Guinea-pig Common Bile Duct Research Articles

Cholecystokinin and cholecystokinin-A receptor: An attractive treatment strategy for biliary dyskinesia?

Biliary dyskinesia is a relatively common gastrointestinal disease that is increasing in incidence as living standards improve. However, its underlying pathogenesis remains unclear, hindering the development of therapeutic drugs. Recently, "Expression and functional study of cholecystokinin-A receptors on the interstitial Cajal-like cells of the guinea pig common bile duct" demonstrated that cholecystokinin (CCK) regulates the contractile function of the common bile duct through interaction with the CCK-A receptor in interstitial Cajal-like cells, contributing to improving the academic understanding of biliary tract dynamics and providing emerging directions for the pathogenesis and clinical management of biliary dyskinesia. This letter provides a brief overview of the role of CCK and CCK-A receptors in biliary dyskinesia from the perspective of animal experiments and clinical studies, and discusses prospects and challenges for the clinical application of CCK and CCK-A receptors as potential therapeutic targets.

Expression and functional study of cholecystokinin-A receptors on the interstitial Cajal-like cells of the guinea pig common bile duct.

Many studies have shown that interstitial Cajal-like cell (ICLC) abnormalities are closely related to a variety of dynamic gastrointestinal disorders. ICLCs are pacemaker cells for gastrointestinal movement and are involved in the transmission of nerve impulses. To elucidate the expression profile and significance of cholecystokinin-A (CCK-A) receptors in ICLCs in the common bile duct (CBD), as well as the role of CCK in regulating CBD motility through CCK-A receptors on CBD ICLCs. The levels of tyrosine kinase receptor (c-kit) and CCK-A receptors in CBD tissues and isolated CBD cells were quantified using the double immunofluorescence labeling technique. The CCK-mediated enhancement of the movement of CBD muscle strips through CBD ICLCs was observed by a muscle strip contraction test. Immunofluorescence showed co-expression of c-kit and CCK-A receptors in the CBD muscularis layer. Observations of isolated CBD cells showed that c-kit was expressed on the surface of ICLCs, the cell body and synapse were colored and polygonal, and some cells presented protrusions and formed networks adjacent to the CBD while others formed filaments at the synaptic terminals of local cells. CCK-A receptors were also expressed on CBD ICLCs. At concentrations ranging from 10-6 mol/L to 10-10 mol/L, CCK promoted CBD smooth muscle contractility in a dose-dependent manner. In contrast, after ICLC removal, the contractility mediated by CCK in CBD smooth muscle decreased. CCK-A receptors are highly expressed on CBD ICLCs, and CCK may regulate CBD motility through the CCK-A receptors on ICLCs.

Effects of trypsin, thrombin and proteinase-activated receptors on guinea pig common bile duct motility

Trypsin and thrombin activate proteinase-activated receptors (PARs), which modulate gastrointestinal motility. The common bile duct is exposed to many proteinases that can activate PARs, especially during infection and stone obstruction. We investigated PAR effects on common bile duct motility in vitro. Contraction and relaxation of isolated guinea pig common bile duct strips caused by PAR1, PAR2 and PAR4 agonists were measured using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of PAR1 and PAR2. Thrombin and two PAR1 peptide agonists, TFLLR-NH2 and SFLLRN-NH2, evoked moderate relaxation of the carbachol-contracted common bile duct in a concentration-dependent manner. Trypsin and three PAR2 peptide agonists, 2-furoyl-LIGRLO-NH2, SLIGKV-NH2 and SLIGRL-NH2, generated moderate to marked relaxation as well. The existence of PAR1 and PAR2 mRNA in the common bile duct was identified by RT-PCR. Moreover, two PAR4-selective agonists, AYPGKF-NH2 and GYPGQV-NH2, produced relaxation of the common bile duct. In contrast, all PAR1, PAR2 and PAR4 inactive control peptides did not elicit relaxation. This indicates that PAR1, PAR2 and PAR4 mediate common bile duct relaxation. The thrombin, TFLLR-NH2, trypsin, and AYPGKF-NH2-induced responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. Our findings provide the first evidence that PAR1 and PAR2 mediate whereas agonists of PAR4 elicit relaxation of the guinea pig common bile duct. Trypsin and thrombin relax the common bile duct. PARs may play an important role in the control of common bile duct motility.

Proteinase-Activated Receptor-1 (PAR1), PAR2 and PAR4 Mediate Relaxation of Guinea-Pig Common Bile Duct

Proteinase-Activated Receptor-1 (PAR1), PAR2 and PAR4 Mediate Relaxation of Guinea-Pig Common Bile Duct

Healing Process of the Guinea Pig Common Bile Duct after End-to-End Anastomosis: Pathological Evaluation after 6 Months

Background: The healing process of an injured bile duct is always associated with stricture formation, the specific mechanism of which remains unclear. Our objective in this study was to establish a guinea pig common bile duct end-to-end anastomosis (EEA) model after total transection of the common bile duct and to assess the postoperative healing process after 6 months. Materials and Methods: Thirty-two male guinea pigs were randomly divided into 4 groups with or without operation (a control group without operation and 3 groups examined 2, 3 and 6 months after biliary EEA reconstruction, respectively). Histological, immunohistochemical (proliferating cell nuclear antigen, α-smooth muscle actin, h-caldesmon, and Bcl-2 expression) and ultrastructural characteristics were examined and evaluated. Results: Myofibroblasts (MFBs) were differentiated from smooth muscle cells by α-SMA and h-caldesmon staining. Gross inspection of operated bile ducts revealed coarctation formation at the anastomotic stoma. Histological and immunohistochemical examinations showed significant inflammatory reaction, the presence of MFBs, epithelial proliferation and glandular element hyperplasia. Bcl-2 expression decreased in groups 2 and 3, indicating an epithelial self-protecting mechanism. Conclusions: A guinea pig common bile duct EEA model was successfully established. Glandular elements, bile duct epithelial cells and MFBs all played crucial roles in the bile duct healing process. It seems important to elucidate the pathomechanisms of these components for restoring bile duct physiological function.

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct.

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct.

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

Morpho-functional evidence for tachykinins as major non-cholinergic excitatory transmitters in the guinea pig common bile duct (CBD)

Morpho-functional evidence for tachykinins as major non-cholinergic excitatory transmitters in the guinea pig common bile duct (CBD)

The effects of dopaminergic and adrenergic drugs on twitch response of guinea-pig common bile duct

1. 1. Effects of various doses of bromocriptine and apomorphine, dopamine receptor agonists, on electrically evoked contractile responses of guinea pig common bile duct (CBD) were investigated in this study. 2. 2. Bromocriptine and apomorphine produced a concentration-dependent reduction of the twitch heights. IC 50 values for bromocriptine and apomorphine were 2.75±0.715 × 10 −6M, and 1.69±0.11 × 10 −5M, respectively. 3. 3. Pretreatment with sulpiride, 0.1 and 1.0 μM, prevented bromocriptine- and apomorphine-induced inhibition. pA 2 values of sulpiride against bromocriptine and apomorphine were 8.26 and 6.29, respectively. 4. 4. Clonidine, 0.1–100 μM, produced 49.3±2.5% inhibition of twitch responses. Its effect was partially antagonized by 1 μM of yohimbine. 5. 5. It would appear that both dopamine D 2- and α 2-adrenergic presynaptic receptors are present on the guinea-pig CBD cholinergic nerve.

Functional Evidence for the Presence of &amp;beta;&lt;sub&gt;3&lt;/sub&gt;-Adrenoceptors in the Guinea Pig Common Bile Duct and Colon

To determine the existence of beta 3-adrenoceptors in functional assays in isolated preparations for which data are lacking, we compared the effects of SR 58611A, a selective beta 3-adrenoceptor agonist, and isoprenaline in the guinea pig common bile duct, distal colon and urinary bladder. SR 58611A and isoprenaline relaxed the common bile duct (EC50: 6.85 and 0.41 mumol/l, respectively). The effect of SR 58611A was resistant to CGP 20712A, ICI 118551, propranolol and tetrodotoxin, but was antagonized by alprenolol (pA2 = 6.86), while the effect of isoprenaline was antagonized by CGP 20712A, ICI 118551, propranolol and alprenolol (pA2 = 7.04, in the presence of propranolol to saturate beta 1- and beta 2-adrenoceptors). In colonic preparations, SR 58611A and isoprenaline relaxed circular muscle strips (EC50: 5.48 and 0.49 mumol/l, respectively). The effect of SR 58611A was resistant to CGP 20712A, ICI 118551, propranolol and tetrodotoxin, but was antagonized by alprenolol (pA2 = 7.01). The effect of isoprenaline was resistant to CGP 20712A, but was antagonized by ICI 118551, propranolol and alprenolol (pA2 = 6.88, in the presence of propranolol). In urinary bladder strips, SR 58611A had no effect, whereas isoprenaline reduced resting tone (EC50:0.87 mumol/l), an effect antagonized by alprenolol (pA2 = 8.14). These data provide functional evidence for the presence of beta 3-adrenoceptors in the guinea pig common bile duct and colon, but not in the urinary bladder. At the concentrations used, the effect of SR 58611A was probably mediated solely by activation of beta 3-adrenoceptors located on smooth muscle cells, whereas the effects of isoprenaline were due to beta 3- and also to beta 1-and/or beta 2-adrenoceptor activation.

Microangioarchitecture of the guinea pig common bile duct and duodenal papilla: A scanning electron and light microscopic study

The microvascular pattern of the duodenal papilla is unknown. Since the duodenal papilla is located in the transition zone between the stomach and duodenum, and because it regulates bile transfer into the duodenum, a particular microangioarchitecture can be expected. Therefore, we examined the microvasculature of the papilla using guinea pigs as a model. The microvascularization of the duodenal papilla and common bile duct was studied in 26 adult guinea pigs (Cavia porcellus), using scanning electron microscopy of microvascular corrosion casts and critical point dried specimens, and light microscopy of tissue sections. The duodenal papilla is located in the cranial portion of the duodenum, approximately 5 mm beyond the pyloric valve. At the most luminal aspect of the cast papilla, ring-shaped capillaries, resembling those of the cast gastric mucosa, are present. Deeper parts of the papilla are provided with villi. Subepithelial capillaries of the papilla are 15 microns thick in average. These capillaries have a dual blood supply either via the straight long arterioles arising from the submucosa or by the pericryptal capillaries. The common bile duct comprises numerous mucoid glands with their pits surrounded by ring-shaped capillaries in corresponding casts. The special arrangement of different capillary patterns, together with their luminal size and the dual blood supply, favor their protective role from the gastric chyme.

Inhibition of the cholinergic twitch response in guinea-pig common bile duct: an evaluation of the effects of noradrenaline, clonidine and fentanyl.

Presynaptic inhibitory effects of noradrenaline, clonidine and fentanyl on twitch contractions evoked by transmural electrical stimulation were investigated in guinea-pig common bile duct (CBD). Antagonism of the response to fentanyl by naloxone and to noradrenaline and clonidine by yohimbine confirmed the involvement of presynaptic opiate and alpha 2-adrenoceptors respectively. Clonidine caused desensitization of presynaptic alpha 2-adrenoceptors. No cross desensitization between clonidine and fentanyl was observed. Furthermore, partial agonistic activity of clonidine at presynaptic alpha 2-adrenoceptors was also observed by the antagonism of noradrenaline response in this preparation.

Comparison of various spasmolytic drugs on guinea-pig isolated common bile duct

Guinea-pig common bile duct preparations were used to quantify the spasmolytic potency of N-butylscopolamine (NBS), papaverine, gallopamil and nifedipine. A method was developed which allowed the measurement of intraluminal pressure changes in vitro. Barium chloride, carbachol and a solution with elevated potassium concentrations were used to stimulate smooth muscles. Concentration-response relationships for the spasmogens as well as for the spasmolytic drugs were evaluated in a cumulative manner. Furthermore, non-cumulative concentration-response curves were constructed for carbachol in the absence and presence of NBS. The -log EC50-values of the spasmogens were found to be 3.28 +/- 0.08 (BaCl2), 6.46 +/- 0.07 (carbachol) and 1.31 +/- 0.08 (KCl), respectively. The Emax values of carbachol and potassium were comparable, and were twice as high as the Emax of BaCl2. Papaverine was less potent than the calcium antagonists gallopamil and nifedipine, but proved capable of completely suppressing elevated muscular tone of the common bile duct preparation, independent of the stimulus used. NBS showed a high potency in suppressing only a carbachol-induced pressure increase, while it was rather ineffective when BaCl2 or a high potassium solution was used as the spasmogen. The concentration-response-curve for carbachol was shifted to the right in a parallel manner by NBS. Only a slight depression of Emax was observed. From the results it is concluded that NBS acts mainly as a muscarinic receptor antagonist. The high potency found for the calcium antagonists, gallopamil and nifedipine, in this model may indicate a possible role for these compounds in the treatment of biliary colic.

Effects of cholecystokinin octapeptide on contractile motility of guinea pig common bile duct.

Cholecystokinin octapeptide (CCK-8)-induced contraction and [3H]acetylcholine ([3H]-ACh) release from the longitudinal muscle strip of the guinea pig common bile duct were studied by a standard organ bath method and superfusion system. CCK-8 (10(-7) M) caused a biphasic contraction, a rapid phasic contraction followed by a fading tonic contraction. Hexamethonium, propranolol, and phentolamine had no effects on CCK-8-evoked contraction. Atropine or tetrodotoxin significantly reduced CCK-8-evoked initial phasic contraction and produced a rightward shift of the dose-response curve for CCK-8 (10(-10)-3 x 10(-7) M). CCK-8 provoked a significant [3H]ACh release in a dose-dependent manner, which was almost completely abolished by tetrodotoxin or Ca2+-free medium. These results suggest that CCK-8 receptors are present not only on the smooth muscle cells but also on the intramural cholinergic nerve cell bodies of the guinea pig common bile duct.

The Effect of Meperidine on the Guinea Pig Extrahepatic Biliary Tract

The effect of meperidine on the mechanical activity of isolated guinea pig common bile duct (CBD) and gallbladder was studied in vitro. The effect was found to consist of inhibitory and excitatory phases. The inhibitory phase, characterized by a decrease in the response of the CBD and gallbladder to carbachol and electrical stimulation, was seen with concentrations of 10(-6) M or higher. The excitatory phase, seen at high concentrations of meperidine (5 X 10(-5)-10(-4) M), involved an increase in spontaneous contractions of CBD and in the tone of the gallbladder. Neither effect of meperidine was affected by 0.5 X 10(-5) M naloxone. These data indicate that meperidine acts on the biliary tract by a mechanism different from that associated with the effect of morphine.