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

Abstract

Acetylcholine potentiates secretin-stimulated ductal secretion by Ca 2؉ -calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)-␥. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of ␣-1A/1C, -1␤ and ␤-1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the ␣-1 and ␤-1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP 3 and Ca 2؉ levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC-␣ inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. ␣-1 and ␤-1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Gö6976. S ecretin stimulates ductal HCO 3Ϫ secretion 1-5 by interacting with receptors expressed only by rat cholangiocytes. 6 This interaction induces an increase in intracellular cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) levels, 4,5,7 which leads to the opening of cystic fibrosis transmembrane conductance regulator channels, 8 followed by activation of the apically located Cl Ϫ /HCO 3 Ϫ exchanger 9 with secretion of HCO 3 Ϫ into bile. 1 Acetylcholine increases intracellular Ca 2ϩ ([Ca 2ϩ ] i ) levels and oscillations in rat cholangiocytes due to influx of extracellular Ca 2ϩ and mobilization of thapsigarginsensitive [Ca 2ϩ ] i stores. 10 It also increases secretin-stimulated ductal HCO 3 Ϫ secretion via Ca 2ϩ -calcineurin mediated modulation of adenylyl cyclase. 9 The D2 dopaminergic receptor agonist quinelorane inhibits secretinstimulated ductal secretion via activation of the Ca 2ϩ -dependent protein kinase C (PKC)-␥. 11 Adrenergic nerve stimulation causes a decrease in bile flow in the isolated perfused rat liver via interaction with ␣-1 adrenergic receptors. 12 In isolated perfused rat liver, adrenaline induces

Follicle-stimulating hormone increases cholangiocyte proliferation by an autocrine mechanism via cAMP-dependent phosphorylation of ERK1/2 and Elk-1

“…3A). In agreement with previous studies (24,38), secretin significantly increased cAMP levels of cholangio- Table 2. Original magnification ϫ20.…”

γ-Aminobutyric Acid Inhibits Cholangiocarcinoma Growth by Cyclic AMP–Dependent Regulation of the Protein Kinase A/Extracellular Signal-Regulated Kinase 1/2 Pathway

“…/IP 3 -related pathways has been described in cholangiocytes (40,41). We propose that cAMP-and Ca 2+ /IP 3 -dependent pathways are both activated in GABA-treated cholangiocarcinoma cells and have a synergistic effect in the inhibition of cholangiocarcinoma proliferation.…”

Knockout of α-calcitonin gene-related peptide reduces cholangiocyte proliferation in bile duct ligated mice

“…24 Protein loading was normalized by immunoblots for b-actin. 30 Images of the bands were obtained using a phospho-imager, Storm 860, Amersham Biosciences (Piscataway, NJ, USA) and subsequently densitometry was carried out using Scion Imaging Software (Frederick, MD, USA).…”

“…We evaluated the in vitro effects of a-and b-CGRP (10 À9 M at RT for 5 min) on cAMP and IP 3 levels of cholangiocytes from BDL mice by RIA kits. 30 We measured the effects of a-and b-CGRP (10 À9 M at 371C for 90 min) on the activity of PKA in WT BDL mouse cholangiocytes with the PepTag assay for nonradioactive detection of PKA according to the manufacturer's protocol (Promega, Madison, WI, USA). 24,37 Finally, we measured the effect of a-and b-CGRP (10 À9 M at 371C for 90 min) on the activity and phosphorylation of CREB in purified WT BDL cholangiocytes in the absence/ presence of pretreatment with H-89 (PKA inhibitor, 10 mM).…”