, reaching its plateau level within 5 min at both 22 and 48C. At 228C, this was 1.5–2 times greater specific binding than was achieved at 48C, perhaps reflecting internalization of bound ligand. Binding of the secretin analogue was inhibited by competing native secretin in a concentration-dependent manner on cholangiocytes from both normal and BDL rats (Fig. 3). Specific binding ranged between 50 and 60%. There was no significant difference in the affinity of the ligand (Ki ) for the receptor on cholangiocytes from normal versus BDL rats; 2168 and 27615 nM, respectively (P 5 0.4) (determined using ligand analysis, with two-site model not statistically better than a one-site model). Binding specificity was affirmed by a lack of displacement by somatostatin and with VIP displaying a much lower binding affinity than secretin, with 1 mM VIP competing for only 11% of saturable binding (Fig. 4). Receptor density per cell was significantly greater on cholangiocytes from BDL than those from normal rats; representing a
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Fig. 2. Time- and temperature-dependence of binding of 125 I-labeled secretin analogue to intact cholangiocytes isolated from BDL rats. Data are expressed as a percentage of control at 48C and room temperature (228C). Data are mean6S.E. for n > 3 animals.
Fig. 4. Affinity labeling of cholangiocyte plasma membranes isolated from BDL rats with 125 I-labeled secretin analogue and increasing concentrations of secretin and VIP up to 1 mM. Samples were reduced prior to electrophoresis on a 10% SDS–polyacrylamide gel and autoradiograms analyzed by quantitative densitometric scanning (Molecular Analyst). Data (n 5 2) are expressed as densitometric units, percent of maximum.
5-fold increase from 8,800 (6760) receptor molecules per cell in normal cholangiocytes to 34,400 (62,800) following BDL (P , 0.05).
3.4. Biochemical characterization of the cholangiocyte secretin receptor Photoaffinity labeling identified specific labeling at an Mr 5 57,000–62,000 band. The specificity of labeling was confirmed by the complete displacement by secretin, and poor displacement by VIP. Quantitative densitometric scanning of affinity-labeled receptor showed an IC 50 value of 1 and 600 nM for secretin and VIP, respectively. Fig. 3. Competition-binding curves for binding of 125 I-labeled secretin analogue to cholangiocytes isolated from normal and BDL rats. Assays were incubated for 60 min at room temperature; 100% binding represents the counts bound in the absence of competing peptide. Data are expressed as radioactivity bound (percent of control). Data are mean6S.E. for cells from n $ 3 animals for the secretin curves.
4. Discussion The major findings reported here relate to observed changes in the secretin receptor protein on cholangiocytes
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following BDL in the rat. Studies in vivo showed that both native secretin and a photolabile secretin analogue used to probe the structure of the secretin receptor caused an increase in bile flow in BDL, but not in sham-operated, rats. Receptor-binding studies in isolated cholangiocytes from both normal and BDL rats using this biologically active analogue of secretin showed time- and temperaturedependence, similar binding affinities, and similar biochemical characteristics. Receptor density calculations showed a significant increase in the number of secretin receptors on cholangiocytes after BDL. We previously reported that secretin induces a hypercholeresis in rats after BDL, an observation confirmed here with both secretin and secretin analogue [9]. We further suggested that this hypercholeresis might be attributable to either alterations in cholangiocyte function, an increased number of cells (since cholangiocytes are the only endogenous liver cells that proliferate after BDL), or both [3]. To address the first possibility, we employed the biologically active secretin analogue described here and isolated cholangiocytes. Compared to cholangiocytes from normal rats, BDL did not alter the affinity of the receptor for secretin. In contrast, the receptor density per cell was significantly greater on cholangiocytes from BDL than those from normal rats. These data provide the first evidence that the hypercholeresis seen after BDL in response to secretin reflects an increased number of secretin receptors per cell. To our knowledge, the cholangiocyte is the only epithelial cell in which the number of secretin receptors per cell is known to be increased after a proliferative stimulus. The possible physiological relevance of this observation is discussed below. For reasons that are yet unclear, the selective proliferation of cholangiocytes that occurs following BDL is accompanied by a selective upregulation of certain genes, including those for the secretin receptor [3]. In our previous studies, we found that the transcript for the secretin receptor increased 7- to 9-fold in cholangiocytes from BDL rats compared with controls [3]. The addition of our current data provides the first direct evidence that there is also an increased number of receptor molecules per cell following BDL. These data also show that the messenger RNA for the secretin receptor is, in fact, translated into a functional protein. The mechanism by which BDL increases the expression of secretin receptor message and protein remains unclear. Our observations relating to differences between normal and BDL cholangiocytes in response to secretin agree with and extend the findings of Farouk and co-workers [6,12]. Employing 125 I-labeled secretin receptor autoradiography on frozen sections of normal and BDL rat liver, they found that saturable secretin binding as measured by quantitative densitometry was increased after BDL. Scatchard analysis of the binding showed a one-site receptor model with a binding affinity constant of 5.361.1 nM. In agreement with our findings, they observed little displacement by
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other secretin family peptides such as VIP. Subsequent studies by Farouk et al. using 3-week BDL rats showed that 125 I-labeled secretin bound saturably to crude plasma membranes prepared from cholangiocytes and was competed off in a concentration-dependent manner (Ki 5 1.360.1 nM, Bmax 5 273623 fmol / mg protein). However, this group did not address the issue of receptor density. Not unexpectedly, based on our findings and those of others [3,6,12], the secretin receptor characterized on cholangiocytes closely resembles the secretin receptor, expressed in the pancreas [8]. We know that several conditions that lead to an increased number of cholangiocytes (e.g., ANIT feeding, partial hepatectomy, and BDL) result in an increase in both spontaneous bile flow and secretin-induced hypercholeresis [13–16]. In addition, we have now shown that BDL increases steady-state levels of secretin receptor mRNA, increases the rate of secretin receptor transcription, and increases the number of secretin receptor molecules per cholangiocyte. We propose that the increase in the rate of secretin transcription and the subsequent increase in the number of secretin receptor sites per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion is amplified by an increase in secretin receptor expression. These findings raise not only intriguing questions about the regulatory signals for selective gene expression that are apparently triggered by BDL but also about the relationship between the selective proliferation of individual cholangiocytes and the increase in secretin receptor gene transcription and mRNA translation. With the diversity of experimental models to study cholangiocyte biology available to us, we are now in a position to further explore these questions.
Acknowledgements This work was supported by Grants DK24031 (Dr. LaRusso) and DK46577 (Dr. Miller) from the National Institutes of Health and by the Mayo Foundation.
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