scholarly journals Acid secretion-associated translocation of KCNJ15 in gastric parietal cells

2011 ◽  
Vol 301 (4) ◽  
pp. G591-G600 ◽  
Author(s):  
Wenjun He ◽  
Wensheng Liu ◽  
Catherine S. Chew ◽  
Susan S. Baker ◽  
Robert D. Baker ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Western Blot ◽  
Acid Secretion ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Apical Membrane ◽  
Parietal Cells ◽  
Immunofluorescence Staining ◽  
Gastric Glands ◽  
Gastric Parietal Cells

Potassium ions are required for gastric acid secretion. Several potassium channels have been implicated in providing K+ at the apical membrane of parietal cells. In examining the mRNA expression levels between gastric mucosa and liver tissue, KCNJ15 stood out as the most highly specific K+ channel in the gastric mucosa. Western blot analysis confirmed that KCNJ15 is abundant in the stomach. Immunofluorescence staining of isolated gastric glands indicated that KCNJ15 was expressed in parietal cells and chief cells, but not in mucous neck cells. In resting parietal cells, KCNJ15 was mainly found in puncta throughout the cytoplasm but was distinct from H+-K+-ATPase. Upon stimulation, KCNJ15 and H+-K+-ATPase become colocalized on the apical membranes, as suggested by immunofluorescence staining. Western blot analysis of the resting and the stimulated membrane fractions confirmed this observation. From nonsecreting preparations, KCNJ15-containing vesicles sedimented after a 4-h centrifugation at 100,000 g, but not after a 30-min spin, which did sediment most of the H+-K+-ATPase-containing tubulovesicles. Most of the KCNJ15 containing small vesicle population was depleted upon stimulation of parietal cells, as indicated by the fact that the KCNJ15 signal was shifted to a large membrane fraction that sedimented at 4,000 g. Our results demonstrate that, in nonsecreting parietal cells, KCNJ15 is stored in vesicles distinct from the H+-K+-ATPase-enriched tubulovesicles. Furthermore, upon stimulation, KCNJ15 and H+-K+-ATPase both translocate to the apical membrane for active acid secretion. Thus KCNJ15 can be added to the family of apical K+ channels in gastric parietal cells.

scholarly journals Polarized Distribution of IQGAP Proteins in Gastric Parietal Cells and Their Roles in Regulated Epithelial Cell Secretion

2003 ◽  
Vol 14 (3) ◽  
pp. 1097-1108 ◽  
Author(s):  
Rihong Zhou ◽  
Zhen Guo ◽  
Charles Watson ◽  
Emily Chen ◽  
Rong Kong ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Actin Cytoskeleton ◽  
Acid Secretion ◽  
Rho Gtpase ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Parietal Cells ◽  
Actin Dynamics ◽  
Cell Secretion ◽  
Gastric Parietal Cells

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.

Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells

1989 ◽  
Vol 256 (6) ◽  
pp. G1082-G1089 ◽  
Author(s):  
D. K. Hanzel ◽  
T. Urushidani ◽  
W. R. Usinger ◽  
A. Smolka ◽  
J. G. Forte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Secretion ◽  
Parietal Cell ◽  
Blood Cells ◽  
Apical Membrane ◽  
Staining Pattern ◽  
Parietal Cells ◽  
Entire Cell ◽  
Gastric Parietal Cells ◽  
Stimulation Of

Monoclonal antibodies were raised against an 80-kDa phosphoprotein (80K) that is phosphorylated upon stimulation of gastric acid secretion and that copurifies with the acid-forming H+-K+-ATPase isolated from stimulated tissue. These antibodies were used to demonstrate that in the gastric mucosa 80K is limited to parietal cells and not found in surface, mucous neck, or chief cells. 80K was also found in other transporting epithelia, including intestine and kidney, but was not found in brain, liver, red blood cells, or colon. Immunohistological localization of 80K in resting glands revealed a fine network, projecting from the gland lumen and anastomosing throughout the parietal cell. This network is quite similar to the staining pattern for F-actin contained in microvilli that line the apical membrane of parietal cells. Stimulation of acid secretion rearranges 80K to a more rugose pattern filling the entire cell. In stimulated cells the distribution pattern of 80K is indistinguishable from that stained with antibodies against the H+-K+-ATPase. These data strongly suggest that 80K is an apical membrane protein of the parietal cell.

State of actin in gastric parietal cells

1998 ◽  
Vol 274 (1) ◽  
pp. C97-C104 ◽  
Author(s):  
John G. Forte ◽  
Bernice Ly ◽  
Qinfen Rong ◽  
Shoji Ogihara ◽  
Marlon Ramilo ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Parietal Cell ◽  
Parietal Cells ◽  
Secretory Process ◽  
Cell Protein ◽  
Cell Secretion ◽  
Gastric Glands ◽  
Western Blot Assay ◽  
Deoxyribonuclease I ◽  
Gastric Parietal Cells

Remodeling of the apical membrane-cytoskeleton has been suggested to occur when gastric parietal cells are stimulated to secrete HCl. The present experiments assayed the relative amounts of F-actin and G-actin in gastric glands and parietal cells, as well as the changes in the state of actin on stimulation. Glands and cells were treated with a Nonidet P-40 extraction buffer for separation into detergent-soluble (supernatant) and detergent-insoluble (pellet) pools. Two actin assays were used to quantitate actin: the deoxyribonuclease I binding assay to measure G-actin and F-actin content in the two pools and a simple Western blot assay to quantitate the relative amounts of actin in the pools. Functional secretory responsiveness was assayed by aminopyrine accumulation. About 5% of the total parietal cell protein is actin, with about 90% of the actin present as F-actin. Stimulation of acid secretion resulted in no measurable change in the relative amounts of G-actin and cytoskeletal F-actin. Treatment of gastric glands with cytochalasin D inhibited acid secretion and resulted in a decrease in F-actin and an increase in G-actin. No inhibition of parietal cell secretion was observed when phalloidin was used to stabilize actin filaments. These data are consistent with the hypothesis that microfilamentous actin is essential for membrane recruitment underlying parietal cell secretion. Although the experiments do not eliminate the importance of rapid exchange between G- and F-actin for the secretory process, the parietal cell maintains actin in a highly polymerized state, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

scholarly journals Glycyrrhizin inhibits LPS-induced inflammatory responses in goat ruminal epithelial cells in vitro

2020 ◽  
Author(s):  
Xuanxuan Pu ◽  
Pattygouri Mullahred ◽  
Junfeng Liu ◽  
Xuefeng Guo ◽  
Jian Gao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Cell Viability ◽  
Mrna Expression ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Staining ◽  
Dependent Manner ◽  
Relative Mrna Expression

AbstractA long-term of high concentration feeding in ruminants can bring huge economic profits, but it also impose ruminants into great threat of suffering subacute ruminal acidosis (SARA). SARA is a kind of disease which attenuate the health, feed intake and production of ruminants, and when ruminants suffer SARA, the concentration of lipopolysaccharide (LPS) increase largely. Glycyrrhizin is reported to have anti-inflammation effects, and the study was conducted to investigate effects of glycyrrhizin on LPS-induced goat ruminal epithelial cells (GRECs) to provide evidence for using glycyrrhizin as a treatment for SARA. Effects of LPS, and glycyrrhizin on cell viability of GRECs were investigated, respectively. Then GRECs were stimulated with LPS (50 mg/L) for 2 h, and glycyrrhizin were added at the concentration of 0, 50, 75, 100 and 125 mg/L for 24 h to investigate the expression of inflammatory cytokines (by Elisa kits), the mRNA expression of NF-κB and inflammatory cytokines (by qRT-PCR), the distribution of Zo-1 and Occludin (by immunofluorescence staining), the expression of Occludin (by Western blot analysis), and the morphology of GRECs. The results showed that: (1) Glycyrrhizin at the concentration of 50, 75, 100, and 125 mg/L had no cytotoxic effects on GRECs, and LPS at the concentration of 50 mg/L significantly decreased the cell viability of GRECs. (2) Glycyrrhizin attenuated the expression and relative mRNA expression of TNF-α, IL-1β, IL-6, IL-8 and IL-12 by a dose-dependent manner, and significantly attenuated the relative mRNA expression of NF-κB. (3) Immunofluorescence staining and Western blot analysis showed that the quantity of Zo-1 and Occludin, and the expression of Occludin all increased with the treatment of glycyrrhizin. (4) Glycyrrhizin attenuated LPS-induced autophagy and protected the structural integrity of GRECs. In conclusion, glycyrrhizin significantly inhibited the inflammatory response in LPS-stimulated GRECs, and it may be used as a potential agent for the treatment of SARA.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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