- Email: [email protected]
Dipeptidyl Peptidase IV Inhibitor Potentiates Amino Acid- and Bile Acid-Induced Duodenal Bicarbonate Secretion in Rats
129
SD rats were used after 18 h fasting. Under urethane anesthesia, an ex-vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3- secretion was measured at pH 7.0 using a pH stat-method. Lubiprostone (1~100 μM) was applied topically to the mucosa or the loop for 10 min. Indomethacin (5 mg/kg), ONO-8711 (EP1 antagonist: 10 mg/kg) or AE5-599 (EP3 antagonist: 10 mg/kg) was given SC or ID, respectively, 1 h before lubiprostone, while AE3-208 (EP4 antagonist: 3 mg/kg) or CFTRinh-172 (CFTR inhibitor: 1 mg/kg) was given IP 30 min before. Results: lubiprostone dose-dependently and significantly increased HCO3- secretion in both the stomach (>30 μM) and duodenum (>1 μM), although the dose required to stimulate the secretion was higher in the stomach than the duodenum. The stimulatory action of lubiprostone in the stomach was significantly abrogated by prior administration of the EP1 antagonist ONO-8711 but not by the EP3/EP4 antagonists or the CFTR inhibitor CFTRinh-172. By contrast, the stimulatory action in the duodenum was significantly attenuated by the EP3 antagonist AE5-599 and the EP4 antagonist AE3-208 as well as CFTRinh-172. Indomethacin had no effect on the HCO3- response to lubiprostone in both tissues. Conclusion: These results suggest that lubiprostone stimulates HCO3secretion in the stomach and duodenum mediated by different EP receptor subtypes; the former mediated by EP1 receptors and the latter mediated by both EP3 and EP4 receptors. It is also assumed that CFTR is involved in modulating HCO3- secretion in the duodenum but not the stomach. Although the difference in the effective dose of lubiprostone in the stomach and duodenum remains unexplained, it may be due to the different affinities to EP receptor subtypes; higher affinity to both EP3/EP4 receptors than EP1 receptors.
AGA Abstracts
Carbon Monoxide Involved in Modulating HCO3- Secretion in Rat Duodenum Koji Takeuchi, Kikuko Amagase, Eitaro Aihara, Shusaku Hayashi Background/Aims: Carbon monoxide (CO) with free iron and biliverdin are generated from degradation of heme catalyzed by constitutive heme oxygenase (HO-2) and inducible HO1 in the body. In the gastrointestinal tract, CO reportedly exerts various effects, such as the relaxation of smooth muscle, gastroprotection, and the stimulation of Cl- secretion. However, it remains unclear whether endogenous CO participates in the regulation of duodenal HCO3secretion. In the present study, we examined the effect of a CO donor, the tricarbonyldichlororuthenium (II) dimer (CO-releasing molecule; CORM-2), on duodenal HCO3- secretion in rats and investigated whether endogenous CO produced by heme oxygenase (HO) is involved in the regulation of this secretion. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and HCO3secretion was measured at pH 7.0 using a pH-stat method. CORM-2 (0.5-2 mM), biliverdin or ruthenium (III) chloride (RuCl3; a negative control), FeCl2 was applied to the loop for 5 min. Mucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Indomethacin or L-NAME was given SC 1 h before the administration of agonists, while Sn (IV) protoporphyrin IX dichloride (SnPP; an inhibitor of HO), CuPP (a negative inhibitor) or hemin was given IP 1 h before. Expression of HO protein was examined by western blotting. Results: We confirmed that CORM-2 dissolved in DMSO dose-dependently generated CO In Vitro at the doses used in this study. The mucosal application of CORM2 dose-dependently increased HCO3- secretion, while neither RuCl3, FeCl2 nor biliverdin had an effect. The stimulatory effect of CORM-2 at 2 μM was significantly attenuated by indomethacin but not L-NAME. The application of CORM-2 increased the mucosal PGE2 content in the duodenum. The acid-induced HCO3- response was markedly inhibited by indomethacin and SnPP but not CuPP, and the inhibitory effect of SnPP was significantly reversed by co-administration of hemin, a substrate of HO. The acid-induced duodenal damage (perfusion with100 mM HCl for 2 h) was significantly worsened by the prior administration of SnPP as well as indomethacin, and the former response was significantly reversed by co-administration of hemin. The expression of HO-1 but not HO-2 protein was up-regulated in the duodenum following the acid treatment, although both isozymes were expressed in the normal mucosa. Conclusion: These results suggest that CO stimulates HCO3- secretion in the duodenum, and this effect is mediated by endogenous PGs. It is assumed that HO/CO plays an important role in maintaining the integrity of the duodenal mucosa.
132 Dipeptidyl Peptidase IV Inhibitor Potentiates Amino Acid- and Bile AcidInduced Duodenal Bicarbonate Secretion in Rats Takuya Inoue, Yasutada Akiba, Eli Engel, Paul H. Guth, Jonathan D. Kaunitz Intestinal endocrine cells release gut hormones, including the glucagon-like peptides (GLPs), in response to luminal nutrients. We have reported that luminal co-perfusion of L-glutamate (L-Glu) and 5'-inosine monophosphate (IMP) synergistically increases duodenal bicarbonate secretion (DBS) via GLP-2 release, suggesting the presence of amino acid taste receptor heterodimers T1R1/T1R3. Intestinal endocrine L cells express numerous GPCRs including the bile acid receptor TGR5. Since dipeptidyl peptidase (DPP) IV rapidly degrades GLP, we hypothesized that activation of L-cell GPCRs by amino acids or bile acids stimulates DBS via GLP-2 release and that DPPIV inhibition enhances GLP-2 plasma concentrations and DBS. Methods: We measured DBS with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or bolus iv injection (1 or 3 μmol/ kg) of the DPPIV inhibitor NVP-728. The duodenal loop was also perfused with 10 μM taurocholic acid (TCA), taurolithocholic acid (TLCA), taurodeoxycholic acid (TDCA), or a selective TGR5 agonist betulinic acid (BTA) with or without NVP-728 iv injection. DPPIV activity was measured in situ on duodenal frozen sections using the fluorogenic substrate H-Gly-Pro-7-amino-4-methylcoumarin. Results: DPPIV activity was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP-728 (0.1 mM). Luminal perfusion of L-Glu/IMP increased DBS, inhibited by the GLP-2 receptor antagonist GLP-2(3-33) (3 nmol/kg, iv), accompanied by the increased GLP-2 release into portal plasma. IV injection of NVP-728 enhanced L-Glu/IMP-induced DBS with the enhanced plasma GLP-2 concentration, whereas luminal perfusion of NVP-728 had no effect, suggesting that the submucosal, but not brush border DPPIV degrades GLP-2. TCA or BTA at 10 μM slightly increased DBS, whereas TLCA or TDCA had no effect. IV injection of NVP-728 markedly enhanced TCA- or BTA-induced DBS with the enhanced plasma GLP-2 concentration. Immunofluorescence confirmed the presence of TGR5, T1R1, T1R3, and GLP-2 in duodenal endocrine-like cells. Conclusions: DPPIV inhibition potentiated luminal L-Glu/ IMP-induced and TGR5 agonist-induced DBS via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal bicarbonate secretion.
130 Capsiate, a Non-Pungent Capsinoid, Enhances Mucosal Defenses via Activation of TRPV1 and TRPA1 in Rat Duodenum Yasutada Akiba, Jonathan D. Kaunitz Capsiate, a natural capsaicin analog capsinoid, was identified from the fruit of non-pungent chili pepper cultivar, CH-19 Sweet. Capsiate, despite its non-pungent nature, has effects, similar to capsaicin on energy metabolism and inflammation, since capsiate is equipotent to capsaicin with regard to transient receptor potential vanilloid 1 (TRPV1) activation. Thus, a non-pungent TRPV1 agonist may be a useful therapeutic tool for the stimulation of capsaicin-sensitive afferent nerves without producing pain. Another natural pungent compound, allyl isothiocyanate (AITC), a component of mustard oil, stimulates another TRP family member TRPA1, expressed on afferent nerves. We hypothesized that luminallyperfused capsiate stimulates TRPV1 or TRPA1 expressed on capsaicin-sensitive afferent nerves, enhancing mucosal defense mechanisms in rat duodenum. Methods: Under isoflurane anesthesia, the duodenal mucosa was topically superfused with pH 7.0 Krebs buffer containing vehicle, capsiate (0.1 - 3 mM), capsaicin (0.03 - 1 mM) or AITC (0.1 - 3 mM) with or without the TRPV1 antagonist capsazepine (CPZ, 0.5 mM) or the TRPA1 antagonist HC030031 (0.1 mM). We measured epithelial intracellular pH (pHi), blood flow and mucus gel thickness (MGT) using In Vivo microscopy. Results: Superfusion of capsiate dosedependently increased duodenal blood flow with EC50 ~ 1 mM, similar to AITC, but ~ 3 times less potent than capsaicin. Capsiate (1 mM) increased blood flow and MGT with no effect on pHi. Capsiate-induced hyperemia was abolished by CPZ and reduced by HC030031, whereas the increased MGT was attenuated by CPZ or HC030031. De-afferentation by capsaicin pretreatment (125 mg/kg, sc) abolished the capsiate-induced hyperemic response and reduced the MGT increase whereas indomethacin pretreatment (5 mg/kg, sc) abolished the MGT increase and partially reduced the hyperemic response. A progressively diminished hyperemic response was observed after repeated exposure (10 min, 3 times) to capsaicin (0.3 mM), whereas no tolerance was observed with capsiate (1 mM). Immunofluorescence localized TRPA1 to the lamina propria and myenteric plexus of the duodenum, similar to TRPV1. Conclusions: Capsiate, similar to capsaicin enhances duodenal mucosal defenses via the capsaicin and prostaglandin pathways, although capsiate may additionally activate TRPA1. Capsiate may thus enhance mucosal protective mechanisms without the pungency or the tolerance associated with capsaicin.
133 Luminal ATP Release is Enhanced in Multidrug Resistant Protein Mdr1a Knockout Mouse Duodenum Yasutada Akiba, Takuya Inoue, Eli Engel, Paul H. Guth, Jonathan D. Kaunitz Extracellular non-lytic ATP release is an essential component of the luminal purinergic signaling system, which along with P2Y receptors and alkaline phosphatase (AP) helps regulate the rate of duodenal bicarbonate secretion (DBS). We have reported that the potent CFTR inhibitor CFTRinh-172 reduces ATP release and that ATP release is impaired in murine intestinal AP Akp3 knockout (KO) duodenum with downregulation of the multidrug resistant proteins Mdr1a and Mrp2. We thus hypothesized that Mdr1a knockout (KO) decreases ATP release. Methods: We measured DBS with pH and CO2 electrodes and ATP output with bioluminescence in a perfused duodenal loop in male wild type FVB mice (WT) or Mdr1a KO mice, or in rats under isoflurane anesthesia. The duodenal loop was perfused with the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the P2 receptor antagonist/ecto-nucleoside triphosphate diphosphohydrolase inhibitor suramin (1 mM), the multidrug resistant protein (MRP) inhibitor MK571 (50 μM) or the pannexin (Panx) inhibitor carbenoxolone (CBX, 0.1 mM). Results: AP inhibition with GP increased DBS and nonlytic ATP release in WT mouse and rat duodena. Co-perfusion of suramin inhibited GPinduced DBS, but enhanced ATP output, consistent with the dual action of suramin. In KO duodenum, GP or suramin further enhanced ATP output without a parallel release of the cytosolic enzyme LDH. The enhanced ATP output in KO duodenum was inhibited by the pretreatment with CFTRinh-172 (1 mg/kg, ip). Expression of Panx1 and MRP1 as measured by real time PCR was selectively upregulated in KO duodenum. In rat duodenum, luminal perfusion of MK571 and CBX reduced GP-induced ATP output, further decreased by the addition of CFTRinh-172 pretreatment. Conclusions: MDR1a (or MRP), CFTR and Panx1 may coordinately contribute to duodenal epithelial ATP release, which regulates DBS. Enhanced ATP release may account for the induction of diarrhea followed by colitis as part of the Mdr1a KO intestinal phenotype.
131 Lubiprostone Stimulates HCO3- Secretion in Rat Stomach and Duodenum Mediated by Different EP Receptor Subtypes Masafumi Koyama, Koji Dogishi, Shusaku Hayashi, Koji Takeuchi Background/Aim: Lubiprostone, a prostaglandin (PG) E1 derivative, is considered a possible chloride channel type-2 (ClC-2) opener and used for the treatment of chronic constipation. A recent study showed that lubiprostone stimulated CFTR-dependent duodenal HCO3secretion in rat, and this action was mediated by the activation of EP4 receptors. We also reported that this compound stimulated HCO3- secretion in isolated mouse stomachs In Vitro via the activation of EP1 receptors. However, it remains unexplored whether EP3 receptors are involved in the HCO3- response to lubiprostone. In the present study, we examined the stimulatory effect of lubiprostone on HCO3- secretion in the rat stomach and duodenum, focusing on the EP receptor subtypes involved in this action. Methods: Male
AGA Abstracts
S-32
SD rats were used after 18 h fasting. Under urethane anesthesia, an ex-vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3- secretion was measured at pH 7.0 using a pH stat-method. Lubiprostone (1~100 μM) was applied topically to the mucosa or the loop for 10 min. Indomethacin (5 mg/kg), ONO-8711 (EP1 antagonist: 10 mg/kg) or AE5-599 (EP3 antagonist: 10 mg/kg) was given SC or ID, respectively, 1 h before lubiprostone, while AE3-208 (EP4 antagonist: 3 mg/kg) or CFTRinh-172 (CFTR inhibitor: 1 mg/kg) was given IP 30 min before. Results: lubiprostone dose-dependently and significantly increased HCO3- secretion in both the stomach (>30 μM) and duodenum (>1 μM), although the dose required to stimulate the secretion was higher in the stomach than the duodenum. The stimulatory action of lubiprostone in the stomach was significantly abrogated by prior administration of the EP1 antagonist ONO-8711 but not by the EP3/EP4 antagonists or the CFTR inhibitor CFTRinh-172. By contrast, the stimulatory action in the duodenum was significantly attenuated by the EP3 antagonist AE5-599 and the EP4 antagonist AE3-208 as well as CFTRinh-172. Indomethacin had no effect on the HCO3- response to lubiprostone in both tissues. Conclusion: These results suggest that lubiprostone stimulates HCO3secretion in the stomach and duodenum mediated by different EP receptor subtypes; the former mediated by EP1 receptors and the latter mediated by both EP3 and EP4 receptors. It is also assumed that CFTR is involved in modulating HCO3- secretion in the duodenum but not the stomach. Although the difference in the effective dose of lubiprostone in the stomach and duodenum remains unexplained, it may be due to the different affinities to EP receptor subtypes; higher affinity to both EP3/EP4 receptors than EP1 receptors.
AGA Abstracts
Carbon Monoxide Involved in Modulating HCO3- Secretion in Rat Duodenum Koji Takeuchi, Kikuko Amagase, Eitaro Aihara, Shusaku Hayashi Background/Aims: Carbon monoxide (CO) with free iron and biliverdin are generated from degradation of heme catalyzed by constitutive heme oxygenase (HO-2) and inducible HO1 in the body. In the gastrointestinal tract, CO reportedly exerts various effects, such as the relaxation of smooth muscle, gastroprotection, and the stimulation of Cl- secretion. However, it remains unclear whether endogenous CO participates in the regulation of duodenal HCO3secretion. In the present study, we examined the effect of a CO donor, the tricarbonyldichlororuthenium (II) dimer (CO-releasing molecule; CORM-2), on duodenal HCO3- secretion in rats and investigated whether endogenous CO produced by heme oxygenase (HO) is involved in the regulation of this secretion. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and HCO3secretion was measured at pH 7.0 using a pH-stat method. CORM-2 (0.5-2 mM), biliverdin or ruthenium (III) chloride (RuCl3; a negative control), FeCl2 was applied to the loop for 5 min. Mucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Indomethacin or L-NAME was given SC 1 h before the administration of agonists, while Sn (IV) protoporphyrin IX dichloride (SnPP; an inhibitor of HO), CuPP (a negative inhibitor) or hemin was given IP 1 h before. Expression of HO protein was examined by western blotting. Results: We confirmed that CORM-2 dissolved in DMSO dose-dependently generated CO In Vitro at the doses used in this study. The mucosal application of CORM2 dose-dependently increased HCO3- secretion, while neither RuCl3, FeCl2 nor biliverdin had an effect. The stimulatory effect of CORM-2 at 2 μM was significantly attenuated by indomethacin but not L-NAME. The application of CORM-2 increased the mucosal PGE2 content in the duodenum. The acid-induced HCO3- response was markedly inhibited by indomethacin and SnPP but not CuPP, and the inhibitory effect of SnPP was significantly reversed by co-administration of hemin, a substrate of HO. The acid-induced duodenal damage (perfusion with100 mM HCl for 2 h) was significantly worsened by the prior administration of SnPP as well as indomethacin, and the former response was significantly reversed by co-administration of hemin. The expression of HO-1 but not HO-2 protein was up-regulated in the duodenum following the acid treatment, although both isozymes were expressed in the normal mucosa. Conclusion: These results suggest that CO stimulates HCO3- secretion in the duodenum, and this effect is mediated by endogenous PGs. It is assumed that HO/CO plays an important role in maintaining the integrity of the duodenal mucosa.
132 Dipeptidyl Peptidase IV Inhibitor Potentiates Amino Acid- and Bile AcidInduced Duodenal Bicarbonate Secretion in Rats Takuya Inoue, Yasutada Akiba, Eli Engel, Paul H. Guth, Jonathan D. Kaunitz Intestinal endocrine cells release gut hormones, including the glucagon-like peptides (GLPs), in response to luminal nutrients. We have reported that luminal co-perfusion of L-glutamate (L-Glu) and 5'-inosine monophosphate (IMP) synergistically increases duodenal bicarbonate secretion (DBS) via GLP-2 release, suggesting the presence of amino acid taste receptor heterodimers T1R1/T1R3. Intestinal endocrine L cells express numerous GPCRs including the bile acid receptor TGR5. Since dipeptidyl peptidase (DPP) IV rapidly degrades GLP, we hypothesized that activation of L-cell GPCRs by amino acids or bile acids stimulates DBS via GLP-2 release and that DPPIV inhibition enhances GLP-2 plasma concentrations and DBS. Methods: We measured DBS with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or bolus iv injection (1 or 3 μmol/ kg) of the DPPIV inhibitor NVP-728. The duodenal loop was also perfused with 10 μM taurocholic acid (TCA), taurolithocholic acid (TLCA), taurodeoxycholic acid (TDCA), or a selective TGR5 agonist betulinic acid (BTA) with or without NVP-728 iv injection. DPPIV activity was measured in situ on duodenal frozen sections using the fluorogenic substrate H-Gly-Pro-7-amino-4-methylcoumarin. Results: DPPIV activity was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP-728 (0.1 mM). Luminal perfusion of L-Glu/IMP increased DBS, inhibited by the GLP-2 receptor antagonist GLP-2(3-33) (3 nmol/kg, iv), accompanied by the increased GLP-2 release into portal plasma. IV injection of NVP-728 enhanced L-Glu/IMP-induced DBS with the enhanced plasma GLP-2 concentration, whereas luminal perfusion of NVP-728 had no effect, suggesting that the submucosal, but not brush border DPPIV degrades GLP-2. TCA or BTA at 10 μM slightly increased DBS, whereas TLCA or TDCA had no effect. IV injection of NVP-728 markedly enhanced TCA- or BTA-induced DBS with the enhanced plasma GLP-2 concentration. Immunofluorescence confirmed the presence of TGR5, T1R1, T1R3, and GLP-2 in duodenal endocrine-like cells. Conclusions: DPPIV inhibition potentiated luminal L-Glu/ IMP-induced and TGR5 agonist-induced DBS via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal bicarbonate secretion.
130 Capsiate, a Non-Pungent Capsinoid, Enhances Mucosal Defenses via Activation of TRPV1 and TRPA1 in Rat Duodenum Yasutada Akiba, Jonathan D. Kaunitz Capsiate, a natural capsaicin analog capsinoid, was identified from the fruit of non-pungent chili pepper cultivar, CH-19 Sweet. Capsiate, despite its non-pungent nature, has effects, similar to capsaicin on energy metabolism and inflammation, since capsiate is equipotent to capsaicin with regard to transient receptor potential vanilloid 1 (TRPV1) activation. Thus, a non-pungent TRPV1 agonist may be a useful therapeutic tool for the stimulation of capsaicin-sensitive afferent nerves without producing pain. Another natural pungent compound, allyl isothiocyanate (AITC), a component of mustard oil, stimulates another TRP family member TRPA1, expressed on afferent nerves. We hypothesized that luminallyperfused capsiate stimulates TRPV1 or TRPA1 expressed on capsaicin-sensitive afferent nerves, enhancing mucosal defense mechanisms in rat duodenum. Methods: Under isoflurane anesthesia, the duodenal mucosa was topically superfused with pH 7.0 Krebs buffer containing vehicle, capsiate (0.1 - 3 mM), capsaicin (0.03 - 1 mM) or AITC (0.1 - 3 mM) with or without the TRPV1 antagonist capsazepine (CPZ, 0.5 mM) or the TRPA1 antagonist HC030031 (0.1 mM). We measured epithelial intracellular pH (pHi), blood flow and mucus gel thickness (MGT) using In Vivo microscopy. Results: Superfusion of capsiate dosedependently increased duodenal blood flow with EC50 ~ 1 mM, similar to AITC, but ~ 3 times less potent than capsaicin. Capsiate (1 mM) increased blood flow and MGT with no effect on pHi. Capsiate-induced hyperemia was abolished by CPZ and reduced by HC030031, whereas the increased MGT was attenuated by CPZ or HC030031. De-afferentation by capsaicin pretreatment (125 mg/kg, sc) abolished the capsiate-induced hyperemic response and reduced the MGT increase whereas indomethacin pretreatment (5 mg/kg, sc) abolished the MGT increase and partially reduced the hyperemic response. A progressively diminished hyperemic response was observed after repeated exposure (10 min, 3 times) to capsaicin (0.3 mM), whereas no tolerance was observed with capsiate (1 mM). Immunofluorescence localized TRPA1 to the lamina propria and myenteric plexus of the duodenum, similar to TRPV1. Conclusions: Capsiate, similar to capsaicin enhances duodenal mucosal defenses via the capsaicin and prostaglandin pathways, although capsiate may additionally activate TRPA1. Capsiate may thus enhance mucosal protective mechanisms without the pungency or the tolerance associated with capsaicin.
133 Luminal ATP Release is Enhanced in Multidrug Resistant Protein Mdr1a Knockout Mouse Duodenum Yasutada Akiba, Takuya Inoue, Eli Engel, Paul H. Guth, Jonathan D. Kaunitz Extracellular non-lytic ATP release is an essential component of the luminal purinergic signaling system, which along with P2Y receptors and alkaline phosphatase (AP) helps regulate the rate of duodenal bicarbonate secretion (DBS). We have reported that the potent CFTR inhibitor CFTRinh-172 reduces ATP release and that ATP release is impaired in murine intestinal AP Akp3 knockout (KO) duodenum with downregulation of the multidrug resistant proteins Mdr1a and Mrp2. We thus hypothesized that Mdr1a knockout (KO) decreases ATP release. Methods: We measured DBS with pH and CO2 electrodes and ATP output with bioluminescence in a perfused duodenal loop in male wild type FVB mice (WT) or Mdr1a KO mice, or in rats under isoflurane anesthesia. The duodenal loop was perfused with the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the P2 receptor antagonist/ecto-nucleoside triphosphate diphosphohydrolase inhibitor suramin (1 mM), the multidrug resistant protein (MRP) inhibitor MK571 (50 μM) or the pannexin (Panx) inhibitor carbenoxolone (CBX, 0.1 mM). Results: AP inhibition with GP increased DBS and nonlytic ATP release in WT mouse and rat duodena. Co-perfusion of suramin inhibited GPinduced DBS, but enhanced ATP output, consistent with the dual action of suramin. In KO duodenum, GP or suramin further enhanced ATP output without a parallel release of the cytosolic enzyme LDH. The enhanced ATP output in KO duodenum was inhibited by the pretreatment with CFTRinh-172 (1 mg/kg, ip). Expression of Panx1 and MRP1 as measured by real time PCR was selectively upregulated in KO duodenum. In rat duodenum, luminal perfusion of MK571 and CBX reduced GP-induced ATP output, further decreased by the addition of CFTRinh-172 pretreatment. Conclusions: MDR1a (or MRP), CFTR and Panx1 may coordinately contribute to duodenal epithelial ATP release, which regulates DBS. Enhanced ATP release may account for the induction of diarrhea followed by colitis as part of the Mdr1a KO intestinal phenotype.
131 Lubiprostone Stimulates HCO3- Secretion in Rat Stomach and Duodenum Mediated by Different EP Receptor Subtypes Masafumi Koyama, Koji Dogishi, Shusaku Hayashi, Koji Takeuchi Background/Aim: Lubiprostone, a prostaglandin (PG) E1 derivative, is considered a possible chloride channel type-2 (ClC-2) opener and used for the treatment of chronic constipation. A recent study showed that lubiprostone stimulated CFTR-dependent duodenal HCO3secretion in rat, and this action was mediated by the activation of EP4 receptors. We also reported that this compound stimulated HCO3- secretion in isolated mouse stomachs In Vitro via the activation of EP1 receptors. However, it remains unexplored whether EP3 receptors are involved in the HCO3- response to lubiprostone. In the present study, we examined the stimulatory effect of lubiprostone on HCO3- secretion in the rat stomach and duodenum, focusing on the EP receptor subtypes involved in this action. Methods: Male
AGA Abstracts
S-32