mouse-human-derived-primary-gastric-epithelial-monolayer-culture-for

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

a-preclinical-human-derived-autologous-gastric-cancer-organoidimmune

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

University of Cincinnati

University of Arizona, Tucson

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF(2alpha) analog; and PGF(2alpha) were examined in HCN-1A cells, a model system for studies of large conductance Ca(2+) activated K(+)(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca(2+)](i) changes and protection against long-term irreversible glutamate-induced [Ca(2+)](i) increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC(50) of 0.6+/-0.2 nM and had no effect on Cl(-) currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF(2alpha) activated a Cl(-) current sensitive to [Ca(2+)](i) chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca(2+) signaling Cl(-) current activation. Latanoprost and PGF(2alpha), but not unoprostone, increased [Ca(2+)](i). Unoprostone, PGF(2alpha) only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca(2+) deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF(2alpha). Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.

Cellular and molecular effects of unoprostone as a BK channel activator.

Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.

Effects of lubiprostone on human uterine smooth muscle cells.

Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out.

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors.

Effects of cis-unoprostone isopropyl, its primary metabolite M1, trans-unoprostone isopropyl, latanoprost free acid, and fluprostenol were studied on Ca(2+)-activated K(+) (BK) channels, plasma membrane potential, [cAMP](i), [cGMP](i), and steady state [Ca(2+)](i), and protection against endothelin-1 (ET-1)-induced steady state [Ca(2+)](i) increases in human cortical neuronal (HCN-1A), trabecular meshwork (HTMC), and pulmonary artery smooth muscle (PASMC) cells. Effects on recombinant human prostaglandin (PG) receptors were determined.

Unoprostone isopropyl and metabolite M1 activate BK channels and prevent ET-1-induced [Ca²⁺]i increases in human trabecular meshwork and smooth muscle.

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Methadone but not morphine inhibits lubiprostone-stimulated Cl- currents in T84 intestinal cells and recombinant human ClC-2, but not CFTR Cl- currents.

It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion. 

Differentiation between human ClC-2 and CFTR Cl- channels with pharmacological agents.

Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]), EP, or EP receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl current activation (increasing ECs) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA/RGEA) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP] in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl currents without increasing [cAMP] while PGE and forskolin-IBMX increased both. Fatty acids were not agonists of EP or EP receptors. L-161,982, a supposed EP-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP], and EP or EP receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.

Identification of the fatty acid activation site on human ClC-2.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ArticleTotal : 2	Year
Publication title Cited by 9	2018
Publication title Cited by 49	2021

Article	Year
Cellular and molecular effects of unoprostone as a BK channel activator. Biochimica et biophysica acta\| PubMed ID: 17307133	2007
Effects of lubiprostone on human uterine smooth muscle cells. Prostaglandins & other lipid mediators\| PubMed ID: 18440264	2008
Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors. BMC pharmacology\| PubMed ID: 22553939	2012
Unoprostone isopropyl and metabolite M1 activate BK channels and prevent ET-1-induced [Ca²⁺]i increases in human trabecular meshwork and smooth muscle. Investigative ophthalmology & visual science\| PubMed ID: 22786902	2012
Methadone but not morphine inhibits lubiprostone-stimulated Cl- currents in T84 intestinal cells and recombinant human ClC-2, but not CFTR Cl- currents. Cell biochemistry and biophysics\| PubMed ID: 22918821	2013
Differentiation between human ClC-2 and CFTR Cl- channels with pharmacological agents. American journal of physiology. Cell physiology\| PubMed ID: 25009109	2014
Identification of the fatty acid activation site on human ClC-2. American journal of physiology. Cell physiology\| PubMed ID: 28424169	2017
CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium. The Journal of pathology\| PubMed ID: 28497484	2017

Jayati Chakrabarti

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