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Long-chain fatty acids activate the free fatty acid receptor 1 (FFA1) and FFA4. In the gastrointestinal system, FFA1 and FFA4 have been found in the pancreas and intestine. Fatty food and decreased lower esophageal sphincter (LES) motility are associated with gastroesophageal reflux disease. The effect of long-chain fatty acids on the esophageal motility is unknown. The purpose of this study is to investigate the effects of long-chain fatty acids on the porcine LES motility ex vivo using isometric transducers. In endothelin 1-precontracted porcine LES strips, the FFA1 selective agonists, fasiglifam, TUG424, and GW9508, caused marked relaxations in a concentration-dependent manner. The relative efficacies to elicit relaxation were GW9508 > TUG424 > fasiglifam in both clasp and sling strips. In contrast, the FFA4 specific agonists, TUG891 and GSK137647, produced mild relaxations. In addition, the endogenous FFA1 agonist DHA caused a mild relaxation whereas GW1100, an FFA1 antagonist, inhibited GW9508 induced relaxation of the porcine LES clasp and sling muscle. Both real-time PCR and immunohistochemistry revealed that FFA1 and FFA4 were expressed in the porcine LES. Real-time PCR analysis showed that the FFA4 expression was much lower than FFA1. Taken together, long-chain fatty acid receptor agonists elicit relaxation of the porcine LES. FFA1 might influence LES motility. [ABSTRACT FROM AUTHOR]

Objective Estrogen interacts with a membrane receptor, G protein-coupled estrogen receptor (GPER). It was reported that 17β-estradiol was able to inhibit contraction of the human colon and cause relaxation of the guinea pig gallbladder, however, the involvement of GPER was not clarified. The aim of the present study was to investigate the effect of estrogen on human gallbladder motility and the possible role of GPER. Materials and Methods Relaxation of human gallbladder strips were measured using isometric transducers. Expression of GPER was evaluated by reverse transcription polymerase chain reaction (PCR), real-time PCR, and immunohistochemistry. Results In human gallbladder strips, 17β-estradiol and G-1 elicited marked and rapid relaxation, whereas tamoxifen produced mild concentration-dependent relaxation. The relative efficacies to cause relaxation were as follows: 17β-estradiol = G-1 > tamoxifen. The relaxant response of 17β-estradiol was not attenuated by tetrodotoxin or conotoxin GVIA. This implies that nerve stimulation was not involved in the 17β-estradiol-induced gallbladder relaxation. Analysis by reverse transcription PCR and real-time PCR showed that GPER was expressed in the human gallbladder. Further analysis by immunohistochemistry revealed that GPER was expressed in the gallbladder muscle. This suggests that 17β-estradiol relaxes the human gallbladder via GPER. Conclusion These results demonstrate for the first time that 17β-estradiol and GPER agonist G-1 cause relaxation of the human gallbladder, probably through GPER. Estrogen might play an important role in the control of human gallbladder motility. [ABSTRACT FROM AUTHOR]

Objectives The internal anal sphincter (IAS) plays an important role in maintaining continence. Atrial natriuretic peptide (ANP) could relax the IAS. The purpose of the present study was to investigate the roles of natriuretic peptide receptors (NPRs) in the IAS. Materials and methods Relaxation of isolated rat IAS strips caused by natriuretic peptides was measured using isometric transducers. Expression of NPRs was evaluated by reverse transcription–polymerase chain reaction (PCR), immunohistochemistry and real-time PCR. Results In the rat IAS, C-type natriuretic peptide (CNP) produced a marked and concentration-dependent relaxation while ANP caused a mild relaxation. By contrast, brain natriuretic peptide, dendroaspis natriuretic peptide, and des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4–23) amide did not generate relaxation. CNP was much more effective than ANP and brain natriuretic peptide in stimulating the relaxation, indicating that NPR-B mediates IAS relaxation. The CNP-induced relaxation was inhibited by the protein kinase G inhibitor Rp-8CPT-cGMPS and potassium channel blocker charybdotoxin but not by protein kinase A inhibitor Rp-cAMPS. This suggests the involvement of cGMP and calcium-activated potassium channels in the CNP-induced IAS relaxation. Reverse transcription PCR and immunohistochemistry revealed the existence of NPR-B in the rat IAS. Furthermore, real-time PCR identified abundant expression of CNP and NPR-B in the rat IAS. Conclusion CNP causes relaxation of the IAS via NPR-B, cGMP, and potassium channel pathways in rats. CNP might regulate IAS motility. NPR-B is a potential therapeutic target in anorectal motility disorders. [ABSTRACT FROM AUTHOR]

Abstract: Activation of proteinase-activated receptor-1 (PAR1) and PAR2 stimulates contraction of the rat but relaxation of the guinea pig colon. The aim of the present study was to investigate PAR effects on internal anal sphincter (IAS) motility. We measured relaxation of isolated muscle strips from the guinea pig IAS caused by PAR agonists using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the existence of PAR. In the IAS, thrombin and PAR1 peptide agonists TFLLR-NH2 and SFLLRN-NH2 evoked moderate to marked relaxation in a concentration-dependent manner. In addition, trypsin and PAR2 peptide agonists 2-furoyl-LIGRLO-NH2, SLIGRL-NH2 and SLIGKV-NH2 produced relaxation. In contrast, both PAR1 and PAR2 inactive control peptides did not elicit relaxation. Furthermore, the selective PAR1 antagonist vorapaxar and PAR2 antagonist GB 83 specifically inhibited thrombin and trypsin-induced relaxations, respectively. RT-PCR revealed the presence of PAR1 and PAR2 in the IAS. This indicates that PAR1 and PAR2 mediate the IAS relaxation. The relaxant responses of TFLLR-NH2 and trypsin were attenuated by N(omega)-Nitro-L-arginine (L-NNA), indicating involvement of NO. These responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. On the other hand, PAR4 agonists GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2 did not cause relaxation or contraction, suggesting that PAR4 is not involved in the sphincter motility. Taken together, these results demonstrate that both PAR1 and PAR2 mediate relaxation of the guinea pig IAS through the NO pathway. PAR1 and PAR2 may regulate IAS tone and might be potential therapeutic targets for anal motility disorders. [Copyright &y& Elsevier]

Abstract: Objectives: Bile acids inhibit contraction of the gallbladder and intestine through the G-protein-coupled bile acid receptor (GPBAR). Perfusion of the esophagus with bile and acid (HCl) decreases lower esophageal sphincter (LES) pressure. The effects of bile acids on LES motility are not clear. The purpose of the present study was to investigate the effects of bile acids on LES motility in vitro. Materials and Methods: We measured the relaxation of muscle strips isolated from guinea pig and rat LES caused by bile acids or the selective GPBAR agonist RG-239. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine GPBAR expression in rat LES. Results: In carbachol-contracted guinea pig LES strips, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and cholic acid (CA) produced relaxation in a concentration-dependent manner. The relative potency was LCA ≥ DCA > CDCA > CA. RG-239 also induced concentration-dependent relaxation. This suggests that GPBAR mediates relaxation in guinea pig LES. DCA-induced LES relaxation was attenuated by the protein kinase A inhibitor KT 5720 but not by the protein kinase G inhibitor KT 5823 or the NO synthase inhibitor L-NNA. This suggests the involvement of cAMP. Separately, in endothelin-1-contracted rat LES strips, bile acids induced relaxation. The relative potency was LCA = DCA > CDCA > CA. RT-PCR revealed GPBAR expression in rat LES. Conclusion: These results demonstrate that bile acids cause relaxation of guinea pig and rat LES through GPBAR. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Background: Three ghrelin gene-derived peptides, acyl ghrelin, des-acyl ghrelin and obestatin, are implicated in the regulation of feeding, adipogenesis and glucose metabolism, as well as gut motility. The effects of ghrelin gene-derived peptides on lower esophageal sphincter motility, however, were unknown. Methods: We investigated the influence of acyl ghrelin, des-acyl ghrelin and obestatin on lower esophageal sphincter motility using the in vitro isolated muscle strip method. Both resting and carbachol-stimulated rat lower esophageal sphincters were tested. Results: Acyl ghrelin, des-acyl ghrelin and obestatin did not alter the resting tone of the lower esophageal sphincter, whereas carbachol increased the force of the muscle strips. Similarly, acyl ghrelin, des-acyl ghrelin and obestatin did not modify carbachol-induced stimulation of the lower esophageal sphincter, whereas papaverine significantly decreased the force of the muscle strips. Conclusion: Since the contractile effects of carbachol and relaxant responses of papaverine on the lower esophageal sphincter were confirmed, our results demonstrate that acyl ghrelin, des-acyl ghrelin and obestatin have no direct effects on rat lower esophageal sphincter motility in vitro. [Copyright &y& Elsevier]

Abstract: Atrial natriuretic peptide (ANP) causes relaxation in the opossum lower esophageal sphincter. The effects of dendroaspis natriuretic peptide (DNP) and other natriuretic peptides in the lower esophageal sphincter were not known. We measured the relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by DNP, ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and a natriuretic peptide receptor-C agonist des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4–23) amide (cANF(4–23)) in vitro. In resting strips of the guinea pig lower esophageal sphincter DNP and BNP caused marked relaxations. Furthermore, in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips, DNP caused marked and BNP caused moderate, concentration-dependent relaxations. ANP as well as CNP caused mild relaxations. In contrast, cANF(4–23) did not cause relaxation. The relative potencies for natriuretic peptides to cause relaxation were DNP>BNP>ANP>=CNP in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips. The DNP and BNP-induced relaxations were not affected by tetrodotoxin or atropine, suggesting that the natriuretic peptide-induced response was not neutrally mediated. In conclusion, these results demonstrate that natriuretic peptides cause the relaxation of the guinea pig lower esophageal sphincter. DNP is the most potent natriuretic peptide to cause lower esophageal sphincter relaxation, which might be mediated by natriuretic peptide receptor-A or a novel DNP-selective natriuretic peptide receptor. [Copyright &y& Elsevier]

Abstract: Objective: Leukotriene D4 (LTD4) causes contraction of the cat lower esophageal sphincter. The effects of leukotrienes in the guinea pig lower esophageal sphincter and the cysteinyl leukotriene receptor (CysLT) subtype that mediate this contraction are not known. The purpose of the present study was to characterize the CysLT receptors in the guinea pig lower esophageal sphincter. Materials and Methods: We measured the contractions of transverse strips from the guinea pig lower esophageal sphincter caused by cysteinyl leukotrienes, LTC4, LTD4 and LTE4, and the dihydroxy leukotriene, LTB4. We also measured LTD4-induced contraction inhibited by CysLT receptor antagonists, tetrodotoxin and atropine. Results: In the guinea pig lower esophageal sphincter strips, LTC4, LTD4 and LTE4, but not LTB4, caused concentration-dependent contractions. The relative potencies for cysteinyl leukotrienes to cause contraction were LTD4 = LTC4 > LTE4. LTE4 was a partial agonist. The LTD4-induced contraction was inhibited by two selective CysLT1 receptor antagonists, montelukast and zafirlukast, and by the dual CysLT1 and CysLT2 receptor antagonist BAY u9773. The combination of both montelukast and BAY u9773 did not potentiate the inhibition caused by montelukast alone. These findings indicate that CysLT1 mediates the contraction in the lower esophageal sphincter. Furthermore, LTD4-induced contraction was not affected by tetrodotoxin or atropine, suggesting a direct effect. Conclusion: These results demonstrate that cysteinyl leukotrienes, but not the dihydroxy leukotriene LTB4, cause contraction of the guinea pig lower esophageal sphincter. The CysLT1 receptor mediates this contraction. [Copyright &y& Elsevier]

Abstract: Natriuretic peptides have been demonstrated to cause relaxation of the human gallbladder muscle through interaction with natriuretic peptide receptor-B (NPR-B/NPR2). Effects of natriuretic peptides in the human esophageal muscle were unknown. To investigate the effects of natriuretic peptides in the human esophagus, we measured relaxation of muscularis mucosae strips isolated from the human esophagus caused by C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. In endothelin-1 or carbachol-contracted mucosal muscle strips, CNP caused moderate, sustained and concentration-dependent relaxation. BNP caused a very mild relaxation whereas ANP and cANP(4-23) did not cause any relaxation. CNP was much more potent than BNP and ANP in causing relaxation. These suggest the existence of NPR-B mediating relaxation. The CNP-induced relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted esophageal strips and not by tetrodotoxin in carbachol-contracted strips, indicating a direct effect of CNP on the human esophageal muscularis mucosae. Taken together, these results demonstrate that natriuretic peptides cause relaxation of the muscularis mucosae of the human esophagus and suggest that the relaxation is through interaction with NPR-B. Natriuretic peptides may play an important role in the control of human esophageal motility. [Copyright &y& Elsevier]

Abstract: To characterize endothelin (ET) receptors in the lower esophageal sphincter, we measured contraction of transverse strips from the guinea-pig lower esophageal sphincter induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the lower esophageal sphincter circular muscle. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 or ET-2 alone did not cause contraction or relaxation in resting strips. However, in carbachol precontracted lower esophageal sphincter strips, ET-1 and ET-2 caused marked, tetrodotoxin-insensitive relaxation. The ET-1-induced relaxation was abolished by BQ-123, an ETA receptor selective antagonist, but not inhibited by BQ-788, an ETB receptor selective antagonist. ET-3 and sarafotoxin S6c, a selective ETB receptor agonist, did not cause relaxation in the carbachol precontracted muscle strips. These clearly indicate that ETA receptors mediate relaxation. On the other hand, ET-3 and sarafotoxin S6c caused tetrodotoxin and atropine-insensitive contraction in the resting strips. The sarafotoxin S6c-induced contraction was inhibited by BQ-788, but not by BQ-123. Furthermore, ET-1 and ET-2 caused contraction of the resting muscle strips after pretreatment with BQ-123. This ET-1-induced contraction was also inhibited by BQ-788. Taken together, these indicate that ETB receptors mediate contraction. Autoradiography localized 125I-ET-1 binding to the lower esophageal sphincter circular muscle as well as longitudinal muscle of the esophagus. Binding of 125I-ET-1 to cell membranes prepared from the circular smooth muscle was saturable and specific. Analysis of dose–inhibition curves indicated the presence of two classes of receptors, ETA and ETB receptors. These results demonstrate that the guinea-pig lower esophageal sphincter possesses ETA receptors mediating relaxation and ETB receptors mediating contraction. [Copyright &y& Elsevier]

Endothelin (ET) causes contraction of the muscularis mucosae in the guinea pig esophagus, but its role in the human esophagus remains unknown. To investigate effects of ET in the human esophagus, we measured contraction of isolated human esophageal muscularis mucosae strips caused by ET related peptides and binding of 125I-ET-1 to cell membranes prepared from the human esophageal muscularis mucosae. Autoradiography demonstrated specific binding of 125I-ET-1 to the muscularis mucosae and muscularis propria (muscularis externa) of the human esophagus. ET-1 caused tetrodotoxin and atropine-insensitive contraction of muscularis mucosae strips. In terms of the maximal tension of contraction, ET-1 and ET-2 were equal in efficacy. The relative potencies for ET related peptides to cause contraction were ET-1=ET-2>ET-3>sarafotoxin S6c (SX6c), an ETB receptor agonist. ET-1 caused contraction was mildly inhibited by BQ-123, an ETA receptor antagonist, and not by BQ-788, an ETB receptor antagonist. It was moderately inhibited by the combination of both antagonists, indicating synergistic inhibition. Furthermore, desensitization to SX6c with SX6c pretreatment failed to abolish the contractile response to ET-1, which was completely inhibited by BQ-123. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose–inhibition curves demonstrated the presence of ETA and ETB receptors. This study demonstrates that, the muscularis mucosae of the human esophagus, similar to that of the guinea pig esophagus, possesses both ETA and ETB receptors mediating muscle contraction. [Copyright &y& Elsevier]

Endothelin (ET) causes contraction of the gallbladder. To investigate effects of ET in the common bile duct, we measured contraction of longitudinal muscle strips from guinea pig common bile ducts induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the common bile duct. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 caused tetrodotoxin and atropine-insensitive contraction. In terms of maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy. However, sarafotoxin S6c, a selective ETB receptor agonist, caused only a negligible contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. The ET-1-induced contraction was inhibited by BQ-123, an ETA-receptor-selective antagonist, but not by BQ-788, an ETB-receptor-selective antagonist. In addition, the combination of both antagonists, BQ-123 and BQ-788, inhibited ET-1 induced contraction but did not potentiate the inhibition caused by BQ-123 alone. These indicate that ETA but not ETB receptors mediate the contraction. Autoradiography localized 125I-ET-1 binding to the smooth muscle layer. Binding of 125I-ET-1 to the smooth muscle cell membranes was saturable and specific. Analysis of dose–inhibition curves indicated the presence of ETA and ETB receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ETA and ETB receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ETA receptor mediates the contraction. [Copyright &y& Elsevier]

Endothelin (ET) causes contraction of the esophageal muscularis mucosae. To characterize the ET receptor subtypes involved in contraction, we measured contraction of isolated muscularis mucosae strips caused by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the guinea pig esophageal muscularis mucosae. Autoradiography demonstrated 125I-ET-1 binding to the muscularis mucosae and muscularis propria. ET-1 caused tetrodotoxin and atropine-insensitive contraction of esophageal muscularis mucosae strips. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. FR-139317, an ETA receptor antagonist, or BQ-788, an ETB receptor antagonist, alone did not alter responses to ET-1. However, the combination of both antagonists almost abolished the ET-1-induced contraction, indicating synergistic inhibition. Desensitization to sarafotoxin S6c, an ETB receptor agonist, failed to abolish the response to ET-1, which was completely inhibited by FR-139317. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose–inhibition curves demonstrated the presence of ETA and ETB receptors.This study demonstrates that the esophageal muscularis mucosae possesses both ETA and ETB receptors mediating muscle contraction. There is cooperation between these two subtypes of ET receptors in the esophagus mediating muscle contraction. [Copyright &y& Elsevier]

Abstract: Atrial natriuretic peptide (ANP) binding sites have been demonstrated in the guinea-pig gallbladder muscle with unclear function. To investigate effects of natriuretic peptides in the gallbladder, we measured relaxation of isolated human and guinea-pig gallbladder strips caused by natriuretic peptides, including C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP) and ANP, as well as des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. Results in the human gallbladder were similar to those in the guinea-pig gallbladder. CNP, BNP, ANP and cANP(4-23) alone did not cause contraction or relaxation in resting gallbladder strips. However, in carbachol or endothelin-1-contracted strips, CNP caused moderate, sustained and concentration-dependent relaxation. The relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted gallbladder strips and not by tetrodotoxin in carbachol-contracted strips. These indicate a direct effect of CNP on the gallbladder muscle. The relative potencies for natriuretic peptides to cause relaxation were CNP>>BNP≥ANP. cANP(4-23) did not cause relaxation. These indicate the existence of the natriuretic peptide receptor-B (NPR-B) mediating the relaxation. Taken together, these results demonstrate that natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with the natriuretic peptide receptor-B. [Copyright &y& Elsevier]