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K+ ATPase, Whereas Kir 4.1 Channels Are Involved in Secretory Membrane Recycling
Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Carbachol- Induced Acid Secretion in Mice: Relation to D Cells/Somatostatin Nobuaki Takahashi, Kohei Yamazaki, Kikuko Amagase, Minoru Matsui, Susumu Okabe, Koji Takeuchi Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1~M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. Although the acid response to acetylcholine is considered to be mainly mediated by the activation of M3-receptors, we recently found that carbachol (CCh)-induced acid secretion was markedly decreased in M4 knockout (KO) mice. In the present study, we demonstrated, using M1~M5 KO mice, the importance of M4 receptors in the cholinergic stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice (12-20 wks old) of wild-type (WT) and M1~M5 KO were used. Under urethane anesthesia, the abdomen was incised, and an acute fistula prepared with a polyethylene tube was provided in the stomach through a pylorus. Then, the stomach was instilled with saline (0.4 ml) through the fistula, and the solution was changed every 20 min. CCh (30 μg/kg) was given SC as a single injection. Atropine (0.3 mg/kg) or CYN154806 [somatostatin-2 receptor (SST2R) antagonist: 0.1-3mg/kg] was given SC 20 min before CCh. Expressions of D cells and M4 receptors was examined immunohistochemically by double staining with anti-somatostatin and anti-M4 receptor antibodies. Results: CCh caused an increase of acid secretion in WT mice, and the effect was completely inhibited by prior administration of atropine. The stimulatory effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice, as compared to WT; especially, the response was all but completely abolished in M4 KO mice. CYN154806, the SST2R antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Somatostatin inhibited the secretion of acid under basal and CCh-stimulated conditions. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. It was also found that serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions and that the somatostatin levels in WT mice were decreased in response to CCh. Conclusion: These results suggest that under cholinergic stimulation the secretion of acid is mediated by the activation of both M3 and M4 receptors but does not involve other muscarinic receptor subtypes, and the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of somatostatin through SST2 receptors, resulting in enhancement of the acid response mediated by M3 receptors on parietal cells.
1062 Expression of the NKCC2 in the Parietal Cells and Its Possible Role On the Gastric H+ Secretion Hong Xue, Tuo Ji, Zhangfeng Dou, Yue Zhang, Jin Song, Yue Xu, Jinxia Zhu Background and aim: Na+-K+-2Cl- cotransporter (NKCC), a cation-chloride cotransporter, plays an important role in the regulation of intracellular Cl− homeostasis and cell volume. Molecular studies have identified two isoforms, NKCC1 and NKCC2, which are encoded by two distinct genes and share 60% identity in their amino acid sequence. Previous studies have demonstrated that NKCC1 expressed at high levels in parietal cell of mouse stomach, but no report on the expression of NKCC2 in the mammalian stomach. In addition, the effect of NKCC on the acid secretion in parietal cell remains controversial. The present study aims to investigate the expression of NKCC2 in the stomach and its possible role on the gastric acid secretion. Methods: RT-PCR and Western blot analysis were used to quantitatively study the expression of NKCC2. Immunohistochemistry and Immunofluorescence were used to investigate the localization of the NKCC2 in the gastric mucosa and primary cultured parietal cells. A noninvasive electrophysiological technique, the scanning ion-selective electrode technique (SIET) was used to measure H+ flux at the surface of the gastric mucosa.Results: The result of RT-PCR and western blot showed that the NKCC2 mRNA and protein were expressed in the rat stomach. The NKCC2 immunoreactivity (IR) was localized to the base of the gastric glands. Double labeling with antibodies specific for the marker of parietal cell (beta subunit of H+-K+ATPase) and NKCC1 or NKCC2 antibody in cryosection of gastric tissue has demonstrated that NKCC1 and NKCC2 co-localized with H+-K+ATPase. The coexistence of NKCC2 with H+-K+ATPase was further confirmed in the primary cultured parietal cells by using confocal microscopy. NKCC2 was present in cytoplasm and membrane of parietal cell in the rest state. After stimulation with histamine, H+-K+-ATPase translocated from the secretory vesicle membrane to the cell membrane. Although NKCC2 distribution in stimulated parietal cell was not signicantly from resting parietal cell, the fluorescence intensity was stronger than the resting cell. By using H+ selective microelectrode the acid secretion generated by the gastric mucosa was further examined. The results showed that the basal H+ flux (outward) was continuous and stable. The H+ flux rate was declined from 0.90 ± 0.01 pmol cm-2 s-1 to 0.68 ±0.01 pmol cm-2 s-1 (n=3) after application of NKCC inhibitor bumetanide (10μM). Conclusions: Taken together, the present results demonstrated that the NKCC2 expressed in the mucosa of rat stomach and localized in the parietal cells. The NKCC2 might be involved in the regulation of gastric acid secretion of rat.
1060 Involvement of Somatostatin/SST2 Receptors in Decreased Acid Secretion of Damaged Stomachs Mediated By Prostacyclin/IP Receptors Maya Honda, Hikaru Nishio, Michitaka Ogura, Shinichi Kato, Koji Takeuchi Background/Aim: We previously reported that the decreased acid response after mucosal exposure to taurocholate (TC) is mediated by endogenous prostaglandins produced by COX1 through prostacyclin (PGI2)/IP receptors, yet it remains unexplored how PGI2 inhibits acid secretion. Since the activation of IP receptors is coupled to Gs protein/cAMP, it is unlikely that PGI2 directly acts to the parietal cells to inhibit acid secretion. In the present study, we examined the effect of a PGI2 analog iloprost on histamine-induced acid secretion and investigated how endogenous PGI2 mediates the decreased acid response in the damaged stomach, in relation to somatostatin and its receptor SST2. Methods: Male C57/BL6 mice (20-30 g), both wild-type (WT) and IP receptor knockout (KO), were used after 18 h of fasting. Under urethane anesthesia, the abdomen was incised, and an acute fistula with a polyethylene tube was provided in the stomach through a pylorus. The stomach was then instilled with saline (0.4 ml) through the fistula, and the solution was changed every 20 min. Acid secretion was stimulated by histamine (0.3-3 mg/kg) given SC as a singe injection. On the other hand, the stomach was exposed to 20 mM TC for 10 min, and the secretion was determined before and after the exposure. Iloprost, a PGI2 analog (1 mg/kg), was given IP 20 min before histamine. CYN154806, a selective somatostatin SST2 receptor antagonist (0.1 mg/kg), was given SC 20 min after histamine or 20 min before TC treatment. Results: Acid secretion in WT mice was dose-dependently increased by histamine, and the same was observed in the animals lacking IP receptors. Iloprost significantly decreased the histamineinduced acid secretion in WT but not IP receptor KO mice. The inhibitory effect of iloprost in WT mice was significantly abrogated by the SST2 antagonist CYN154806. The mucosal exposure to TC decreased the acid secretion in WT mice with an increase of mucosal PGI2 contents, but the reduced acid response was less marked in IP KO mice. Furthermore, the decrease of acid secretion in the damaged stomach of WT mice was also significantly reversed by the prior administration of CYN154806. Somatostatin content in the stomach was reduced after administration of iloprost or the mucosal exposure to TC, while the blood levels increased. Conclusion: These results confirmed the role of PGI2/IP receptor in the reduced acid response in the damaged stomach and further suggested that the inhibitory action of PGI2 is mediated by somatostatin through SST2 receptors. It is assumed that PGI2 releases somatostatin from D cells, which in turn decreases the secretion of acid via the activation of SST2 receptors.
1063 Prospective Assessment of the Appropriateness and Timeliness of Gastrointestinal Inpatient Consultation Requests At a U.S. Teaching Hospital Lukejohn W. Day, John P. Cello, Erin Madden, Mark Segal Background: Subspecialty consultation requests are common. Research on outpatient gastroenterology (GI) consultations indicates that information communicated among physicians may be incorrect. Studies on GI inpatient consultations are lacking. We aimed to assess the appropriateness of GI inpatient consultation requests at a teaching hospital. Methods: A prospective, cross-sectional study of healthcare providers requesting GI consultation was conducted at a large county hospital. A detailed questionnaire was completed by GI fellows upon receiving an inpatient consultation request which assessed 25 specific aspects of the requesting healthcare providers' knowledge and patient documentation. The appropriateness of the consultation was evaluated prospectively by the GI consult attending physician using a linear analog scale and was based on his/her overall assessment of the completed questionnaire. Appropriateness was defined on this linear scale from 0 to 10 (“appropriateness scores”), with higher numbers reflecting more appropriate consultations. Multivariate linear regression was used to assess the association between appropriateness scores and several predictors. A separate analysis was performed for GI bleeding consultations. Results: Data were collected on 278 consultation requests. The most common consultation request was “GI bleeding” (53%). Pertinent patient information was provided to the consultant by the provider in 50% of instances. Also, only 60% of providers were able to provide adequate knowledge about the rationale for the GI consultation. Predictors associated with higher appropriateness scores were requesting providers' knowledge of patient past medical history (p < 0.001), patient laboratory data (p=0.01), rationale for consult request (p=0.05), and the providers' admission notes being present in the patients' charts (p=0.002). Lower appropriateness scores were associated with requests by medical students (p=0.04) and during the first three months of the academic year (p=0.03). In a subgroup analysis of GI bleeding consultation requests, providers' knowledge of patient past medical history (p=0.01), knowledge of patient NSAID usage (p=0.003), and providers' notes being present in patients' charts (p=0.002) were associated with higher appropriateness scores. Conclusions: Our study demonstrates that in 20% to 30% of consultation requests, crucial patient data were missing or unknown, creating a potential nidus for medical errors. Predictors for appropriate consultation included healthcare providers' knowledge, year of clinical training, and time of year. Our data highlights the need for further training in consultation education.
1061 Gastric KNCQ1 Channels Provide Luminal K+ to the H+/K+ ATPase, Whereas Kir 4.1 Channels Are Involved in Secretory Membrane Recycling Penghong Song, Stephanie Groos, Brigitte Riederer, Zhe Feng, Anja Krabbenhöft, Michael P. Manns, Adam J. Smolka, Susan J. Hagen, Clemens Neusch, Ursula Seidler Backround and aim: K+ recycling across the parietal cell (PC) apical membrane is essential for gastric acid secretion, and both Kir4.1 and KCNQ1 K+ channels have been recently found to traffic to the apical membrane during acid stimulation. This study was undertaken to explore the functional role of KCNQ1 and Kir4.1 channels in gastric acid secretion. Results: Forskolin-stimulated acid secretory rates were strongly reduced in KCNQ1-/- gastric
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AGA Abstracts
AGA Abstracts
mucosa of 7 day-old mice in mini-Ussing chamber setups. Application of a high K+ concentration to the luminal membrane restored normal acid secretory rates in the KNCQ1-/- mucosa, indicating that the primary function of KCNQ1 is the supply of K+ to the extracellular K+ binding site of the H+/K+-ATPase. Surprisingly, Kir4.1-/- mucosa secreted significantly more acid and initiated secretion more rapidly. Electrone microscopy revealed the presence of fully elaborate but collapsed canalicular membranes and a lack of tubulovesicles in resting state Kir4.1-/- parietal cells. After In Vitro stimulation by forskolin, massive membrane fusion and canalicular enlargement was seen in the WT but not in the Kir4.1-/- mucosae, suggesting that Kir4.1 is essential for the membrane recycling events that accompany the acid secretory cycle. Parietal cell numbers were equal between wt and both KNCQ1- and Kir4.1-deficient gastric mucosae, but in Kir4.1-/- mucosae, H+/K+ ATPase mRNA was increased while that for AE2 mRNA, the basolateral anion exchanger which is highly expressed in parietal cells and therefore and excellent marker for parietal cell abundance, was normal. Conclusions: KCNQ1 channels are the major pathway to provide K+ as substrate for H+/K+ ATPase. Kir4.1 channels are involved in the control of tubulovesicular fusion and/ or retrieval.
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1062 Expression of the NKCC2 in the Parietal Cells and Its Possible Role On the Gastric H+ Secretion Hong Xue, Tuo Ji, Zhangfeng Dou, Yue Zhang, Jin Song, Yue Xu, Jinxia Zhu Background and aim: Na+-K+-2Cl- cotransporter (NKCC), a cation-chloride cotransporter, plays an important role in the regulation of intracellular Cl− homeostasis and cell volume. Molecular studies have identified two isoforms, NKCC1 and NKCC2, which are encoded by two distinct genes and share 60% identity in their amino acid sequence. Previous studies have demonstrated that NKCC1 expressed at high levels in parietal cell of mouse stomach, but no report on the expression of NKCC2 in the mammalian stomach. In addition, the effect of NKCC on the acid secretion in parietal cell remains controversial. The present study aims to investigate the expression of NKCC2 in the stomach and its possible role on the gastric acid secretion. Methods: RT-PCR and Western blot analysis were used to quantitatively study the expression of NKCC2. Immunohistochemistry and Immunofluorescence were used to investigate the localization of the NKCC2 in the gastric mucosa and primary cultured parietal cells. A noninvasive electrophysiological technique, the scanning ion-selective electrode technique (SIET) was used to measure H+ flux at the surface of the gastric mucosa.Results: The result of RT-PCR and western blot showed that the NKCC2 mRNA and protein were expressed in the rat stomach. The NKCC2 immunoreactivity (IR) was localized to the base of the gastric glands. Double labeling with antibodies specific for the marker of parietal cell (beta subunit of H+-K+ATPase) and NKCC1 or NKCC2 antibody in cryosection of gastric tissue has demonstrated that NKCC1 and NKCC2 co-localized with H+-K+ATPase. The coexistence of NKCC2 with H+-K+ATPase was further confirmed in the primary cultured parietal cells by using confocal microscopy. NKCC2 was present in cytoplasm and membrane of parietal cell in the rest state. After stimulation with histamine, H+-K+-ATPase translocated from the secretory vesicle membrane to the cell membrane. Although NKCC2 distribution in stimulated parietal cell was not signicantly from resting parietal cell, the fluorescence intensity was stronger than the resting cell. By using H+ selective microelectrode the acid secretion generated by the gastric mucosa was further examined. The results showed that the basal H+ flux (outward) was continuous and stable. The H+ flux rate was declined from 0.90 ± 0.01 pmol cm-2 s-1 to 0.68 ±0.01 pmol cm-2 s-1 (n=3) after application of NKCC inhibitor bumetanide (10μM). Conclusions: Taken together, the present results demonstrated that the NKCC2 expressed in the mucosa of rat stomach and localized in the parietal cells. The NKCC2 might be involved in the regulation of gastric acid secretion of rat.
1060 Involvement of Somatostatin/SST2 Receptors in Decreased Acid Secretion of Damaged Stomachs Mediated By Prostacyclin/IP Receptors Maya Honda, Hikaru Nishio, Michitaka Ogura, Shinichi Kato, Koji Takeuchi Background/Aim: We previously reported that the decreased acid response after mucosal exposure to taurocholate (TC) is mediated by endogenous prostaglandins produced by COX1 through prostacyclin (PGI2)/IP receptors, yet it remains unexplored how PGI2 inhibits acid secretion. Since the activation of IP receptors is coupled to Gs protein/cAMP, it is unlikely that PGI2 directly acts to the parietal cells to inhibit acid secretion. In the present study, we examined the effect of a PGI2 analog iloprost on histamine-induced acid secretion and investigated how endogenous PGI2 mediates the decreased acid response in the damaged stomach, in relation to somatostatin and its receptor SST2. Methods: Male C57/BL6 mice (20-30 g), both wild-type (WT) and IP receptor knockout (KO), were used after 18 h of fasting. Under urethane anesthesia, the abdomen was incised, and an acute fistula with a polyethylene tube was provided in the stomach through a pylorus. The stomach was then instilled with saline (0.4 ml) through the fistula, and the solution was changed every 20 min. Acid secretion was stimulated by histamine (0.3-3 mg/kg) given SC as a singe injection. On the other hand, the stomach was exposed to 20 mM TC for 10 min, and the secretion was determined before and after the exposure. Iloprost, a PGI2 analog (1 mg/kg), was given IP 20 min before histamine. CYN154806, a selective somatostatin SST2 receptor antagonist (0.1 mg/kg), was given SC 20 min after histamine or 20 min before TC treatment. Results: Acid secretion in WT mice was dose-dependently increased by histamine, and the same was observed in the animals lacking IP receptors. Iloprost significantly decreased the histamineinduced acid secretion in WT but not IP receptor KO mice. The inhibitory effect of iloprost in WT mice was significantly abrogated by the SST2 antagonist CYN154806. The mucosal exposure to TC decreased the acid secretion in WT mice with an increase of mucosal PGI2 contents, but the reduced acid response was less marked in IP KO mice. Furthermore, the decrease of acid secretion in the damaged stomach of WT mice was also significantly reversed by the prior administration of CYN154806. Somatostatin content in the stomach was reduced after administration of iloprost or the mucosal exposure to TC, while the blood levels increased. Conclusion: These results confirmed the role of PGI2/IP receptor in the reduced acid response in the damaged stomach and further suggested that the inhibitory action of PGI2 is mediated by somatostatin through SST2 receptors. It is assumed that PGI2 releases somatostatin from D cells, which in turn decreases the secretion of acid via the activation of SST2 receptors.
1063 Prospective Assessment of the Appropriateness and Timeliness of Gastrointestinal Inpatient Consultation Requests At a U.S. Teaching Hospital Lukejohn W. Day, John P. Cello, Erin Madden, Mark Segal Background: Subspecialty consultation requests are common. Research on outpatient gastroenterology (GI) consultations indicates that information communicated among physicians may be incorrect. Studies on GI inpatient consultations are lacking. We aimed to assess the appropriateness of GI inpatient consultation requests at a teaching hospital. Methods: A prospective, cross-sectional study of healthcare providers requesting GI consultation was conducted at a large county hospital. A detailed questionnaire was completed by GI fellows upon receiving an inpatient consultation request which assessed 25 specific aspects of the requesting healthcare providers' knowledge and patient documentation. The appropriateness of the consultation was evaluated prospectively by the GI consult attending physician using a linear analog scale and was based on his/her overall assessment of the completed questionnaire. Appropriateness was defined on this linear scale from 0 to 10 (“appropriateness scores”), with higher numbers reflecting more appropriate consultations. Multivariate linear regression was used to assess the association between appropriateness scores and several predictors. A separate analysis was performed for GI bleeding consultations. Results: Data were collected on 278 consultation requests. The most common consultation request was “GI bleeding” (53%). Pertinent patient information was provided to the consultant by the provider in 50% of instances. Also, only 60% of providers were able to provide adequate knowledge about the rationale for the GI consultation. Predictors associated with higher appropriateness scores were requesting providers' knowledge of patient past medical history (p < 0.001), patient laboratory data (p=0.01), rationale for consult request (p=0.05), and the providers' admission notes being present in the patients' charts (p=0.002). Lower appropriateness scores were associated with requests by medical students (p=0.04) and during the first three months of the academic year (p=0.03). In a subgroup analysis of GI bleeding consultation requests, providers' knowledge of patient past medical history (p=0.01), knowledge of patient NSAID usage (p=0.003), and providers' notes being present in patients' charts (p=0.002) were associated with higher appropriateness scores. Conclusions: Our study demonstrates that in 20% to 30% of consultation requests, crucial patient data were missing or unknown, creating a potential nidus for medical errors. Predictors for appropriate consultation included healthcare providers' knowledge, year of clinical training, and time of year. Our data highlights the need for further training in consultation education.
1061 Gastric KNCQ1 Channels Provide Luminal K+ to the H+/K+ ATPase, Whereas Kir 4.1 Channels Are Involved in Secretory Membrane Recycling Penghong Song, Stephanie Groos, Brigitte Riederer, Zhe Feng, Anja Krabbenhöft, Michael P. Manns, Adam J. Smolka, Susan J. Hagen, Clemens Neusch, Ursula Seidler Backround and aim: K+ recycling across the parietal cell (PC) apical membrane is essential for gastric acid secretion, and both Kir4.1 and KCNQ1 K+ channels have been recently found to traffic to the apical membrane during acid stimulation. This study was undertaken to explore the functional role of KCNQ1 and Kir4.1 channels in gastric acid secretion. Results: Forskolin-stimulated acid secretory rates were strongly reduced in KCNQ1-/- gastric
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AGA Abstracts
AGA Abstracts
mucosa of 7 day-old mice in mini-Ussing chamber setups. Application of a high K+ concentration to the luminal membrane restored normal acid secretory rates in the KNCQ1-/- mucosa, indicating that the primary function of KCNQ1 is the supply of K+ to the extracellular K+ binding site of the H+/K+-ATPase. Surprisingly, Kir4.1-/- mucosa secreted significantly more acid and initiated secretion more rapidly. Electrone microscopy revealed the presence of fully elaborate but collapsed canalicular membranes and a lack of tubulovesicles in resting state Kir4.1-/- parietal cells. After In Vitro stimulation by forskolin, massive membrane fusion and canalicular enlargement was seen in the WT but not in the Kir4.1-/- mucosae, suggesting that Kir4.1 is essential for the membrane recycling events that accompany the acid secretory cycle. Parietal cell numbers were equal between wt and both KNCQ1- and Kir4.1-deficient gastric mucosae, but in Kir4.1-/- mucosae, H+/K+ ATPase mRNA was increased while that for AE2 mRNA, the basolateral anion exchanger which is highly expressed in parietal cells and therefore and excellent marker for parietal cell abundance, was normal. Conclusions: KCNQ1 channels are the major pathway to provide K+ as substrate for H+/K+ ATPase. Kir4.1 channels are involved in the control of tubulovesicular fusion and/ or retrieval.
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