Antral Mucous Cells Research Articles

Nitric oxide synthesis stimulated by arachidonic acid accumulation via PPARα in acetylcholine-stimulated gastric mucous cells.

What is the central question of this study? Arachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in [Ca2+ ]i . Given that the intracellular AA concentration is too low to measure, the relationship between AA accumulation and NO production remains uncertain. Is AA accumulation a key step for NO production? What is the main finding and its importance? We demonstrated that AA accumulation is a key step for NO production. The amount of AA released could be measured using fluorescence-HPLC. The intracellular AA concentration was maintained at <1μM. Nitric oxide is produced by AA accumulation in antral mucous cells, not as a direct effect of [Ca2+ ]i . In the present study, we demonstrate that NO production is stimulated by an accumulation of arachidonic acid (AA) mediated via peroxisome proliferation-activated receptorα (PPARα) and that the NO produced enhances Ca2+ -regulated exocytosis in ACh-stimulated antral mucous cells. The amount of AA released from the antral mucosa, measured by fluorescence high-performance liquid chromatography (F-HPLC), was increased by addition of ionomycin (10 μM) or ACh, suggesting that AA accumulation is stimulated by an increase in [Ca2+ ]i . The AA production was inhibited by an inhibitor of cytosolic phospholipaseA2 (cPLA2-inhα). GW6471 (a PPARα inhibitor) and cPLA2-inhα inhibited NO synthesis stimulated by ACh. Moreover, indomethacin, an inhibitor of cyclooxygenase, stimulated AA accumulation and NO production. However, acetylsalicylic acid did not stimulate AA production and NO synthesis. An analogue of AA (AACOCF3) alone stimulated NO synthesis, which was inhibited by GW6471. In antral mucous cells, indomethacin enhanced Ca2+ -regulated exocytosis by increasing NO via PPARα, and the enhancement was abolished by GW6471 and cPLA2-inhα. Thus, AA produced via PLA2 activation is the key step for NO synthesis in ACh-stimulated antral mucous cells and plays important roles in maintaining antral mucous secretion, especially in Ca2+ -regulated exocytosis.

Abstract A67: TFF (Trefoil Factor Family) is a novel tumor suppressor and can be the therapeutic target for pancreatic cancer

Abstract Purpose: TFF (Trefoil Factor Family) are secretory protein abundantly expressed in the mucosa of GI system. The key role of TFF is healing of damaged gastrointestinal mucosa by a process called restitution. Among 3 subtypes of TFF, TFF1 is expressed in normal gastric mucosa in foveolar epithelial surface cells, and TFF2 in deep antral gland mucous cells. Recent reports revealed that loss of TFF1 result in gastric tumorigenesis and that loss of TFF2 accelerates Helicobacter-pylori induced gastric dysplasia, suggesting that TFF play a role as tumor suppressor in gastric carcinogenesis. Here we aimed to clarify whether TFF can also act as tumor suppressor in pancreatic tumorigenesis, and to investigate how to utilize the tumor-suppressive function of TFF against pancreatic cancer. Material and Methods: The expression patterns of TFF1 and TFF2 in surgically resected pancreas (Organ Donor, Pancreatitis, IPMN; Intraductal Papillary Mucinous Neoplasm and PDAC; Pancreatic Ductal Adenocarcinoma) were analyzed by immunohistochemistry. Pancreatic cancer cell lines were transfected with either siRNA against TFF1 or overexpression vector of TFF2 in order to investigate the functional role of TFF. Pancreatic cancer cell lines were treated with estradiol and/or pioglitazone to evaluate the effect of TFF in vitro. Results: In normal and injured pancreas, TFF expression was found only in PDG (Pancreatic Duct Gland), the progenitor compartment of pancreatic ductal epithelium located in the mesenchymal cuff of pancreatic duct. Premalignant lesions of pancreas express abundant TFF; TFF1 throughout tumor cells of PanIN and IPMN, TFF2 in tumor-progenitor compartment PDG. TFF expressions are lost in invasive pancreatic cancer, indicating that TFF1 and TFF2 might act as tumor suppressor in pancreatic carcinogenesis. The suppression of TFF1 in pancreatic cancer cell lines results in the epithelial-mesenchymal transition (EMT) and accelerated invasive ability of cancer cells, while overexpression of TFF2 inhibit proliferative ability of cancer cells. These results suggest that TFF1 and TFF2 have tumor-suppressive function for pancreatic cancer in a different manner. Estradiol has been known to upregulate TFF1, and pioglitazone was found to upregulate TFF2 expression. Either of these drugs has mild tumor suppressive effect; however, combined use of them showed enhanced suppression of cancer cell proliferation in vitro. Conclusion: TFF1 and TFF2 act as tumor suppressor in pancreatic carcinogenesis in a different manner, and they can be a novel therapeutic target for PDAC. Citation Format: Junpei Yamaguchi, Yukihiro Yokoyama, Toshio Kokuryo, Masato Nagino.{Authors}. TFF (Trefoil Factor Family) is a novel tumor suppressor and can be the therapeutic target for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A67.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

胃幽門腺粘液開口放出のアラキドン酸/PPARαによる増強

胃幽門腺粘液開口放出のアラキドン酸/PPARαによる増強

Modelling human development and disease in pluripotent stem-cell-derived gastric organoids

Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

PPARα autocrine regulation of Ca²⁺-regulated exocytosis in guinea pig antral mucous cells: NO and cGMP accumulation.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N(5)-[imino(propylamino)methyl]-L-ornithine hydrochloride (N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca(2+)-regulated exocytosis in antral mucous cells.

267 Macrophages Promote the Progression of Metaplasia in the Stomach Following Acute Parietal Cell Loss

Adenocarcinoma in the human stomach evolves in the setting of Helicobacter pylori-induced oxyntic atrophy and chronic mucous cell metaplasia. Two types of metaplasia associated with development of intestinal-type cancers are observed in the human stomach: intestinal metaplasia (IM; intestinal type goblet cells in the stomach) and Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM; with the presence of antral type TFF2-expressing mucous cells in the oxyntic region). Our previous study showed that SPEM developed from transdifferentiation of mature chief cells, rather than originating from aberrant differentiation of resident progenitor cells. Recent investigations have noted evidence for increased Ras activity in up to a third of gastric cancers. Thus, we have hypothesized that activated Ras, specifically induced in mature chief cells, elicits transdifferentiation of chief cells into SPEM and progression of SPEM to more intestinalized metaplasia. To test this hypothesis, we have examined a novel mouse model of metaplasia in the stomach in Mist1-CreERT2Tg/+;LSLK-Ras(G12D)Tg/+;mT/mGTg/+ mice. Tamoxifen (TAM) treatment induces the expression of activated K-Ras specifically in mature chief cells and marks the K-Ras-induced chief cells with GFP as a lineage tracer. This mouse model rapidly develops SPEM and IM within 3 months, following TAM induction of activated K-Ras expression in mature chief cells. At 1 week post TAM treatment, we found several groups of metaplastic glands between normal oxyntic glands that were positive for SPEM markers. At 3 months post TAM treatment, oxyntic glands were replaced withmetaplastic glands that showed highly proliferative mucous cell populations at the bases and the cells in the metaplastic glands were strongly positive for clusterin and TFF3 with distinct goblet cell morphologies, and also positive for Pdx1. Some glands were positive for IM markers such as villin and Cdx1. The mice at 4 months post TAM treatment developed severe inflammation, metaplasia, dysplasia and the invasive adenocarcinoma. Consistent with active K-Ras expression, cells with nuclear expression of phospho-ERK1/2 were also observed at the bases of metaplastic glands. Interestingly, we observed that the metaplastic glands were positive for GFP, indicating that they were derived from transdifferentiation of Cre-induced chief cells. Therefore, the induction of activated Ras specifically in mature chief cells initially leads to transdifferentiation of chief cells into SPEM followed by evolution of goblet cell IM and eventual neoplastic lesions. These studies demonstrate that expression of activated Ras in Chief cells can lead to the development of the entire spectrum of metaplastic and neoplastic lineages leading to gastric cancer.

266 Active Kras Leads to Rapid Transdifferentiation of Gastric Chief Cells Into Metaplasia With Progression to Dysplasia and Invasive Cancer

Adenocarcinoma in the human stomach evolves in the setting of Helicobacter pylori-induced oxyntic atrophy and chronic mucous cell metaplasia. Two types of metaplasia associated with development of intestinal-type cancers are observed in the human stomach: intestinal metaplasia (IM; intestinal type goblet cells in the stomach) and Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM; with the presence of antral type TFF2-expressing mucous cells in the oxyntic region). Our previous study showed that SPEM developed from transdifferentiation of mature chief cells, rather than originating from aberrant differentiation of resident progenitor cells. Recent investigations have noted evidence for increased Ras activity in up to a third of gastric cancers. Thus, we have hypothesized that activated Ras, specifically induced in mature chief cells, elicits transdifferentiation of chief cells into SPEM and progression of SPEM to more intestinalized metaplasia. To test this hypothesis, we have examined a novel mouse model of metaplasia in the stomach in Mist1-CreERT2Tg/+;LSLK-Ras(G12D)Tg/+;mT/mGTg/+ mice. Tamoxifen (TAM) treatment induces the expression of activated K-Ras specifically in mature chief cells and marks the K-Ras-induced chief cells with GFP as a lineage tracer. This mouse model rapidly develops SPEM and IM within 3 months, following TAM induction of activated K-Ras expression in mature chief cells. At 1 week post TAM treatment, we found several groups of metaplastic glands between normal oxyntic glands that were positive for SPEM markers. At 3 months post TAM treatment, oxyntic glands were replaced withmetaplastic glands that showed highly proliferative mucous cell populations at the bases and the cells in the metaplastic glands were strongly positive for clusterin and TFF3 with distinct goblet cell morphologies, and also positive for Pdx1. Some glands were positive for IM markers such as villin and Cdx1. The mice at 4 months post TAM treatment developed severe inflammation, metaplasia, dysplasia and the invasive adenocarcinoma. Consistent with active K-Ras expression, cells with nuclear expression of phospho-ERK1/2 were also observed at the bases of metaplastic glands. Interestingly, we observed that the metaplastic glands were positive for GFP, indicating that they were derived from transdifferentiation of Cre-induced chief cells. Therefore, the induction of activated Ras specifically in mature chief cells initially leads to transdifferentiation of chief cells into SPEM followed by evolution of goblet cell IM and eventual neoplastic lesions. These studies demonstrate that expression of activated Ras in Chief cells can lead to the development of the entire spectrum of metaplastic and neoplastic lineages leading to gastric cancer.

Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum

ObjectiveThe glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical...

Inhibition of Ca2+-regulated exocytosis by levetiracetam, a ligand for SV2A, in antral mucous cells of guinea pigs

Levtiracetam (Lev), an inhibitor of SV2A (synaptic vesicle protein A2), affected the ATP-dependent priming of Ca2+-regulated exocytosis in antral mucous cells of guinea pig. In antral mucous cells, the Ca2+-regulated exocytosis, which is activated by acetylcholine (ACh), consists of an initial peak that declines rapidly (initial phase) followed by a second slower decline (late phase). Dinitrophenol (DNP), which depletes ATP, inhibits the ATP-dependent priming. DNP abolished the initial phase by reducing the number of primed granules, Lev decreased the frequency of initial phase, but not in the presence of DNP. Moreover, 8-bromoguanosine 3′5′-cyclic monophosphate (8BrcGMP) accelerates the ATP-dependent priming. 8BrcGMP enhances the frequency of initial phase by increasing the number of primed granule. Lev added prior to 8BrcGMP addition decreased the frequency of initial phase, but Lev added after 8BrcGMP addition did not. Thus, Lev affected the granules in the process of priming, but it did not affect the granules already primed. Lev did not affect [Ca2+]i in unstimulated or ACh-stimulated antral mucous cells. Immunohistochemistry and western blotting demonstrated that SV2A exists in antral mucous cells. The results suggest that SV2A plays an essential role in maintaining the process of ATP-dependent priming in antral mucous cells. In conclusion, Lev decreases the frequency of Ca2+-regulated exocytosis the number of primed granules by inhibiting SV2A functions, leading to a decrease in antral mucous cells.

A PKG inhibitor increases Ca2+-regulated exocytosis in guinea pig antral mucous cells: cAMP accumulation via PDE2A inhibition

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of an initial peak that declines rapidly (initial transient phase) followed by a second slower decline (late phase) lasting during ACh stimulation. The addition of 8-bromo-cGMP (8-BrcGMP) enhanced the initial phase, which was inhibited by the protein kinase G (PKG) inhibitor guanosine 3',5'-cyclic monophosphorothoiate, β-phenyl-1,N(2)-etheno-8-bromo, Rp-isomer, sodium salt (Rp-8-BrPETcGMPS, 100 nM). However, Rp-8-BrPETcGMPS produced a delayed, but transient, increase in the exocytotic frequency during the late phase that was abolished by a protein kinase A (PKA) inhibitor (PKI-amide), suggesting that Rp-8-BrPETcGMPS accumulates cAMP. The cGMP-dependent phosphodiesterase 2 (PDE2), which degrades cAMP, may exist in antral mucous cells. The PDE2 inhibitor BAY-60-7550 (250 nM) mimicked the effect of Rp-8-BrPETcGMPS on ACh-stimulated exocytosis. Measurement of the cGMP and cAMP contents in antral mucosae revealed that ACh stimulates the accumulation of cGMP and that BAY-60-7550 accumulates cAMP similarly to Rp-8-BrPETcGMPS during ACh stimulation. Analyses of Western blot and immunohistochemistry demonstrated that PDE2A exists in antral mucous cells. In conclusion, Rp-8-BrPETcGMPS accumulates cAMP by inhibiting PDE2 in ACh-stimulated antral mucous cells, leading to the delayed, but transient, increase in the frequency of Ca(2+)-regulated exocytosis. PDE2 may prevent antral mucous cells from excessive mucin secretion caused by the cAMP accumulation.

Inhibition of Ca2+‐regulated exocytosis by levetiracetam in guinea‐pig antral mucous cells: role of synaptic vesicle protein 2A, SV2A

Levetiracetam, an antiepileptic drug, a ligand for the SV2A, which contributes the priming of synaptic vesicles for neurotransmitter release. In this study, we investigated the effects of levetiracetam on Ca2+‐regulated exocytosis activated by acetylcholine (ACh) in antral mucous cells of guinea pigs. Because the priming dependent on ATP is the final step for releasing granular contents in the antral mucous cells. The Ca2+‐regulated exocytosis in isolated antral mucous cells was observed by videomicroscopy. Dinitrophenol (DNP) inhibits the priming resulting in a decrease in the frequency of Ca2+‐regulated exocytosis and 8‐bromoguanosine 3′, 5′‐cyclic monophosphate (8BrcGMP) enhances the priming resulting in an increase in its frequency. Levetiracetam inhibited the frequency of Ca2+‐regulated exocytosis by 40%, but it had no effects on the frequency in DNP‐treated cells. When we first added levetiracetam and then 8BrcAMP, 8BrcAMP did not enhance the frequency of Ca2+‐regulated exocytosis. In the next, when we first added 8BrcGMP and then levetiracetam, 8BrcGMP still enhanced the frequency. These results suggest that levetiracetam inhibits the priming of Ca2+‐regulated exocytosis in antral mucous cells. In conclusion, in antral mucous cells, the SV2A, which exists on the mucous granules, plays a crucial role in the priming.

Requirement of the Epithelium-specific Ets Transcription Factor Spdef for Mucous Gland Cell Function in the Gastric Antrum

Mucus-secreting cells of the stomach epithelium provide a protective barrier against damage that might result from bacterial colonization or other stimuli. Impaired barrier function contributes to chronic inflammation and cancer. Knock-out mice for the epithelium-specific transcription factor Spdef (also called Pdef) have defects in terminal differentiation of intestinal and bronchial secretory cells. We sought to determine the physiologic function of Spdef in the stomach, another site of significant levels of Spdef expression. We used in situ hybridization and immunohistochemistry to localize Spdef-expressing cells in the mouse stomach; targeted gene disruption to generate mice lacking Spdef; and histologic, immunologic, and transcriptional profiling approaches to determine the requirements of Spdef in stomach epithelial homeostasis. In wild-type mice, Spdef RNA and protein are expressed predominantly in mucous gland cells of the antrum and in mucous neck cells of the glandular corpus. Within 1.5 years, nearly half of homozygous mutant mice developed profound mucosal hyperplasia of the gastric antrum. Submucosal infiltration of inflammatory cells preceded antral hyperplasia by several weeks. The absence of Spdef impaired terminal maturation of antral mucous gland cells, as reflected in reduced expression of Muc6 and Tff2 and reduced numbers of secretory granules. Antral gene expression abnormalities overlapped significantly with those in Spdef(-/-) colon, including genes implicated in secretory granule traffic and functions. Spdef is required for terminal maturation of antral mucous gland cells to protect animals from gastric inflammation and resulting hyperplasia. These requirements parallel Spdef functions in secretory intestinal cells and suggest a common molecular mechanism for maturation of gastrointestinal secretory lineages.

Peroxisome proliferation activation receptor α modulation of Ca2+‐regulated exocytosis via arachidonic acid in guinea‐pig antral mucous cells

Indomethacin (IDM, 10 microm), not aspirin (ASA; 10 microm), enhanced the Ca(2+)-regulated exocytosis stimulated by 1 microm acetylcholine (ACh) in guinea-pig antral mucous cells. Indomethacin inhibits prostaglandin G/H (PGG/H) and 15R-hydroperoxy-eicosatetraenoic acid (15R-HPETE) production from arachidonic acid (AA), while ASA inhibits PGG/H production but accelerates 15R-HPETE production. This suggests that IDM accumulates AA. Arachidonic acid (2 microm) enhanced Ca(2+)-regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF(3)), also enhanced Ca(2+)-regulated exocytosis, indicating that AA, not products from AA, enhances Ca(2+)-regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor alpha (PPARalpha), because AA is a natural ligand for PPARalpha. A PPARalpha agonist (WY14643; 1 microm) enhanced Ca(2+)-regulated exocytosis, and a PPARalpha blocker (MK886; 50 microm) abolished the enhancement of Ca(2+)-regulated exocytosis induced by AA, IDM, AACOCF(3) and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARalpha exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca(2+)-regulated exocytosis activated by 1 microm ACh or 2 microm thapsigargin alone by 25-30%. Thus, ACh stimulates AA accumulation via an [Ca(2+)](i) increase, which activates PPARalpha, leading to enhancement of Ca(2+)-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARalpha enhances Ca(2+)-regulated exocytosis in guinea-pig antral mucous cells.

Arachidonic Acid/ppara Enhancement of Ca2+-Regulated Exocytosis in Antral Mucous Cells of Guinea Pig

Indomethacin (IDM, 10 μM) enhanced the Ca2+-regulated exocytosis stimulated by 1 μM ACh in guinea-pig antral mucous cells, but not aspirin (ASA, 10 μM). The differences in pharmacological actions between IDM and ASA suggest that IDM accumulates arachidonic acid (AA), which enhances Ca2+-regulated exocytosis. AA (2 μM) enhanced Ca2+-regulated exocytosis in antral mucous cells similarly to IDM, moreover, an analogue of AA, AACOCF3 (Arachidonyltrifluoromethyl ketone, a PLA2 blocker) also enhanced it. These indicate that the Ca2+-regulated exocytosis is directly enhanced by AA, not by the products of the AA cascade, such as PGs, LXs and LTs. We examined the effects of MK886 (an inhibitor of peroxisome proliferation activation receptor α, PPARα) on the AA-induced enhancement of Ca2+-regulated exocytosis, because AA is a natural ligand for PPARα. MK-886 (40 μM) abolished the enhancement of Ca2+-regulated exocytosis induced by AA, IDM and AACOCF3. Moreover, WY14643 (a PPARα agonist) enhanced the Ca2+-regulated exocytosis, similarly to AA. MK-886 decreased the frequency of the Ca2+-regulated exocytosis activated by 1 μM ACh or thapsigargin by 25-30 %. Western blotting and immunohistochemical examinations demonstrated that PPARα exists in antral mucous cells. Thus, ACh stimulates AA accumulation via increases in [Ca2+]i, and then, AA activates PPARα, which enhances Ca2+-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARα maintains Ca2+-regulated exocytosis of the antral mucous cells of guinea pig.

Ca2+‐regulated exocytosis modulated by [Cl−]i in ACh‐stimulated antral mucous cells of guinea pig

The effects of [Cl−]i on ACh‐stimulated exocytosis were studied in guinea pig antral mucous cells. ACh activated Ca2+‐regulated exocytosis (initial phase followed by a sustained phase). Bumetanide or an NO3− solution enhanced it, in contract, NPPB decreased it and eliminated the enhancement. ATP depletion studies demonstrated that exposure of NO3− solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3− solution after ATP depletion induced only a sustained phase. Measurements of [Cl−]i revealed that ACh decreases [Cl−]i. and that bumetanide and NO3− solution decreased [Cl−]i and enhanced the ACh‐evoked [Cl−]i decrease; in contrast, NPPB increased [Cl−]i and inhibited the [Cl−]i decrease induced by ACh, bumetanide or NO3− solution. [Ca2+]i measurements showed that a decrease in [Cl−]i enhanced the ACh‐stimulated [Ca2+]i increase. In conclusion, a decrease in [Cl−]i accelerates ATP‐dependent priming and [Ca2+]i increase, which enhance Ca2+‐regulated exocytosis in ACh‐stimulated antral mucous cells.

Cl−]imodulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.

Arachidonic acid modulation of Ca2+-regulated exocytosis via PPARα/cGMP in gastric antral mucous cells of guinea pig

Arachidonic acid modulation of Ca2+-regulated exocytosis via PPARα/cGMP in gastric antral mucous cells of guinea pig

A monoclonal antibody, PGM34, against 6‐sulfated blood‐group H type 2 antigen, on the carbohydrate moiety of mucin

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

ACh‐stimulated exocytosis enhanced by arachidonic acid in guinea pig antral mucous cells: cGMP accumulation via PPARα

Arachidonic acid (AA, 10 nM – 2 μM) enhanced Ca2+-regulated exocytosis in acetylcholine (ACh) stimulated antral mucous cells of guinea pig. The enhancement in ACh-stimulated exocytosis induced by AA was not mediated via PGE2 accumulation. The AA actions were inhibited by MK886 (50 μM, an inhibitor of by peroxisome proliferation activation receptor ? (PPAR?), and were mimicked by PPAR? agonists (Eicosatetraynoic acid (ETYA) and WY14643). The enhancement of ACh-stimulated exocytosis induced by AA, ETYA and WQY14643 was eliminated by a PKG inhibitor (Rp8BrPET-cGMPS) and was mimicked by 8Br-cGMP. Nitro-L-arginine methyl ester (L-NAME) also inhibited the enhancement induced by AA, ETYA and WY14643, and 1-Hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12) enhanced ACh-stimulated exocytosis similar to AA, ETYA and WY14643, which was inhibited by Rp8BrPET-cGMPS. AA, ETYA or WY14643 did not increase intracellular Ca2+ concentration ([Ca2+]i) and did not enhance ACh-stimulated increases in [Ca2+]i. Measurements of cGMP contents in antral mucosa demonstrated that stimulation with ACh, AA and PPAR? agonists stimulate cGMP accumulation, which is inhibited by MK886 and L-NAME. In conclusion, in ACh-stimulated antral mucous cells, AA accumulated stimulates PPAR?, which activates the NO/cGMP cascade, and PKG activated by cGMP enhances Ca2+-regulated exocytosis in antral mucous cells by increasing number of the primed granule.μ μ μ μ μ