Dataset Information

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium.

ABSTRACT:

Background and purpose

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H?S) affects ion secretion across rat distal colonic epithelium.

Experimental approach

Changes in short-circuit current induced by the H?S-donor, sodium hydrosulphide (NaHS; 10 mmol·L?¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²(+) concentration ([Ca²(+)](i)) and Ca²(+) in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

Key results

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na(+) -K(+)-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²(+)-dependent) basolateral K(+) channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²(+)](i) , an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²(+). Initial falls in [Ca²(+)](i) were not mediated by a sequestration of Ca²(+) in intracellular Ca²(+) storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²(+)](i) were inhibited by 2',4'-dichlorobenzamil, an inhibitor of the Na(+)-Ca²(+)-exchanger, and attenuated in Na(+)-free buffer, suggesting a transient stimulation of Ca²(+) outflow by this transporter, directly demonstrated by Mn²(+) quenching experiments.

Conclusions and implications

ATP-sensitive and Ca²(+)-dependent basolateral K(+) conductances, the basolateral Na(+)-K(+)-pump as well as Ca²(+) transporters were involved in the action of H?S in regulating colonic ion secretion.

SUBMITTER: Pouokam E

PROVIDER: S-EPMC3031060 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium.

Pouokam E E Diener M M

British journal of pharmacology 20110101 2

<h4>Background and purpose</h4>The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.<h4>Experimental approach</h4>Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²(+) concentration ([Ca²(+)](i)) and Ca²(+) in intracellular stores were measured with f ...[more]

PMID: 20840536

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Project description:Comparison of global gene expression in the proximal and distal colonic epithelium in azoxymethane treated rats. Experiment Overall Design: Ten male Sprague Dawley rats weighing approximately 245 Â± 14.5 g were housed in wire-bottomed caging in a temperature controlled room (22-24Â°C) with a 12 h light/dark cycle. They were randomly allocated into two groups (n=5) with approximately equal body weights. They were given free access to water and modified AIN-93G diet for 10 days when they were injected with 15 mg of AOM/kg subcutaneously (Sigma Chemical Co., St. Louis, MO, USA). They were anaesthetised with isoflurane and killed by exsanguination six hours after injection. The large bowel (excluding the rectum) was removed, opened longitudinally along the mesenteric border and digesta removed. The colon was rinsed clean with PBS and transferred to a chilled ceramic plate. The most distal and the most proximal 0.5 cm were discarded. Mucosal samples for gene expression and protein analyses were collected by scraping the next 4 cm of proximal and distal colon with new microscope slides. Experiment Overall Design: The mucosal scraping was transferred into RNAlater (Sigma Chemical Co., St. Louis, MO, USA) and stored at -80oC for later processing. All instruments were replaced or cleaned thoroughly between animals. All procedures involving animals were approved by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Human Nutrition Animal Ethics Committee and complied with the Australian code of practice (2004). Experiment Overall Design: The distal and proximal colonic mucosal samples were removed from the RNAlater stabilisation reagent (Sigma, Australia) and placed in 1ml of TRIzolÂ® Reagent (Invitrogen, Sydney, N.S.W., Australia). Samples were then homogenised using beads (mix of 2.5mm glass and 0.1 - 1.0mm diameter silicon-zirconian beads) in a MiniBeadbeater-8â¢ (BioSpec Products Inc. Oklahoma, US). Total RNA was then extracted according to the TRIzolÂ® Reagent manufacturerâs instruction after which samples were further purified using RNAeasy mini spin columns (QIAGEN, Doncaster, Victoria, Australia) with a DNase on-column digestion as per the manufactureâs instructions. The integrity of the RNA was checked using a Bioanalyzer 2100 (Agilent Technologies) and quantified using a NanoDropÂ® ND-1000 Spectrophotometer. Experiment Overall Design: RNA (4.5ug) samples were processed for Microarray expression analysis using high-density oligonucleotide arrays (AffymetrixÂ® GeneChip array, AffymetrixÂ®, Santa Clara, CA, USA) commensurate with the manufacturerâs instructions.

Project description:IntroductionHydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer.ObjectivesThis study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs.MethodsEighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively.ResultsThe H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells.ConclusionThese findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Dataset Information

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium.

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium.

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