To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca2+-dependent PKC isoforms and2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca2+-dependent PKC-γ but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.
- intrahepatic biliary epithelium
- bile flow
- secretin receptor
ductal secretion is regulated by gastrointestinal hormones/peptides (3, 7,19, 20, 33, 37, 64), bile salts (4), and nerves (12, 34, 38, 39). Although secretin does not increase bile flow in normal rats (7, 29, 41), recent studies (29) have shown that secretin increases biliary bicarbonate in a dose-dependent fashion and is much more effective when administered via the hepatic artery. In addition to bile duct-ligated (BDL) rats (7, 24, 43, 64), in which there is enhanced ductal mass (7), secretin has also been shown to stimulate bicarbonate secretion in isolated intrahepatic bile duct units (IBDU) (12) and purified cholangiocytes (9, 41) of normal rats. The stimulatory effect of secretin on ductal secretion occurs by interaction with receptors expressed only by cholangiocytes (11) through an increase in intracellular cAMP levels (3, 37, 38, 40, 41, 43, 64), which leads to the opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel (22) and activation of the Cl−/HCO exchanger (3, 12,41) with subsequent secretion of bicarbonate into bile (7,24, 38, 41, 43, 64). Bombesin and vasoactive intestinal peptide stimulate ductal secretion (19, 20). Gastrin, somatostatin, and endothelin-1 inhibit secretin-stimulated choleresis in BDL rats (5, 18, 24, 64).
Cholangiocytes proliferate in response to liver injury/toxins including BDL (5, 7, 23, 43), partial hepatectomy (41), acute carbon tetrachloride administration (43), or chronic feeding of α-naphthylisothiocyanate (40) or bile salts (6). Cholangiocyte proliferation is associated with enhanced basal and secretin-stimulated ductal secretion (5, 7, 11, 23, 24, 33, 38). The BDL rat model has been widely used for evaluating basal and secretin-stimulated ductal secretion (5, 7, 11, 23, 24, 33, 38). In these studies, we used the BDL rat model, because it allows: 1) the isolation of a greater number of pure cholangiocytes compared with normal rats (7, 8) and 2) the in vivo evaluation of the stimulatory effect of intravenous administration of secretin on bile flow (7, 8, 38, 43, 64), which is absent in normal rats (7, 8, 23, 41).
There is growing information regarding the role of nerves in the regulation of ductal secretion (12, 34, 38, 39). ACh induces intracellular Ca2+ concentration ([Ca2+]i) increases and oscillation in rat cholangiocytes due to both influx of extracellular Ca2+ and mobilization of thapsigargin-sensitive [Ca2+]i stores (49). Other studies (12) have shown that ACh, by interaction with M3 ACh receptor subtypes, increases secretin-stimulated Cl−/HCO exchanger activity in cholangiocytes by Ca2+-calcineurin-mediated, PKC-independent modulation of adenyl cyclase. Interruption of the cholinergic system of BDL rats by vagotomy decreases cholangiocyte cAMP levels and inhibits secretin-stimulated ductal secretion (38). The α2-adrenergic receptor agonist UK-14304 inhibits cholangiocarcinoma growth through time course-dependent modulation of Raf-1 and B-Raf activities (34). The α1-adrenergic agonist phenylephrine potentiates secretin-stimulated ductal secretion through a Ca2+- and PKC-dependent amplification of the adenylyl cyclase system (39).
In a number of cells, D1 and D2 dopaminergic receptors modulate adenylyl cyclase activity with a stimulatory effect mediated by D1 dopaminergic receptors and inhibitory effects regulated by D2 dopaminergic receptors (26, 50, 59), the latter acting through d-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and Ca2+-dependent intracellular pathways (28). Dopaminergic receptors regulate secretory activity of several epithelia (15, 21,56). The intrahepatic biliary epithelium displays dopaminergic innervation (45, 46); however, no information exists regarding the role and mechanisms of action of the dopaminergic system in the regulation of ductal secretion and adenyl cyclase activity in cholangiocytes. We addressed the following questions. First, are functional D1, D2, and D3 dopaminergic receptors expressed by cholangiocytes? Second, do D1, D2, and D3 dopaminergic receptor agonists/antagonists regulate basal and secretin-stimulated ductal secretion? Third, are dopaminergic effects on ductal bile secretion associated with increased protein expression of Ca2+-dependent (α, βI, βII, and γ) and/or Ca2+-independent novel (ε and θ) and atypical (η and ζ) PKC isoforms? Fourth, are quinelorane effects on ductal secretion associated with cytoskeleton-to-membrane distribution of PKC-γ, whose protein expression is increased by quinelorane? Finally, is increased protein expression of PKC-γ (after treatment with a dopaminergic receptor agonist) associated with downregulation of PKA activity leading to inhibition of secretin-stimulated ductal secretion?
MATERIALS AND METHODS
Male Fischer 344 rats (150–175 g) were purchased from Charles River (Wilmington, MA). The animals were kept in a temperature-controlled environment (22°C) with a 12:12-h light-dark cycle and fed ad libitum with standard rat chow. The studies were performed in rats with cholangiocyte proliferation induced by BDL (7) [for isolation of cells (5, 18, 23, 24, 38, 43,64) or IBDU (3, 47, 55)] or bile duct incannulation [BDI, for bile collection (7)] for 1 wk. BDL and BDI were performed as described (7). Before each procedure, animals were anesthetized with pentobarbital sodium (50 mg/kg body wt).
Reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated. The substrate for γ-glutamyltranspeptidase (γ-GT),N-(γ-l-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA). Rabbit antibodies reacting with the D1, D2, or D3 dopaminergic receptors were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Radioimmunoassay (RIA) kits for the determination of intracellular cAMP and Ins(1,4,5)P3 levels were purchased from Amersham (Arlington Heights, IL). The antibodies (IgG) against the rat Ca2+-dependent PKC-α, PKC-βI, PKC-βII, PKC-γ, and the Ca2+-independent novel (ε and θ) and atypical (η and ζ) PKC isoforms were purchased from Santa Cruz Biotechnology. The D1 dopaminergic receptor agonist, [(±)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide 6-chloro-N-allyl-SKF-38393 hydrobromide] or (±)-SKF-82958 hydrobromide (48), the D2 dopaminergic receptor agonist quinelorane (51), the D2 dopaminergic receptor antagonist eticlopride (31, 62), and the D3 dopaminergic receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) (44) were obtained from Research Biochemicals International (Natick, MA).
Purification of Cholangiocytes
Pure cholangiocytes were obtained from BDL rats by immunoaffinity separation (4-6, 18, 23, 24, 30, 37, 38, 40,41, 43, 64) by using a mouse monoclonal antibody against an unidentified membrane antigen expressed by all rat intrahepatic cholangiocytes (30). Purity of isolated cholangiocytes was evaluated by histochemistry for γ-GT (58), a specific histochemical marker for cholangiocytes in rat liver (7,41). Cell viability (∼97%) was determined by trypan blue exclusion.
Expression of D1, D2, and D3 Dopaminergic Receptors in Purified Cholangiocytes, Liver Sections, and Apical and Basolateral Cholangiocyte Membranes
The expression of D1, D2, and D3 dopaminergic receptors was evaluated by immunoblots in pure cholangiocytes from both normal and BDL rats. The expression of the D2 dopaminergic receptor (the only receptor subtype expressed by cholangiocytes by immunoblots of whole cell lysate) was also determined by immunohistochemistry in liver sections, cytospin smears of pure cholangiocytes, and immunoblots in apical and basolateral cholangiocyte membranes from BDL rats.
Immunoblots for D1, D2, or D3 dopaminergic receptors in pure cholangiocytes from BDL rats was performed as follows. Proteins (100 μg) were resolved by SDS-7.5% polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was incubated overnight with rotation at 4°C with a rabbit antibody for the D1, D2, or D3 dopaminergic receptor diluted 1:3,000 with Tris-buffered saline (TBST; 50 mM Tris, 150 mM NaCl, and 0.05% Tween 20)-5% milk. The membrane was washed with TBST and incubated for 1 h at room temperature with a secondary antibody, an anti-rabbit IgG peroxidase conjugate [enhanced chemiluminescence (ECL) kit, Amersham Life Science, Little Chalfont, England] diluted 1:100,000 with TBST. The membrane was washed with TBST, and proteins were visualized by using chemiluminescence (ECL Plus kit, Amersham Life Science). The intensity of the bands was determined by scanning video densitometry by using the ChemiImager 4000 low-light imaging system (Alpha Innotech, San Leandro, CA).
Immunohistochemistry for D2 dopaminergic receptor was performed in fresh frozen liver sections (5-μm thick) from BDL rats. Sections were incubated for 30 min with primary affinity-purified goat polyclonal antibody against the D2 dopaminergic receptor (N-19, Santa Cruz Biotechnology) diluted 1:50. The sections were incubated for 30 min with rabbit-anti-goat antibody (DAKO, Glostrup, Denmark), followed by incubation for 30 min with rabbit EnVision (DAKO). All reagents were diluted in PBS (pH 7.2). Each incubation was followed by a wash in three changes of PBS, pH 7.2, for 15 min. The reaction product was developed with the use of 3-amino-9-ethylcarbazole and H2O2. Sections that were not incubated with a primary antibody served as negative controls. The sections were examined with a microscope (model BX 40; Olympus Optical).
Immunohistochemistry for D2 dopaminergic receptor was performed in cytospin smears of cholangiocytes. Cell smears were fixed with ice-cold acetone for 5 min. After smears were washed with PBS, endogenous peroxidase activity was blocked with 70% methanol containing 3% H2O2. After being blocked with normal rabbit serum, the cells were incubated with the primary antibody (dilution 1:100) at 4°C overnight. After washes with PBS, the conjugated antibody was detected by using a commercially available kit [Histofine SAB-PO(G) kit; Nichirei, Tokyo, Japan] according to the vendor's instructions. The reaction product was visualized with diaminobenzidine. The negative control was performed by using normal goat serum instead of primary antibody.
To determine the subcellular distribution (apical vs. basolateral) of the D2 dopaminergic receptor in cholangiocytes, we evaluated by immunoblots (23) protein expression for the D2 dopaminergic receptor in membranes obtained from the basolateral or apical domain of pure cholangiocytes from BDL rats. Cholangiocyte apical and basolateral membranes were prepared by isopycnic centrifugation on sucrose gradients by using a technique previously used by Tietz et al. (63) in cholangiocytes from BDL rats.
We measured intracellular Ins(1,4,5)P3 and [Ca2+]i levels, second messenger system-activated by D2 dopaminergic innervation (17, 65). Cholangiocytes from BDL rats were incubated for 1 h at 37°C (37) and subsequently stimulated for 15 min at 22°C with 0.2% BSA (basal value) or quinelorane (100 μM) with 0.2% BSA. Intracellular Ins(1,4,5)P3 levels were assessed by the [3H]Ins(1,4,5)P3 kit (Amersham) according to the instructions provided by the manufacturer. Data were expressed as picomoles per million cells.
Calcium fluorescence measurements in purified cholangiocytes from BDL rats were performed by using fluo 3-AM (Molecular Probes, Eugene, Oregon) and a microplate reader (Fluoroskan Ascent FL Thermolabsystems, Helsinki, Finland) equipped with three injectors (36). Isolated cholangiocytes (4 × 104 per well) were loaded for 1 h at room temperature with 5 μM of fluo 3-AM in Tyrode's salt solution (TSS) [(in mM): 137 NaCl, 2.7 KCl, 1 MgCl2, 0.2 NaH2PO4, 12 NaHCO3, and 5.5 glucose] with 0.1% Pluronic F-127 (Molecular Probes). Loaded cells were washed two times with TSS. The loaded cells were subsequently resuspended in TSS and incubated for an additional 30 min at room temperature. The loaded cells were then pelleted and resuspended at 4 × 104 cells per 100 μl of TSS. The 4 × 104 cells per well were added to a 96-well black microplate. The baseline fluorescence was measured 50 times at 2-s intervals. TSS alone or quinelorane (100 μM) dissolved in buffer were injected sequentially into separate wells, and the fluorescence intensity was measured at 538 nm for 3 min at 1-s intervals. The excitation wavelength was 485 nm. [Ca2+]i was calculated as follows: [Ca2+]i =K d(F − Fmin)/(Fmax − F). Fmax is fluorescence intensity measured after permeabilization of the cells with 1% NP-40. EGTA (0.1 M) was then added to chelate Ca2+and minimum fluorescence intensity (Fmin) was obtained. TheK d of fluo 3-AM was 390 nm (35).
Effect of Dopaminergic Receptor Agonists on Ductal Secretion
Measurement of bile flow, bicarbonate concentration, and secretion.
BDI rats were surgically prepared for bile collection (7). One jugular vein was incannulated with a polyethylene-50 cannula to infuse Krebs-Ringer-Henseleit (KRH), secretin, or the selected dopaminergic receptor agonist/antagonist. Body temperature (37°C) was monitored with a rectal thermometer. When steady-state bile flow was achieved (60 min of KRH infusion from the beginning of bile collection), we subsequently infused 1) secretin (100 nM) for 30 min; 2) KRH until steady state; 3) quinelorane (100 μM) for 30 min; 4) KRH; or 5) quinelorane (100 μM) + secretin (100 nM) for 30 min, followed by a final infusion of KRH. In separate experiments, after steady-state bile flow was reached, rats were subsequently infused with1) secretin (100 nM) for 30 min; 2) KRH until steady state; 3) eticlopride (100 μM) for 30 min;4) KRH until steady state; or 5) eticlopride (a D2 dopaminergic receptor antagonist; 100 μM) + quinelorane (100 μM) + secretin (100 nM) for 30 min, followed by a final infusion of KRH. We evaluated the effects of a D1 [(±)-SKF-82958 hydrobromide; 100 μM (48)] or a D3 (7-OH-DPAT; 100 nM) (44) dopaminergic receptor agonist on basal and secretin-stimulated bile flow and on bicarbonate concentration and secretion. Bile was collected every 10 min, and bile flow was determined by weight, assuming a density of 1.0 g/ml. Biliary bicarbonate concentration (measured as CO2) was determined in bile by a Natelson microgasometer apparatus (Scientific Industries, Bohemia, NY).
Effect of Quinelorane on Ductal Lumen Expansion in IBDU
IBDU were isolated as described (3, 47, 55). IBDU were incubated for 24 h at 37°C in minimum essential medium (GIBCO-BRL, Grand Island, NY) with 10% fetal calf serum to allow complete sealing of bile duct lumen (3, 47, 55). Ductal fluid secretion was estimated from the changes in the area of IBDU lumen space, as previously described (3, 47, 55), after perfusion with 1) 0.2% BSA (basal); 2) secretin (positive control, 100 nM) (3) with 0.2% BSA;3) quinelorane (100 μM) in the absence or presence of secretin (100 nM) with 0.2% BSA; or 4) BAPTA-AM (50 μM; an [Ca2+]i chelator) (12, 23), chelerythrine (1 μM) or H7 (20 μM), two Ca2+-dependent PKC inhibitors (23, 32), or rottlerin (10 μM; a Ca2+-independent PKC-δ inhibitor) (54) + quinelorane (100 μM) + secretin (100 nM) with 0.2% BSA.
Intracellular cAMP Levels in Purified Cholangiocytes
After isolation, pure cholangiocytes from BDL rats were incubated for 1 h at 37°C (37) and subsequently incubated at room temperature (4-6, 18, 23, 24, 37, 38, 40,41, 43) with 1) 0.2% BSA (basal) for 5 min;2) secretin (100 nM) with 0.2% BSA for 5 min; 3) quinelorane (100 μM for 5 min) in the absence or presence of secretin (100 nM for 5 min) with 0.2% BSA; 4) eticlopride (100 μM for 5 min) + quinelorane (100 μM for 5 min) + secretin (100 nM for 5 min) with 0.2% BSA; or 5) eticlopride (100 μM for 5 min) in the absence or in the presence of secretin (100 nM for 5 min) with 0.2% BSA. We evaluated the effect of quinelorane (100 μM for 5 min) on secretin-stimulated cAMP levels in the absence or presence of BAPTA-AM (50 μM for 5 min), chelerythrine (1 μM for 5 min), H7 (20 μM for 5 min), or rottlerin (10 μM for 5 min) with 0.2% BSA. We evaluated the effect of BAPTA-AM, H7, chelerythrine, or rottlerin on basal cAMP levels. Intracellular cAMP levels were determined by RIA by using commercially available kits.
Are Quinelorane Effects on Secretin-Stimulated Ductal Secretion Associated with Increased Expression of Ca2+-dependent and Ca2+-independent PKC Isoforms?
We evaluated whether the inhibitory effects of quinelorane on secretin-stimulated ductal bile secretion are associated with increased protein expression of Ca2+-dependent (α, βI, βII, γ) and/or Ca2+-independent novel (δ, ε, θ) and atypical (η, ζ) PKC isoforms, which regulate cell functions in several epithelia including cholangiocytes (10, 14, 23, 32, 61,68).
Cholangiocytes were stimulated for 90 min (14, 23) at 37°C with 0.2% BSA (basal) or quinelorane (100 μM) with 0.2% BSA and were subsequently analyzed for protein expression for PKC-α, -βI, -βII, -γ, -δ, -ε, -θ, -η, and -ζ by immunoblots (23). The intensity of the bands was determined by scanning video densitometry by using the ChemiImager 4000.
Because quinelorane increases only the protein expression for the Ca2+-dependent PKC-γ, we then determined whether the effects of quinelorane on secretin-stimulated ductal secretion were associated with intracellular redistribution of PKC-γ. To achieve this, we treated in vitro pure cholangiocytes from 1-wk-old BDL rats at 37°C for 90 min (14, 23) with 1) 0.2% BSA (basal) or 2) quinelorane (100 μM) in 0.2% BSA. Subsequently, we evaluated protein expression for PKC-γ in a triton-soluble (containing cytoplasm and membrane) fraction and a triton-insoluble (containing cytoskeleton) fraction by immunoblots (23). Both triton-soluble and triton-insoluble fractions were obtained from cholangiocytes as described (14, 23).
Is Quinelorane-Induced Increase in PKC-γ Protein Expression Associated with Downregulation of PKA Activity in Cholangiocytes?
We evaluated in pure cholangiocytes whether increased expression of PKC-γ (induced by in vitro treatment with 100 μM quinelorane) causes inhibition of secretin-stimulated PKA activity, which regulates ductal secretion (13). PKA assay was performed according to the manufacturer's instructions by using PepTag assay protein kinase kits (Promega, Madison, WI) for PKA. Purified cholangiocytes (5 × 106) from BDL rats were stimulated at 37°C with 1) 0.2% BSA (basal) for 30 min; 2) secretin (30 min at 100 nM) with 0.2% BSA; 3) quinelorane (30 min at 100 μM) with 0.2% BSA; 4) quinelorane (10 min at 100 μM) before stimulation with secretin (30 min at 100 nM) with 0.2% BSA; or 5) chelerythrine, BAPTA-AM, H7, or rottlerin (10 min each) before treatment with quinelorane (100 μM; 10 min) followed by secretin (100 nM; 30 min) stimulation with 0.2% BSA. After stimulation of cholangiocytes with the appropriate agonist/antagonist, samples were centrifuged at 1,400 rpm for 5 min. Samples were resuspended in 1 ml of a lysis buffer (in mM: 20 Tris · HCl, pH 7.4, 150 NaCl, 5 EDTA, pH 8.0, and 1 PMSF with 10 μM leupeptin and 10 μM aprotinin). The samples were then sonicated with a cell membrane disrupter for three 30-s bursts. Samples were centrifuged at 2,000 rpm for 5 min, and the supernatant was saved. The supernatant was analyzed with the PepTag assay for nonradioactive detection of PKA activity from Promega. For PKA, the positive control was performed by using the same assay conditions as the samples with the positive control containing 10 ng of PKA provided with the kit. The negative controls (deionized water) were performed by using the same assay conditions as the samples. Phosphorylated and nonphosphorylated peptide bands were visualized on a 0.8% agarose gel. Phosphorylated peptide bands were quantitated by scanning densitometry by using the ChemiImager 4000. Densitometric values were normalized on the basis of cell number.
All data are expressed as means ± SE. Differences between groups were analyzed by Student's t-test when two groups were analyzed or by ANOVA if more than two groups were analyzed.
Cholangiocytes Express D2 Dopaminergic Receptors
Immunoblotting analysis shows that a band (migrating at 55 kDa) (16) for the D2 dopaminergic receptor was expressed by cholangiocytes from both normal and BDL rats (Fig.1 A), whereas D1 and D3 dopaminergic receptors were absent (Fig. 1 A). Immunohistochemistry shows positive staining for D2 dopaminergic receptors in liver sections (Fig. 1 B) and in cholangiocyte smears from BDL rats (Fig. 1 C). Negative controls for D2 dopaminergic receptors for sections and cells from BDL rats are shown in Fig. 1, B and C, respectively. At the subcellular level, we found that a band (migrating at 55 kDa) (16) for the D2 dopaminergic receptor was present in the basolateral but not the apical domain of cholangiocytes (Fig.1 D).
Consistent with the concept that cholangiocytes contain functional D2 dopaminergic receptors, quinelorane increased intracellular Ins(1,4,5)P3 levels [0.37 ± 0.059 vs. 0.18 ± 0.018 (basal) pmol/1 × 106 cells, P< 0.05] (Table 1). Parallel with changes in Ins(1,4,5)P3 levels, quinelorane significantly (P < 0.05) increased [Ca2+]ilevels of cholangiocytes compared with its corresponding basal value (Table 1).
Quinelorane Inhibits Secretin-Stimulated Bile and Bicarbonate Secretion
Secretin increased bile flow and bicarbonate concentration and secretion compared with its corresponding basal value (Table2). When infused alone, the D2 dopaminergic receptor agonist quinelorane did not alter basal bile flow and bicarbonate concentration and secretion, but it decreased secretin-stimulated bile flow and bicarbonate concentration and secretion of BDL rats (Table 2). Consistent with the expression of D2 dopaminergic receptors in cholangiocytes, the inhibitory effect of quinelorane on secretin-stimulated bicarbonate-rich choleresis was abolished by eticlopride, a D2 dopaminergic receptor antagonist (Table2). A potential shortcoming of these in vivo results is that the effect of quinelorane on secretin-stimulated bile flow and bicarbonate secretion of BDL rats (Table 2) may be influenced by the in vivo vascular effects of the D2 dopaminergic receptor agonist quinelorane (53, 66). Eticlopride alone did not alter basal bile flow and bicarbonate concentration and secretion (Table 2). Due to the lack of expression of D1 and D3 dopaminergic receptors, neither (±)-SKF-82958 hydrobromide (a D1 dopaminergic receptor agonist) nor 7-OH-DPAT (a D3 dopaminergic receptor agonist) altered basal or secretin-stimulated bile flow and bicarbonate concentration and secretion (Table 2).
Quinelorane Inhibition of Secretin-Stimulated IBDU Lumen Expansion Is Blocked by BAPTA-AM, Chelerythrine, and H7 but not Rottlerin
Secretin increased the area of IBDU luminal spaces in IBDU preparations from BDL rats (Fig. 2). Quinelorane alone did not alter the area of IBDU luminal spaces (Fig.2 A). However, pretreatment of IBDU with quinelorane before the addition of secretin inhibited the stimulatory effect of secretin on the expansion of IBDU luminal spaces (Fig. 2,B–F). The inhibitory effect of quinelorane on secretin-induced ductal lumen expansion was abolished by pretreating purified IBDU with BAPTA-AM (Fig. 2 C), chelerythrine (Fig.2 D), and H7 (Fig. 2 E) but not rottlerin (Fig.2 F). A possible shortcoming of our studies is represented by the fact that the use of rottlerin excludes the role of PKC-δ but not of other calcium-independent PKC isoforms in quinelorane inhibition of secretin-stimulated ductal secretion.
Quinelorane Inhibition of Secretin-Stimulated cAMP Levels Is Blocked by BAPTA-AM, Chelerythrine, and H7 but not Rottlerin
Secretin increased intracellular cAMP levels of cholangiocytes from BDL rats (Fig. 3). Quinelorane alone did not alter basal cAMP levels but inhibited secretin-stimulated cAMP levels of cholangiocytes from BDL rats (Fig. 3). The inhibitory effect of quinelorane on secretin-stimulated cAMP levels was blocked by the D2 dopaminergic receptor antagonist eticlopride (Fig. 3). Consistent with the concept that the Ca2+-dependent PKC system regulates the effects of D2 dopaminergic receptor agonists on cholangiocyte cAMP levels, quinelorane inhibition of secretin-stimulated cAMP synthesis was ablated by BAPTA-AM, chelerythrine, and H7 but not rottlerin (Fig.3). Due to the lack of expression of D1 and D3 dopaminergic receptors, neither (±)-SKF-82958 hydrobromide (a D1 dopaminergic receptor agonist) nor 7-OH-DPAT (a D3 dopaminergic receptor agonist) altered basal or secretin-stimulated cAMP levels (results not shown).
Quinelorane Inhibition of Secretin-Stimulated Ductal Secretion Is Associated with Increased Protein Expression and Membrane Translocation of PKC-γ
Immunoblotting analysis shows that the protein for the Ca2+-dependent PKC (α, βI, βII, γ) and the Ca2+-independent novel (δ, ε, θ) and atypical (η, ζ) PKC isoforms was expressed by cholangiocytes from BDL rats (Fig.4 A). Quinelorane did not enhance protein expression for the Ca2+-dependent PKC-α, -βI, and -βII but increased PKC-γ protein expression (Fig.4 A). Quinelorane alone did not increase protein expression of the Ca2+-independent novel (δ, ε, θ) and atypical (η, ζ) PKC isoforms (Fig. 4 A).
Similar to that shown in other epithelia (14), PKC-γ protein expression in cholangiocytes was significantly higher in the triton-soluble fraction compared with the triton-insoluble fraction (Fig. 4 B). Quinelorane induces cytoskeleton-to-membrane distribution of PKC-γ, which was evidenced by increased PKC-γ protein expression only in the triton-soluble fraction (Fig.4 B). Consistent with PKC-γ translocation to cholangiocyte membranes, there was a corresponding loss of PKC-γ protein expression in the triton-insoluble fraction of cholangiocytes stimulated with quinelorane (Fig. 4 B).
Quinelorane-Induced Increase in PKC-γ Expression Is Associated with Downregulation of PKA Activity in Cholangiocytes
We evaluated whether quinelorane-induced increase in PKC-γ expression causes inhibition of secretin-stimulated PKA activity. We found that secretin increases PKA activity and that quinelorane inhibits basal and secretin-induced increases in PKA activity (Fig.5). The inhibitory effect of quinelorane on secretin-stimulated PKA activity was ablated by eticlopride, BAPTA-AM, chelerythrine, and H7 but not rottlerin (Fig. 5).
The present study shows that cholangiocytes express D2 (but not D1 and D3) dopaminergic receptors as demonstrated by immunohistochemistry in liver sections and both immunoblots and immunohistochemistry in purified cholangiocytes. Immunoblots show that the D2 dopaminergic receptor was present in the basolateral but not the apical domain of cholangiocyte membranes. Stimulation of D2 dopaminergic receptors with the D2/D3 dopaminergic receptor agonist quinelorane (27) induces 1) an increase in intracellular Ins(1,4,5)P3 and Ca2+ levels in purified cholangiocytes; 2) inhibition of secretin-stimulated bicarbonate-rich choleresis in BDL rats and secretin-induced fluid secretion in IBDU; and 3) inhibition of secretin-induced cAMP levels and secretin-stimulated PKA activity in purified cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion were blocked by the selective D2 dopaminergic receptor antagonist eticlopride (27, 62), supporting the concept that quinelorane acts only through D2 dopaminergic receptors in cholangiocytes. These findings indicate that cholangiocytes express functionally active D2 dopaminergic receptors, which are involved in the counterregulation of secretin-stimulated, bicarbonate-rich ductal choleresis. We evaluated the signal transduction pathways involved in quinelorane inhibition of secretin-stimulated ductal secretion by demonstrating that1) quinelorane effects are blocked by BAPTA-AM (a [Ca2+]i chelator), chelerythrine, and H7 (two PKC inhibitors) but not rottlerin (a PKC-δ inhibitor); 2) quinelorane effects on secretin-stimulated ductal secretion are associated with increased protein expression of the Ca2+-dependent PKC-γ but not the Ca2+-independent novel (δ, ε, θ) and atypical (η, ζ) PKC isoforms; and 3) quinelorane-induced increase in protein expression and cytoskeleton to membrane distribution of PKC-γ was associated with inhibition of secretin-stimulated PKA activity.
A number of studies (12, 34, 38, 49) have shown that the nervous system is involved in the regulation of cholangiocyte pathophysiology. We have demonstrated (12) that the cholinergic system regulates cholangiocyte secretory functions. By acting on M3 ACh receptor subtypes, ACh potentiates secretin-stimulated bicarbonate secretion through an intracellular pathway involving Ca2+, calcineurin, and calmodulin but not PKC (12). The final target of this pathway is adenylyl cyclase, whose activity is positively modulated, leading to amplification of secretin-induced cAMP levels (12). This results in marked stimulation of CFTR and Cl−/HCO exchanger activity, which leads to maximal excretion of bicarbonate in bile (12). Furthermore, surgical interruption of parasympathetic nerves (by vagotomy) of BDL rats decreased cholangiocyte cAMP levels and inhibits secretin-stimulated ductal secretion and cholangiocyte proliferation (38).
In this study, we demonstrated that dopaminergic innervation is involved in the counterregulation of secretin-stimulated choleresis. We first demonstrated in bile fistula BDL rats that quinelorane has no effect on basal bile flow and biliary bicarbonate concentration and secretion. However, quinelorane almost completely abolished the stimulatory effect of secretin on bile flow and biliary bicarbonate concentration and secretion. The inhibition of secretin-stimulated choleresis in bile fistula rats could be the consequence of an indirect systemic effect of the dopaminergic system as well as effect on acetylcholine or prolactin release (1, 53, 66, 67). For these reasons, we directly evaluated the effect of quinelorane on isolated IBDU from BDL rats and demonstrated that the expansion of luminal space induced by secretin was completely blocked by quinelorane, indicating a direct action on cholangiocytes by a specific interaction with D2 dopaminergic receptors. This finding was also confirmed in pure cholangiocytes, in which we found that quinelorane (but not D1 or D3 dopaminergic receptor agonists) blocked secretin-stimulated cAMP synthesis.
In IBDU and cholangiocytes, we evaluated the intracellular mechanisms involved in quinelorane inhibition of secretin-stimulated ductal secretion. We found that quinelorane increased intracellular cholangiocyte Ins(1,4,5)P3 and Ca2+ levels. In addition, by the use of the [Ca2+]i chelator BAPTA-AM, the PKC antagonists chelerythrine and H7, and the PKC-δ-specific antagonist rottlerin, we provided evidence that PKC-γ plays a role in quinelorane inhibition of secretin-stimulated ductal secretion. This is supported by previous findings showing that, in many different cell types, D2 dopaminergic receptors are coupled to Ca2+, Ins(1,4,5)P3, and PKC activation (17, 52, 60, 65). The intracellular mechanisms by which quinelorane inhibits secretin-stimulated ductal secretion was demonstrated in isolated cholangiocytes in which both cAMP levels and PKA activity induced by secretin were abolished by pretreatment with quinelorane. This is in keeping with the known functional role of D2, which is a Gi-coupled receptor whose activation or overexpression results in the inhibition of adenyl cyclase activity, as demonstrated in many different central and peripheral cells (25).
Although the involvement of the PKC pathway in the regulation of cell function is widely described in different cell types including cholangiocytes (10, 14, 23, 61, 68), a novel aspect of our findings is the demonstration that the dopaminergic agonist inhibits secretin-stimulated ductal secretion by increasing protein expression and inducing cytoskeleton-to-membrane distribution of PKC-γ. In support of the concept that different PKC isoforms can be activated differentially, we have shown (23) that gastrin inhibits cholangiocyte proliferation and secretin-stimulated ductal secretion through activation of PKC-α. Similarly, insulin ursodeoxycholate or tauroursodeoxycholate inhibition of secretin-stimulated choleresis is associated with activation of PKC-α (2, 42). Feeding of taurocholate and taurolithocholate to normal rats stimulates cholangiocyte proliferation and secretin-stimulated secretion through activation of PKC-α (10). The α1-adrenergic agonist phenylephrine increases secretin-stimulated ductal secretion through a Ca2+- and PKC-dependent amplification of adenylyl cyclase (39). The different cross-talk between intracellular Ca2+ and cAMP (which leads to stimulatory or inhibitory effects on secretin-stimulated ductal secretion) (2, 10, 12, 23, 38, 39,42) likely depends on the type of receptor [M3 acetylcholine (12), D2 dopaminergic, insulin (42), gastrin (23), or α1-adrenergic (39)] or transporter (e.g., Na+-dependent apical bile acid transporter) (2, 10) that is up- or downregulated. For example, whereas there is activation of intracellular Ca2+but not PKC (insensitivity to staurosporine) with acetylcholine (12), there is activation of intracellular Ins(1,4,5)P3 and Ca2+- and specific isoforms of PKC with quinelorane (PKC-γ), gastrin (PKC-α) (23), insulin (PKC-α) (42), taurocholate (PKC-α) (10), ursodeoxycholate (PKC-α) (2), or tauroursodeoxycholate (PKC-α) (2). Studies aimed to evaluate the type of PKC isoform activated by the α1-adrenergic receptor agonist phenylephrine are ongoing. These interactions may result in a different cross-talk between [Ca2+]i and specific adenylate cyclase isoforms, leading to inhibition or stimulation of adenylate cyclase and of secretin-stimulated ductal secretion.
Thus the dopaminergic system plays a role in the complex mechanisms regulating the secretory activities of the intrahepatic biliary epithelium. By opposing the cholinergic system (which potentiates secretin-stimulated ductal secretion and adenylate cyclase activity) (12), the dopaminergic system inhibits secretin-induced choleresis through inhibition of adenylate cyclase activity. Although both systems exert their functions through increased intracellular Ins(1,4,5)P3 and Ca2+, the cholinergic system acts via calmodulin and calcineurin but without recruitment of PKC (12), whereas the D2 dopaminergic system inhibits secretin-stimulated ductal secretion by increasing protein expression and inducing cytoskeleton-to-membrane distribution of PKC-γ. However, a possible shortcoming of our studies is that we would like to emphasize that PKC-γ may be not the only player in the regulation of dopaminergic effects on secretin choleresis and that other PKC isoforms (e.g., α, β1, β2, δ, ε, θ, η, and ζ), although decreased or unchanged by quinelorane, may have a role in dopaminergic regulation of secretin-stimulated ductal secretion. Our studies have pathophysiological implications, because modulation of the dopaminergic system may play an important role in the regulation of cholangiocyte proliferation and ductal secretion in cholestatic liver diseases. This concept is supported by studies showing that 1) ACh potentiates secretin-stimulated ductal bicarbonate secretion (12); 2) interruption of the parasympathetic system (by vagotomy) inhibits cholangiocyte proliferation and secretin-stimulated choleresis of BDL rats (38); and3) proliferating cholangiocytes acquire phenotypic features of neuroendocrine tissue and expresses the neural adhesion molecule (57).
We thank S. Ambrus for technical assistance with immunohistochemistry.
This work was supported by a Scott & White Hospital grant and the Texas A&M University System (to G. LeSage and G. Alpini), by a Scott & White Hospital grant (to S. Glaser), by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54208 (to G. LeSage), by a Veterans Affairs Merit Award and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58411 (to G. Alpini), and by MURST Grant MM06215421/2 progetto nazionale 2000 (to D. Alvaro).
Address for reprint requests and other correspondence: G. Alpini, Associate Professor, Internal Medicine and Medical Physiology, The Texas A&M University System, Health Sciences Center COM and Central Texas Veterans Health Care System, 702 SW H. K. Dodgen Loop, Temple, TX 76504 (E-mail:).
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First published December 27, 2002;10.1152/ajpgi.00302.2002
- Copyright © 2003 the American Physiological Society